Effect of Introduced Pseudomonas fluorescens Strains on Soil Nematode and Protozoan Populations in the Rhizosphere of Wheat and Pea

Abstract Previous studies have shown that inoculation of pea seeds with Pseudomonas fluorescens strains F113lacZY or F113G22 increased mineralization of organic nitrogen in the rhizosphere. In contrast, inoculation of the same strains onto wheat seeds reduced mineralization of N from organic residues incorporated into soil. In the present study, we report on a likely explanation of this phenomenon, which appears to be governed by the effect of plant-microbe interactions on bacterial-feeding nematodes and protozoa. In soil microcosm tests, inoculation of pea seeds with Pseudomonas fluorescens strains F113lacZY or F113G22 resulted in an increase in the number of nematodes and protozoa in the rhizosphere as compared to noninoculated controls. This trend was repeated using a model sand system into which the bacteriophagous nematode Caenorhabditis elegans was introduced. It was subsequently found that non-inoculated germinating pea seeds exerted a nematicidal effect on C. elegans, which was remedied by inoculation with either strain F113lacZY or F113G22. This suggests that nematicidal compounds released by the germinating pea seeds were metabolized by the microbial inoculants before they affected nematode populations in the spermosphere or rhizosphere of pea. In contrast, inoculation of wheat plants resulted in significantly lower nematode populations in the rhizosphere, whereas protozoan numbers were unaffected. No nematicidal effects of inoculated or noninoculated wheat seeds could be found, suggesting that microfaunal populations were affected at a later stage during plant growth. Because of their key roles in accelerating the turnover of microbially immobilized N and organic matter, plants that support a larger microfaunal population are likely to benefit from a higher availability of inorganic nitrogen. Therefore, an understanding of plant-microbe interactions and their effects on soil microfaunal populations is essential in order to assess the effects of microbial inocula on plant mineral nutrition. Received: 27 May 1999; Accepted: 15 July 1999; Online Publication: 17 December 1999     

Effect of Parental Growth on Dynamics of Conjugative Plasmid Transfer in the Pea Spermosphere

Plasmid transfer rates for the conjugative plasmid R388::Tn1721 from Pseudomonas cepacia (donor) to Pseudomonas fluorescens (recipient) on agar media, in broth, and in microcosms containing sterile or nonsterile soil, in the presence or absence of germinating pea seeds, were determined. Donors, recipients, and transconjugants were enumerated on selective media after 1 day on agar or in broth culture and over a 7-day period in soil or pea spermosphere microcosms. Donor and recipient growth rates and plasmid transfer rate constants [(gamma), where (gamma) = transconjugants (middot) (donors (middot) recipients)(sup-1) (middot) h(sup-1)] were calculated for three initial parental densities (10(sup4), 10(sup6), or 10(sup8) CFU/g or ml) in each system. For all initial density levels, values of (gamma) in agar and broth matings were higher than those in soil or in the pea spermosphere-rhizosphere microcosms. Values of (gamma) were not influenced by the pea spermosphere or by sterile or nonsterile conditions of the soil. However, (gamma) values in microcosm experiments were inversely related to initial parental density and were directly related to donor growth rates. Values of (gamma) averaged 4 x 10(sup-10), 4 x 10(sup-12), and 3 x 10(sup-14) when initial donor and recipient cell densities were 10(sup4), 10(sup6), and 10(sup8) CFU/g, respectively. These results suggest that the plasmid transfer rate constant is independent of parental cell density only when parental growth is not limited. In a resource-limited environment, intra- or interspecific competition may reduce the transfer rate by limiting parental growth.     

噬藻体和蓝藻间的基因转移及协同进化作用

生物物种之间的水平基因转移广泛存在于细菌、古生菌和真核生物中,并能造成同一生境中种群的快速协同进化。噬藻体是感染蓝藻的专一性病毒,近年研究表明其在蓝藻水华生消中发挥了重要作用,使人们认识到了噬藻体的重要生态地位。综述了物种间的水平基因转移,介绍了噬藻体遗传多样性研究中常用的光合作用基因、结构蛋白基因等靶标基因所介导的基因转移以及基因转移引起的病毒和宿主的协同进化,并介绍了研究基因转移所用到的试验技术以及今后所要面临的问题。     

Horizontal Gene Transfer and the History of Life

Microbes acquire DNA from a variety of sources. The last decades, which have seen the development of genome sequencing, have revealed that horizontal gene transfer has been a major evolutionary force that has constantly reshaped genomes throughout evolution. However, because the history of life must ultimately be deduced from gene phylogenies, the lack of methods to account for horizontal gene transfer has thrown into confusion the very concept of the tree of life. As a result, many questions remain open, but emerging methodological developments promise to use information conveyed by horizontal gene transfer that remains unexploited today.The discovery of the existence of prokaryotic microbes dates back more than 300 years. Since then, our picture of our distant microscopic relatives has undergone several revolutions: from being the living “proofs” of the existence of spontaneous generation, they became later the “archaic” representatives of our distant ancestors, to finally be legitimately recognized as exceptionally diverse organisms, keystone to any ecosystem, including the most familiar and the most hostile environments on Earth. Similarly, although they were first seen as elementary and unbreakable bricks of life, they are now seen as genetically composite bodies, heavyweight champions of “gene robbery.” The most recent of these revolutions has indeed been the realization of their unparalleled ability to integrate genetic material coming from more or less evolutionarily distant organisms. This mechanism is called “horizontal gene transfer” as opposed to vertical transmission from mother to daughter cell.     

Stability of a Pseudomonas putida KT2440 Bacteriophage-Carried Genomic Island and Its Impact on Rhizosphere Fitness

The stability of seven genomic islands of Pseudomonas putida KT2440 with predicted potential for mobilization was studied in bacterial populations associated with the rhizosphere of corn plants by multiplex PCR. DNA rearrangements were detected for only one of them (GI28), which was lost at high frequency. This genomic island of 39.4 kb, with 53 open reading frames, shows the characteristic organization of genes belonging to tailed phages. We present evidence indicating that it corresponds to the lysogenic state of a functional bacteriophage that we have designated Pspu28. Integrated and rarely excised forms of Pspu28 coexist in KT2440 populations. Pspu28 is self-transmissible, and an excisionase is essential for its removal from the bacterial chromosome. The excised Pspu28 forms a circular element that can integrate into the chromosome at a specific location, att sites containing a 17-bp direct repeat sequence. Excision/insertion of Pspu28 alters the promoter sequence and changes the expression level of PP_1531, which encodes a predicted arsenate reductase. Finally, we show that the presence of Pspu28 in the lysogenic state has a negative effect on bacterial fitness in the rhizosphere under conditions of intraspecific competition, thus explaining why clones having lost this mobile element are recovered from that environment.     

Phylogenetic Analysis of Pseudomonas syringae Pathovars Suggests the Horizontal Gene Transfer of argK and the Evolutionary Stability of hrp Gene Cluster

Pseudomonas syringae are differentiated into approximately 50 pathovars with different plant pathogenicities and host specificities. To understand its pathogenicity differentiation and the evolutionary mechanisms of pathogenicity-related genes, phylogenetic analyses were conducted using 56 strains belonging to 19 pathovars. gyrB and rpoD were adopted as the index genes to determine the course of bacterial genome evolution, and hrpL and hrpS were selected as the representatives of the pathogenicity-related genes located on the genome (chromosome). Based on these data, NJ, MP, and ML phylogenetic trees were constructed, and thus 3 trees for each gene and 12 gene trees in total were obtained, all of which showed three distinct monophyletic groups: Groups 1, 2 and 3. The observation that the same set of OTUs constitute each group in all four genes suggests that these genes had not experienced any intergroup horizontal gene transfer within P. syringae but have been stable on and evolved along with the P. syringae genome. These four index genes were then compared with another pathogenicity-related gene, argK (the phaseolotoxin-resistant ornithine carbamoyltransferase gene, which exists within the argK–tox gene cluster). All 13 strains of pv. phaseolicola and pv. actinidiae used had been confirmed to produce phaseolotoxin and to have argK, whose sequences were completely identical, without a single synonymous substitution among the strains used (Sawada et al. 1997a). On the other hand, argK were not present on the genomes of the other 43 strains used other than pv. actinidiae and pv. phaseolicola. Thus, the productivity of phaseolotoxin and the possession of the argK gene were shown at only two points on the phylogenetic tree: Group 1 (pv. actinidiae) and Group 3 (pv. phaseolicola). A t test between these two pathovars for the synonymous distances of argK and the tandemly combined sequence of the four index genes showed a high significance, suggesting that the argK gene (or argK–tox gene cluster) experienced horizontal gene transfer and expanded its distribution over two pathovars after the pathovars had separated, thus showing a base substitution pattern extremely different from that of the noncluster region of the genome. Received: 18 January 1999 / Accepted: 25 May 1999     

Genomic Analysis of Xanthomonas translucens Pathogenic on Wheat and Barley Reveals Cross-Kingdom Gene Transfer Events and Diverse Protein Delivery Systems

In comparison to dicot-infecting bacteria, only limited numbers of genome sequences are available for monocot-infecting and in particular cereal-infecting bacteria. Herein we report the characterisation and genome sequence of Xanthomonas translucens isolate DAR61454 pathogenic on wheat and barley. Based on phylogenetic analysis of the ATP synthase beta subunit (atpD) gene, DAR61454 is most closely related to other X. translucens strains and the sugarcane- and banana- infecting Xanthomonas strains, but shares a type III secretion system (T3SS) with X. translucens pv. graminis and more distantly related xanthomonads. Assays with an adenylate cyclase reporter protein demonstrate that DAR61454''s T3SS is functional in delivering proteins to wheat cells. X. translucens DAR61454 also encodes two type VI secretion systems with one most closely related to those found in some strains of the rice infecting strain X. oryzae pv. oryzae but not other xanthomonads. Comparative analysis of 18 different Xanthomonas isolates revealed 84 proteins unique to cereal (i.e. rice) infecting isolates and the wheat/barley infecting DAR61454. Genes encoding 60 of these proteins are found in gene clusters in the X. translucens DAR61454 genome, suggesting cereal-specific pathogenicity islands. However, none of the cereal pathogen specific proteins were homologous to known Xanthomonas spp. effectors. Comparative analysis outside of the bacterial kingdom revealed a nucleoside triphosphate pyrophosphohydrolase encoding gene in DAR61454 also present in other bacteria as well as a number of pathogenic Fusarium species, suggesting that this gene may have been transmitted horizontally from bacteria to the Fusarium lineage of pathogenic fungi. This example further highlights the importance of horizontal gene acquisition from bacteria in the evolution of fungi.     

Generation of Genic Diversity among Streptococcus pneumoniae Strains via Horizontal Gene Transfer during a Chronic Polyclonal Pediatric Infection

Although there is tremendous interest in understanding the evolutionary roles of horizontal gene transfer (HGT) processes that occur during chronic polyclonal infections, to date there have been few studies that directly address this topic. We have characterized multiple HGT events that most likely occurred during polyclonal infection among nasopharyngeal strains of Streptococcus pneumoniae recovered from a child suffering from chronic upper respiratory and middle-ear infections. Whole genome sequencing and comparative genomics were performed on six isolates collected during symptomatic episodes over a period of seven months. From these comparisons we determined that five of the isolates were genetically highly similar and likely represented a dominant lineage. We analyzed all genic and allelic differences among all six isolates and found that all differences tended to occur within contiguous genomic blocks, suggestive of strain evolution by homologous recombination. From these analyses we identified three strains (two of which were recovered on two different occasions) that appear to have been derived sequentially, one from the next, each by multiple recombination events. We also identified a fourth strain that contains many of the genomic segments that differentiate the three highly related strains from one another, and have hypothesized that this fourth strain may have served as a donor multiple times in the evolution of the dominant strain line. The variations among the parent, daughter, and grand-daughter recombinant strains collectively cover greater than seven percent of the genome and are grouped into 23 chromosomal clusters. While capturing in vivo HGT, these data support the distributed genome hypothesis and suggest that a single competence event in pneumococci can result in the replacement of DNA at multiple non-adjacent loci.     

The Conflict Between Horizontal Gene Transfer and the Safeguard of Identity: Origin of Meiotic Sexuality

Contrary to a widespread opinion, horizontal gene transfer (HGT) between distantly related microorganisms (such as Bacteria and Archaea) has not been demonstrated to occur on a large scale. Except for transfer of mobile elements between closely related organisms, most alleged HGT events reflect phylogenetic discrepancies that can be explained by a variety of artefacts or by the differential loss of paralogous gene copies either originally present in the Last Universal Common Ancestor (LUCA) to the three Domains (a sophisticated, genetically redundant and promiscuous community of protoeukaryotes), or created by duplications having occurred at later times. Besides, (i) there is no experimental evidence for the facile acquisition of foreign DNA between distant taxa and (ii) important biological constraints operate on the phenotypic success of genetic exchange at several levels, including protein–protein interactions involved in metabolic channelling; stable integration and expression of foreign DNA is, therefore, expected to require strong selection. Explaining phylogenetic discrepancies by artefacts or loss of paralogs does not eliminate difficulties in retracing species genealogy but maintains the picture of a universal tree of life, HGT between distant organisms being reduced to a trickle. We illustrate our thesis by the phylogenetic analysis of carbamoyltransferases, a family of paralogous proteins. Among higher eukaryotes HGT appears of limited scope except in asexual organisms. We suggest that meiotic sexuality (a hallmark of eukaryotes) emerged in the genetically redundant and protoeukaryotic LUCA as a molecular identity check providing a defence mechanism against the deleterious effects of HGT.     

Positive Selection and Horizontal Gene Transfer in the Genome of a Male-Killing Wolbachia

Wolbachia are a genus of widespread bacterial endosymbionts in which some strains can hijack or manipulate arthropod host reproduction. Male killing is one such manipulation in which these maternally transmitted bacteria benefit surviving daughters in part by removing competition with the sons for scarce resources. Despite previous findings of interesting genome features of microbial sex ratio distorters, the population genomics of male-killers remain largely uncharacterized. Here, we uncover several unique features of the genome and population genomics of four Arizonan populations of a male-killing Wolbachia strain, wInn, that infects mushroom-feeding Drosophila innubila. We first compared the wInn genome with other closely related Wolbachia genomes of Drosophila hosts in terms of genome content and confirm that the wInn genome is largely similar in overall gene content to the wMel strain infecting D. melanogaster. However, it also contains many unique genes and repetitive genetic elements that indicate lateral gene transfers between wInn and non-Drosophila eukaryotes. We also find that, in line with literature precedent, genes in the Wolbachia prophage and Octomom regions are under positive selection. Of all the genes under positive selection, many also show evidence of recent horizontal transfer among Wolbachia symbiont genomes. These dynamics of selection and horizontal gene transfer across the genomes of several Wolbachia strains and diverse host species may be important underlying factors in Wolbachia’s success as a male-killer of divergent host species.     

Genotypic and Phenotypic Diversity of phlD-Containing Pseudomonas Strains Isolated from the Rhizosphere of Wheat

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent Pseudomonas spp. results in the suppression of root diseases caused by certain fungal plant pathogens. In this study, fluorescent Pseudomonas strains containing phlD, which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands. To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2,4-DAPG producers. Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR. Representatives of this group were isolated from eight different soils taken from four different geographic locations. ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates. In most cases, a single genotype predominated among isolates obtained from each soil. Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized. Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates. Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others. Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates. Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses.     

Quantification of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens Strains in the Plant Rhizosphere by Real-Time PCR

A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing (phlD⁺) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD⁺ P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (C_Ts) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5 phlD⁺ strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD⁺ pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phlD-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r² = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.     

Nitrogen Availability to Pseudomonas fluorescens DF57 Is Limited during Decomposition of Barley Straw in Bulk Soil and in the Barley Rhizosphere

The availability of nitrogen to Pseudomonas fluorescens DF57 during straw degradation in bulk soil and in barley rhizosphere was studied by introducing a bioluminescent reporter strain (DF57-N3), responding to nitrogen limitation, to model systems of varying complexity. DF57-N3 was apparently not nitrogen limited in the natural and sterilized bulk soil used for these experiments. The soil was subsequently amended with barley straw, representing a plant residue with a high carbon-to-nitrogen ratio (between 60 and 100). In these systems the DF57-N3 population gradually developed a nitrogen limitation response during the first week of straw decomposition, but exclusively in the presence of the indigenous microbial population. This probably reflects the restricted ability of DF57 to degrade plant polymers by hydrolytic enzymes. The impact of the indigenous population on nitrogen availability to DF57-N3 was mimicked by the cellulolytic organism Trichoderma harzianum Rifai strain T3 when coinoculated with DF57-N3 in sterilized, straw-amended soil. Limitation occurred concomitantly with fungal cellulase production, pointing to the significance of hydrolytic activity for the mobilization of straw carbon sources, thereby increasing the nitrogen demand. Enhanced survival of DF57-N3 in natural soil after straw amendment further indicated that DF57 was cross-fed with carbon/energy sources. The natural barley rhizosphere was experienced by DF57-N3 as an environment with restricted nitrogen availability regardless of straw amendment. In the rhizosphere of plants grown in sterilized soil, nitrogen limitation was less severe, pointing to competition with indigenous microorganisms as an important determinant of the nitrogen status for DF57-N3 in this environment. Hence, these studies have demonstrated that nitrogen availability and gene expression in Pseudomonas is intimately linked to the structure and function of the microbial community. Further, it was demonstrated that the activities of cellulolytic microorganisms may affect the availability of energy and specific nutrients to a group of organisms deficient in hydrolytic enzyme activities.     

Construction and Screening of Barley Genomic Library with a B1-Hordein Gene Probe

Barley genomic library from cv NP113 was made in a replacement vector EMBL-3. The titre was found to be 1.25 x 10⁶ per μg of genomic DNA. The recombinants were screened using a B₁-hordein DNA probe. One clone contained a positively hybridizing 4 kb fragment.     

盐胁迫下大麦根系多胺代谢与其耐盐性的关系

研究了0～300mmol/LNaCl对大麦(Hordeum-vulgare-L.)幼苗生长速率、根系游离和结合态多胺含量以及多胺生物合成关键酶活性的影响。结果表明,在0～200mmol/L NaCl处理下精氨酸脱羧酶（ADC）、多胺氧化酶(PAO)以及转谷酰胺酶(Tgase)活性明显提高,而在300 mmol/L NaCl处理下活性下降。与之对应,游离腐胺（Put）含量随处理盐浓度的提高一直呈上升趋势,亚精胺(Spd)和在根系内检测到的未知多胺(Pax)在低浓度盐处理时含量上升,随盐浓度的提高含量下降。盐处理前后精胺(Spm)含量变化不明显。低浓度盐处理时游离态（Spd+Pax）/Put上升,随盐浓度的提高比值明显下降。结合态Put、Spd和Pax含量以及结合态多胺总量均在低浓度盐处理时上升,随盐浓度的提高含量明显下降。统计分析显示,大麦相对生长速率与游离态（Spd+Pax）/Put和结合态多胺含量间均呈极显著正相关关系,与游离态多胺和结合态多胺的比值间均呈显著负相关关系,上述结果说明盐胁迫下大麦体内游离态Spd、Pax与Put以及结合态形式之间的平衡与大麦耐盐性关系密切,游离态Put向Spd 、Pax以及结合态形式转化均有利于大麦耐盐性的提高.     

盐胁迫下大麦根系多胺代谢与其耐盐性的关系

研究了0－300mmol/L NaCl对大麦（Hordeum vulgare L.)幼苗生长速率,根系游离和结合态多胺含量以及多胺生物合成关键酶活性的影响。结果表明,在0－200mmol/L NaCl处理下精氨酸脱羧酶（ADC）、多胺氧化酶（PAO）以及转谷酰胺酶（TGase)活性明显提高,而在300mol/L NaCl处理下活性下降,与之对应,游离腐胺（Put)含量随处理盐浓度的提高一直呈上升趋势。亚精胺（Spd)和在根系内检测到的未知多胺（PAx）在低浓度盐处理时含量上升,随盐浓度的提高含量下降,盐处理前后精胺（Spm)含量变化不明显,低浓度盐处理时游离态（Spd PAx)/Put上升,随盐浓度的提高比值明显下降,结合态Put,Spd和PAx含量以及结合态多胺总量均在低浓度盐处理时上升,随盐浓度的提高含量明显下降,统计分析显示,大麦相对生长速率与游离态（Spd PAx）／Put和结合态多胺含量间均呈极显著正相关关系,与游离态多胺和结合态多胺的比值间均呈显著负相关关系,上述结果说明盐胁迫下大麦体内游离态Spd,PAx与Put以及结合态形式之间的平衡与大麦耐盐性关系密切,游离态Put向Spd,PAx以及结合态形式转化均有利于大麦耐盐性的提高。     

Root Colonization by Pseudomonas sp. DSMZ 13134 and Impact on the Indigenous Rhizosphere Bacterial Community of Barley

Over the last few decades, the ability of rhizosphere bacteria to promote plant growth has been considered to be of scientific, ecological, and economic interest. The properties and mechanisms of interaction of these root-colonizing bacteria have been extensively investigated, and plant protection agents that are based on these bacterial strains have been developed for agricultural applications. In the present study, the root colonization of barley by Pseudomonas sp. DSMZ 13134, that is contained in the commercially available plant protection agent Proradix®, was examined using the fluorescence in situ hybridization method with oligonucleotide probes and specific gfp-tagging of the inoculant strain in combination with confocal laser scanning microscopy. In the first phase of root colonization, the inoculant strain competed successfully with seed and soil-borne bacteria (including Pseudomonads) for the colonization of the rhizoplane. Pseudomonas sp. DSMZ 13134 could be detected in all parts of the roots, although it did not belong to the dominant members of the root-associated bacterial community. Gfp-tagged cells were localized particularly in the root hair zone, and high cell densities were apparent on the root hair surface. To investigate the impact of the application of Proradix® on the structure of the dominant root-associated bacterial community of barley, T-RFLP analyses were performed. Only a transient community effect was found until 3 weeks post-application.     

Production of Cyclic Lipopeptides by Pseudomonas fluorescens Strains in Bulk Soil and in the Sugar Beet Rhizosphere

The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere. Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca. 5 μg g⁻¹) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil. When a whole-cell inoculum of P. fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week. By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil. In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for ~2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days. All three CLPs remained detectable for several days in the rhizosphere. Subsequent tests of five other CLP-producing P. fluorescens strains also demonstrated significant production in the young rhizosphere. The results thus provide evidence that production of different CLPs is a common trait among many P. fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.     

A Method for Detection of Pseudobactin, the Siderophore Produced by a Plant-Growth-Promoting Pseudomonas Strain, in the Barley Rhizosphere

Detection in the rhizosphere of the siderophore produced by an inoculated microorganism is critical to determining the role of microbial iron chelators on plant growth promotion. We previously reported the development of monoclonal antibodies (MAb) to ferric pseudobactin, the siderophore of plant-growth-promoting Pseudomonas strain B10. One of these MAb reacted less strongly to pseudobactin than to ferric pseudobactin. The MAb reacted to Al(III), Cr(III), Cu(II), and Mn(II) complexes of pseudobactin at a level similar to the level at which it reacted to ferric pseudobactin and reacted less to the Zn(II) complex, but these metals would make up only a small fraction of chelated pseudobactin in soil on the basis of relative abundance of metals and relative binding constants. Fourteen-day-old barley plants grown in limed and autoclaved soil were inoculated with 10⁹ CFU of Pseudomonas strain Sm1-3, a strain of Pseudomonas B10 Rif^r Nal^r selected for enhanced colonization, and sampled 3 days later. Extraction and analysis of the roots and surrounding soil using the MAb in an immunoassay indicated a concentration of 3.5 × 10^-10 mol of ferric pseudobacting g^-1 (wet weight). This is the first direct measurement of a pseudobactin siderophore in soil or rhizosphere samples.     

Green Fluorescent Protein-Marked Pseudomonas fluorescens: Localization, Viability, and Activity in the Natural Barley Rhizosphere

The gfp-tagged Pseudomonas fluorescens biocontrol strain DR54-BN14 was introduced into the barley rhizosphere. Confocal laser scanning microscopy revealed that the rhizoplane populations of DR54-BN14 on 3- to 14-day-old roots were able to form microcolonies closely associated with the indigenous bacteria and that a majority of DR54-BN14 cells appeared small and almost coccoid. Information on the viability of the inoculant was provided by a microcolony assay, while measurements of cell volume, the intensity of green fluorescent protein fluorescence, and the ratio of dividing cells to total cells were used as indicators of cellular activity. At a soil moisture close to the water-holding capacity of the soil, the activity parameters suggested that the majority of DR54-BN14 cells were starving in the rhizosphere. Nevertheless, approximately 80% of the population was either culturable or viable but nonculturable during the 3-week incubation period. No impact of root decay on viability was observed, and differences in viability or activity among DR54-BN14 cells located in different regions of the root were not apparent. In dry soil, however, the nonviable state of DR54-BN14 was predominant, suggesting that desiccation is an important abiotic regulator of cell viability.