Jan Sørensen

Viscosinamide, a new cyclic depsipeptide with surfactant and antifungal properties produced by Pseudomonas fluorescens DR54

Pseudomonas fluorescens DR54 showed antagonistic properties against plant pathogenic Pythium ultimum and Rhizoctonia solani both in vitro and in planta. Antifungal activity was extractable from spent growth media, and fractionation by semi‐preparative HPLC resulted in isolation of an active compound, which was identified as a new bacterial cyclic lipodepsipeptide, viscosinamide, using 1D and 2D 1H‐, 13C‐NMR and mass spectrometry. The new antibiotic has biosurfactant properties but differs from the known biosurfactant, viscosin, by containing glutamine rather than glutamate at the amino acid position 2 (AA2). No viscosin production was observed, however, when Ps. fluorescens DR54 was cultured in media enriched with glutamate. In vitro tests showed that purified viscosinamide also reduced fungal growth and aerial mycelium development of both P. ultimum and R. solani. Viscosinamide production by Ps. fluorescens DR54 was tightly coupled to cell proliferation in the batch cultures, as the viscosinamide produced per cell mass unit approached a constant value. In batch cultures with variable initial C, N or P nutrient levels, there were no indications of elevated viscosinamide production during starvation or maintenance of the cultures in stationary phase. Analysis of cellular fractions and spent growth media showed that a major fraction of the viscosinamide produced remained bound to the cell membrane of Ps. fluorescens DR54. The isolation, determination of structure and production characteristics of the new compound with both biosurfactant and antibiotic properties have promising perspectives for the application of Ps. fluorescens DR54 in biological control.

Structure, production characteristics and fungal antagonism of tensin – a new antifungal cyclic lipopeptide from Pseudomonas fluorescens strain 96.578

Aim: To study the antagonistic activity by Pseudomonas fluorescens strain 96.578 on the plant pathogenic fungus Rhizoctonia solani.

Direct analysis of tfdA gene expression by indigenous bacteria in phenoxy acid amended agricultural soil.

Expression of the functional gene tfdA involved in degradation of phenoxyacetic acids such as 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA) was investigated during degradation scenarios in natural unseeded soil samples. The results illustrate how messenger RNA (mRNA)-based analysis is well suited to quantitatively study the activity of specific microbial populations in soil using phenoxyacetic acid biodegradation as a model system. Via quantitative real-time PCR, a clear response to the presence of phenoxy acids was shown during degradation in soil amended with 20 mg 2,4-D or MCPA per kg soil. Further, we found a relatively high degree of correlation between expression of the functional gene and the rates of mineralization. Melting curve analyses of real-time PCR products, supported by tfdA-denaturing gradient gel electrophoresis analysis showed that, although only class I tfdA genes were apparent in the indigenous microbial population, class III tfdA genes became predominant during incubation, and were the only genes expressed during degradation of MCPA in soil. In contrast, both classes were expressed during degradation of the structurally similar compound 2,4-D. The ability to quantify microbial transcripts directly in environmental samples will have a profound impact on our understanding of microbial processes in the environment in future studies.

Molecular tools in rhizosphere microbiology—from single-cell to whole-community analysis

It is the aim of this chapter to present an overview of new, molecular tools that have been developed over recent years to study individual, single cells and composite, complex communities of microorganisms in the rhizosphere. We have carefully focused on culture-independent assays and selected methodologies that have already been or will soon be applicable for rhizosphere microbiology. Emphasis is placed on rhizosphere bacteria and the review first describes a number of the new methodologies developed for detection and localization of specific bacterial populations using modern electron and fluorescence microscopy combined with specific tagging techniques. First half of the chapter further comprises a thorough treatise of the recent development of reporter gene technology, i.e. using specific reporter bacteria to detect microscale distributions of rhizosphere compounds such as nutrients, metals and organic exudates or contaminants. Second half of the chapter devoted to microbial community analysis contains a thorough treatise of nucleotide- and PCR-based technologies to study composition and diversity of indigenous bacteria in the natural rhizosphere. Also included are the most recent developments of functional gene and gene expression analyses in the rhizosphere based on specific mRNA transcript or transcriptome analysis, proteome analysis and construction of metagenomic libraries.

Soil and rhizosphere as habitats for Pseudomonas inoculants: new knowledge on distribution, activity and physiological state derived from micro-scale and single-cell studies

Pseudomonas spp. comprise an important group of bacteria used for biological control of microfungi in the plant rhizosphere. Successful performance of microbial inoculants requires both establishment, proliferation and activity under in situ conditions. To identify the factors controlling fate and performance of the inoculants, small-scale analyses are needed due to the heterogeneity characterizing the complex soil and rhizosphere environments. Direct staining techniques and advanced microscopy have provided the first detailed single-cell images of root colonization by these bacteria using fluorescent antibodies, fluorescent in situ hybridization and marker gene technology. These tracking methods have, in conjunction with activity assays, provided high-resolution data on the metabolic activity and growth of the inoculants. Finally, Pseudomonas reporter bacteria constructed to sense phosphorus, nitrogen, iron, and oxygen limitations have provided new insight into the significance of growth-limiting factors in the soil and along the root. The present work reviews the current knowledge on Pseudomonas inoculants in soil and rhizosphere based on these modern techniques. Finally, some perspectives for future studies are discussed.

Production of Cyclic Lipopeptides by Fluorescent Pseudomonads

Members of the genus Pseudomonas produce a large amount of metabolites that are released into the extracellular environment. Among these metabolites are siderophores, phytohormones, biosurfactants and several antibiotic compounds, which may have broad-spectrum activity24, 81. Production of cyclic lipopeptides (CLPs) including compounds referred to as lipodepsipeptides, lipoundecapeptides and so on, has recently been established as a common trait among the pseudomonads. However, CLPs are also produced by a variety of other bacteria as well as fungi. Among the bacterial metabolites the CLP surfactin produced by Bacillus subtilis has received large attention due to its powerful biosurfactant activity and the antifungal, antibacterial and antiviral effects78.

Back to the Future of Soil Metagenomics

Direct extraction and characterization of microbial community DNA through PCR amplicon surveys and metagenomics has revolutionized the study of environmental microbiology and microbial ecology. In particular, metagenomic analysis of nucleic acids provides direct access to the genomes of the “uncultivated majority.” Accelerated by advances in sequencing technology, microbiologists have discovered more novel phyla, classes, genera, and genes from microorganisms in the first decade and a half of the twenty-first century than since these “many very little living animalcules” were first discovered by van Leeuwenhoek (Table 1). The unsurpassed diversity of soils promises continued exploration of a range of industrial, agricultural, and environmental functions. The ability to explore soil microbial communities with increasing capacity offers the highest promise for answering many outstanding who, what, where, when, why, and with whom questions such as: Which microorganisms are linked to which soil habitats? How do microbial abundances change with changing edaphic conditions? How do microbial assemblages interact and influence one another synergistically or antagonistically? What is the full extent of soil microbial diversity, both functionally and phylogenetically? What are the dynamics of microbial communities in space and time? How sensitive are microbial communities to a changing climate? What is the role of horizontal gene transfer in the stability of microbial communities? Do highly diverse microbial communities confer resistance and resilience in soils?

Risk factors and prognosis after discharge for patients admitted because of suspected acute myocardial infarction with and without confirmed diagnosis

The prognosis regarding cardiac events--acute myocardial infarction (AMI) or cardiac death after discharge--was evaluated in 257 patients admitted because of suspected AMI due to chest pain, but in whom AMI was not confirmed (non-AMI patients). The findings and patient prognoses were compared with those of 275 patients with confirmed AMI. All patients were younger than 76 years and free of severe chronic diseases, and no cause of chest pain other than possible ischemic heart disease was found. The patients were followed for cardiac events for 11 to 24 months (median 14). The prognoses for the non-AMI patients were significantly better than those for the AMI patients (p = 0.05). The proportion without a cardiac event after 1 year was estimated at 91% and 86%, respectively. In the non-AMI patients, angina pectoris, previous AMI and electrocardiographic changes on admission (intraventricular block and permanent or transient ST-T changes) were significant predictors of cardiac events by univariate and multivariate analysis. In the AMI patients, female gender, heart failure, previous AMI and angina pectoris were significant predictors of cardiac events by univariate analysis. With use of multivariate analysis, female gender, heart failure and angina pectoris were independent predictors of cardiac events. Thus, non-AMI patients admitted with chest pain have a high risk of cardiac events after discharge. The risk is highest when there is evidence of coronary artery disease (electrocardiographic changes on admission and angina pectoris or previous AMI.

Confocal imaging of living fungal hyphae challenged with the fungal antagonist viscosinamide

The combination of confocal laser scan- ning microscopy (CLSM) and staining with vital fluo- rescence dyes has promising potential for studying subcellular structures in living filamentous fungi and oomycetes. CLSM offers high resolution images and the possibility of optical sectioning of the specimen. Vital dyes make it possible to do this while the cells are still alive and with minimum disturbance of cel- lular processes. In this study confocal microscopy and the vital dyes Nile red, SYTO 13, DIOC7(3), and car- boxy SNARF-1 were used on several species. Subse- quently the technique was used to detect effects of the fungal antagonist viscosinamide. These fluores- cent dyes were expected to stain hydrophobic ele- ments, nuclei, mitochondria, and cytoplasm respec- tively. Nile red and DIOC7(3) were found to be es- pecially well suited for confocal imaging. Nile red stained the vacuolar membranes in Rhizoctonia solani

Hyphae-Colonizing Burkholderia sp.—A New Source of Biological Control Agents Against Sheath Blight Disease ( Rhizoctonia solani AG1-IA ) in Rice

Sheath blight infection of rice by Rhizoctonia solani Kühn AG1-IA often results in serious yield losses in intensive rice cultivation. Biological control agents (BCAs) have previously been isolated but poor efficiency is often observed when applied under field conditions. This study compares a traditional dual-culture plate assay and a new water-surface microcosm assay for isolation of antagonistic soil bacteria. In the water-surface microcosm assay, floating pathogen mycelium is used as a source for isolation of hyphae-colonizing soil bacteria (HCSB), which are subsequently screened for antagonism. Ten antagonistic soil bacteria (ASB) isolated from a variety of Vietnamese rice soils using dual-culture plates were found to be affiliated with Bacillus based on 16S rRNA gene sequencing. However, all the ASB isolates grew poorly and showed no antagonism in the water-surface microcosm assay. In contrast, 11 (out of 13) HCSB isolates affiliated with Burkholderia sp. all grew well by colonizing the hyphae in the microcosms. Two of the Burkholderia sp. isolates, assigned to B. vietnamiensis based on recA gene sequencing, strongly inhibited fungal growth in both the dual-culture and water-surface microcosm assays; HCSB isolates affiliated to other species or species groups showed limited or no inhibition of R. solani in the microcosms. Our results suggest that HCSB obtained from floating pathogen hyphae can be a new source for isolation of efficient BCAs against R. solani, as the isolation assay mimics the natural habitat for fungal-bacterial interaction in the fields.