Identification and potential enzyme capacity of flavobacteria isolated from the rhizosphere of barley (Hordeum vulgare L.).

The diversity of 99 flavobacterium-like isolates from a barley rhizosphere is described. They were identified on 1/10 strength tryptic soy agar by their yellowish colour and a flexirubin reaction after exposure to 10% KOH.16S rDNA partial sequencing identified the majority (70%) of isolates as Flavobacterium. Twelve percent of the isolates belonged to other genera in the phylum Bacteroidetes. Finally 17% of the isolates did not belong to the phylum Bacteroidetes. Most of the Flavobacterium isolates were affiliated to various aquatic, validly named species and likely represent a bulk of undescribed soil Flavobacterium species found in especially high numbers in the rhizosphere of young plant roots. Most Flavobacterium isolates showed gliding motility on CY agar and VY/2 agar, whereas none of the other isolates shared this feature. A high percentage of Flavobacterium strains produced enzymes involved in polysaccharide and protein digestion as well as extracellular phosphatases, compared with strains related to other genera in the phylum Bacteroidetes.

Cultivating previously uncultured soil bacteria using a soil substrate membrane system.

Most bacteria are recalcitrant to traditional cultivation in the laboratory. The soil substrate membrane system provides a simulated environment for the cultivation of previously undescribed soil bacteria as microcolonies. The system uses a polycarbonate membrane as a solid support for growth and soil extract as the substrate. Diverse microcolonies can be visualized using total bacterial staining combined with fluorescence in situ hybridization (FISH) after 7-10-d incubation. Molecular typing shows that the majority of microcolony-forming bacteria recovered using this protocol were resistant to growth using standard methods. The protocol takes <4 h of bench time over the 10-d period.

Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria.

Traditional microbiological methods of cultivation recover less than 1% of the total bacterial species, and the culturable portion of bacteria is not representative of the total phylogenetic diversity. Classical cultivation strategies are now known to supply excessive nutrients to a system and therefore select for fast-growing bacteria that are capable of colony or biofilm formation. New approaches to the cultivation of bacteria which rely on growth in dilute nutrient media or simulated environments are beginning to address this problem of selection. Here we describe a novel microcultivation method for soil bacteria that mimics natural conditions. Our soil slurry membrane system combines a polycarbonate membrane as a growth support and soil extract as the substrate. The result is abundant growth of uncharacterized bacteria as microcolonies. By combining microcultivation with fluorescent in situ hybridization, previously "unculturable" organisms belonging to cultivated and noncultivated divisions, including candidate division TM7, can be identified by fluorescence microscopy. Successful growth of soil bacteria as microcolonies confirmed that the missing culturable majority may have a growth strategy that is not observed when traditional cultivation indicators are used.

Luteibacter rhizovicinus gen. nov., sp. nov., a yellow-pigmented gammaproteobacterium isolated from the rhizosphere of barley (Hordeum vulgare L.).

Three strains of Gram-negative, aerobic, yellow-pigmented, chemo-organotrophic bacteria, motile by a polar flagellum, were isolated from the rhizosphere of spring barley (Hordeum vulgare L.) at a research field near Copenhagen, Denmark. The three strains, LJ79, LJ96T and LJ99, formed visible colonies on one-tenth-strength tryptic soy broth supplemented with agar (1/10 TSBA) after incubation for 6 days at 15 degrees C. The strains hydrolysed starch, casein (skimmed milk), gelatin and various pentoses and hexoses and grew on MacConkey agar and full-strength TSBA. Growth on 1/10 TSBA occurred at 4-30 degrees C, pH 6-9 and 0-3 % (w/v) NaCl. The strains had identical 16S rRNA gene sequences and ERIC (enterobacterial repetitive intergenic consensus sequence) fingerprint profiles, but could be differentiated by their RAPD (random amplified polymorphic DNA) fingerprint patterns. Strain LJ96(T) had a DNA G+C content of 64.3 mol% and the major fatty acids were 15 : 0 iso (23.4 %), 17 : 1 iso omega9c (25.5 %) and 17 : 0 iso (18.1 %). Phylogenetic analysis of the 16S rRNA gene sequences of the three strains showed 96 % sequence similarity to Rhodanobacter lindaniclasticus LMG 18385T, 95 % to Frateuria aurantia DSM 6220T and 96 % to Fulvimonas soli LMG 19981T. Using LJ96T DNA as probe, DNA-DNA hybridizations documented the relationship of the three strains to a single species (87.4-98.7 % relatedness) and showed less than 30 % relatedness to Frateuria aurantia DSM 6220T and Fulvimonas soli DSM 14263T. Rhodanobacter lindaniclasticus LMG 18385T is not extant and the strain not available from any public strain collections, thus DNA-DNA hybridization could not include this strain. On the basis of genotypic and phenotypic characteristics, the three yellow-pigmented strains could also be differentiated from Frateuria aurantia, Fulvimonas soli and Rhodanobacter lindaniclasticus. The name Luteibacter rhizovicinus gen. nov., sp. nov. is proposed, with the type strain LJ96T (=DSM 16549T=ATCC BAA-1015T).

Non-target effects of bacterial biological control agents suppressing root pathogenic fungi.

Microorganisms have knowingly been used during the last century to control plant diseases. During the last decades, research and application of biological control agents (BCAs) as a pest control strategy have gained increasing attention. This review focuses specifically on non-target effects of bacterial BCAs that are used to suppress root pathogenic fungi. It attempts to critically evaluate the strengths and weaknesses of non-target effect studies published to date and relate them to the success of the BCA in fungal pathogen control. Significant non-target effects of BCAs have indeed been observed, but these are generally small in scale and limited to a growth season, and have not been proven to affect soil health. We discuss these studies and point out what we believe are notable deficiencies. Among the modes of disease suppression by BCAs, antibiotic production is believed to be of major importance. But assurances that in situ antibiotic production actually occurs in environmental samples are lacking in the non-target effect studies. Also the effectiveness of the BCA on the target pathogen, the absence of appropriate controls for inoculation effects, and the presence of pathogenic fungi are missing in most studies. In future non-target effect studies we recommend focusing on proven effective BCAs and clearly distinguishing effects of antimicrobial compounds from effects of general microbial activity.

Mercury decreases culturability of Pseudomonas frederiksbergensis JAJ 28 in soil microcosms.

Mercury is a biologically potent heavy metal, which has been found to change the diversity of culturable bacteria. Therefore, we investigated whether Hg kills bacteria in soil or reduces culturability. Soil microcosms were inoculated with Pseudomonas frederiksbergensis JAJ 28 and were sampled regularly during 28 days. The total number of acridine orange-stained cells was relatively constant, and Hg reduced the number on only one sampling day. However, the fraction of culturable cells on 1/10 tryptic soy agar was lowered on days 6, 13, and 21. The number of microcolony forming units, which represents viable cells, was also affected by Hg, but this effect was delayed compared with the effects on CFUs. The amount of headspace CO2 per cell was overall increased by Hg, another indication of the toxic effects of Hg on the bacterial cells. Our results thus emphasize the need to take culturability into account when studying the effects of heavy metals on bacterial diversity.

Isolation of methane oxidising bacteria from soil by use of a soil substrate membrane system.

Abstract A new method for isolation of methane oxidising bacteria (methanotrophs) is presented. Soil samples from a wetland area and a landfill were plated on polycarbonate membranes, which were incubated in a methane-air atmosphere using a non-sterile soil suspension as the medium. The membrane acted as a permeable growth support. The membrane method resulted in selective growth conditions, which allowed isolation of methane oxidising bacteria. The method resulted in isolation of both type I and type II methanotrophs from natural wetland and landfill soils. The isolates obtained from the landfill were dominated by type II methanotrophs and included several isolates carrying the gene for soluble methane monooxygenase (sMMO). Repetitive element sequence-based PCR fingerprinting documented genotypic diversity at the strain level. The presented method is a promising tool for easy and rapid isolation of different indigenous methanotrophs from an environment of interest.