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.

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.

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Nitrogen and oxygen transformations were studied in a bioturbated (reworked by animals) estuarine sediment (Norsminde Fjord, Denmark) by using a combination of '5N isotope (N03), specific inhibitor (C2H2), and microsensor (N20 and 02) techniques in a continuous-flow core system. The estuarine water was NO3rich (125 to 600 ,LM), and N03was consistently taken up by the sediment on the four occasions studied. Total N03 uptake (3.6 to 34.0 mmol of N m-2 day-') corresponded closely to N2 production (denitrification) during the experimental steady state, which indicated that dissimilatory, as well as assimilatory, N03-reduction to NH4' was insignificant. When C2H2 was applied in the flow system, denitrification measured as N20 production was often less (58 to 100%) than the N03 uptake because of incomplete inhibition of N20 reduction. The NO-formed by nitrification and not immediately denitrified but released to the overlying water, uncoupled nitrification, was calculated both from '5NO3dilution and from changes in N03 uptake before and after C2H2 addition. These two approaches gave similar results, with rates ranging between 0 and 8.1 mmol of N m2 day-' on the four occasions. Attempts to measure total nitrification activity by the difference between NH4+ fluxes before and after C2H2 addition failed because of non-steady-state NH4+ fluxes. The vertical distribution of denitrification and oxygen consumption was studied by use of N20 and 02 microelectrodes. The N20 profiles measured during the experimental steady state were often irregularly shaped, and the buildup of N20 after C2H2 was added was much too fast to be described by a simple diffusion model. Only bioturbation by a dense population of infauna could explain these observations. This was corroborated by the relationship between diffusive and total fluxes, which showed that only 19 to 36 and 29 to 62% of the total 02 uptake and denitrification, respectively, were due to diffusion-reaction processes at the regular sediment surface, excluding animal burrows.

The bacterial population in barley field soil was estimated by determining the numbers of (i) cells reducing the artificial electron acceptor 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to CTC-formazan (respiratory activity), (ii) cells dividing a limited number of times (microcolony formation) on nutrient-poor media, (iii) cells dividing many times (colony formation) on nutrient-poor agar media, and (iv) cells stained with acridine orange (total counts). The CTC reduction assay was used for the first time for populations of indigenous soil bacteria and was further developed for use in this environment. The number of viable cells was highest when estimated by the number of microcolonies developing during 2 months of incubation on filters placed on the surface of nutrient-poor media. The number of bacteria reducing CTC to formazan was slightly lower than the number of bacteria forming microcolonies. Traditional plate counts of CFU (culturable cells) yielded the lowest estimate of viable cell numbers. The microcolony assay gave an estimate of both (i) cells forming true microcolonies (in which growth ceases after a few cell divisions) representing viable but nonculturable cells and (ii) cells forming larger microcolonies (in which growth continues) representing viable, culturable cells. The microcolony assay, allowing single-cell observations, thus seemed to be best suited for estimation of viable cell numbers in soil. The efect on viable and culturable cell numbers of a temperature increase from 4 to 17°C for 5 days was investigated in combination with drying or wetting of the soil. Drying or wetting prior to the temperature increase, rather than the temperature increase per se, afected both the viable and culturable numbers of bacteria; both numbers were reduced in predried soil, while they increased slightly in the prewetted soil.