Assessment of oil refinery wastewater and effluent integrating bioassays, mechanistic modelling and bioavailability evaluation.

Water is used in petroleum oil refineries in significant volumes for cooling, steam generation and processing of raw materials. Effective water management is required at refineries to ensure their efficient and responsible operation with respect to the water environment. However, ascertaining the potential environmental risks associated with discharge of refinery effluents to receiving waters is challenging because of their compositional complexity. Recent European research and regulatory initiatives propose a more holistic approach including biological effect methods to assess complex effluents and surface water quality. The study presented here investigated potential effects of effluent composition, particularly hydrocarbons, on aquatic toxicity and was a component of a larger study assessing contaminant removal during refinery wastewater treatment (Hjort et al 2021). The evaluation of effects utilised a novel combination of mechanistic toxicity modelling based on the exposure composition, measured bioavailable hydrocarbons using biomimetic solid phase microextraction (BE-SPME), and bioassays. The results indicate that in the refinery effluent assessments measured bioavailable hydrocarbons using BE-SPME was correlated with the responses in standard bioassays. It confirms that bioassays are providing relevant data and that BE-SPME measurement, combined with knowledge of other known non-hydrocarbon toxic constituents, provide key tools for toxicity identification. Overall, the results indicate that oil refinery effluents treated in accordance to the EU Industrial Emissions Directive requirements have low to negligible toxicity to aquatic organisms and their receiving environments. Low-cost, animal-free BE-SPME represents a compelling tool for rapid effluent characterization.

Solubility Constraints on Aquatic Ecotoxicity Testing of Anionic Surfactants.

In order to develop models that can predict the environmental behavior and effects of chemicals, reliable experimental data are needed. However, for anionic surfactants the number of ecotoxicity studies is still limited. The present study therefore aimed to determine the aquatic ecotoxicity of three classes of anionic surfactants. To this purpose we subjected daphnids (Daphnia magna) for 48 h to alkyl carboxylates (CxCO2-), alkyl sulfonates (CxSO3-), and alkyl sulfates (CxSO4-) with different carbon chain lengths (x). However, all surfactants with x > 11 showed less than 50% immobility at water solubility. Hence, EC50 values for only few surfactants could be gathered: C9CO2- (16 mg L-1), C11CO2- (0.8 mg L-1) and C11SO4- (13.5 mg L-1). Data from these compounds showed an increase in ecotoxicity with a factor 4.5 per addition of a hydrocarbon unit to the alkyl chain, and a factor 20 when replacing the sulfate head group by a carboxylate head group. Unfortunately, we could not test carboxylates with a broader variety of chain lengths because solubility limited the range of chain length that can be tested.

Impact of axial root growth angles on nitrogen acquisition in maize depends on environmental conditions.

BACKGROUNDS AND AIMS: Crops with reduced requirement for nitrogen (N) fertilizer would have substantial benefits in developed nations, while improving food security in developing nations. This study employs the functional structural plant model SimRoot to test the hypothesis that variation in the growth angles of axial roots of maize (Zea mays L.) is an important determinant of N capture. METHODS: Six phenotypes contrasting in axial root growth angles were modelled for 42 d at seven soil nitrate levels from 10 to 250 kg ha(-1) in a sand and a silt loam, and five precipitation regimes ranging from 0·5× to 1·5× of an ambient rainfall pattern. Model results were compared with soil N measurements of field sites with silt loam and loamy sand textures. KEY RESULTS: For optimal nitrate uptake, root foraging must coincide with nitrate availability in the soil profile, which depends on soil type and precipitation regime. The benefit of specific root architectures for efficient N uptake increases with decreasing soil N content, while the effect of soil type increases with increasing soil N level. Extreme root architectures are beneficial under extreme environmental conditions. Extremely shallow root systems perform well under reduced precipitation, but perform poorly with ambient and greater precipitation. Dimorphic phenotypes with normal or shallow seminal and very steep nodal roots performed well in all scenarios, and consistently outperformed the steep phenotypes. Nitrate uptake increased under reduced leaching conditions in the silt loam and with low precipitation. CONCLUSIONS: Results support the hypothesis that root growth angles are primary determinants of N acquisition in maize. With decreasing soil N status, optimal angles resulted in 15-50 % greater N acquisition over 42 d. Optimal root phenotypes for N capture varied with soil and precipitation regimes, suggesting that genetic selection for root phenotypes could be tailored to specific environments.

Genotypic and phenotypic variation among Lysobacter capsici strains isolated from Rhizoctonia suppressive soils.

Four Gram-negative bacterial strains, recovered from clay soils cultivated with different crops in the Netherland, were subjected to a polyphasic taxonomic study in order to clarify their taxonomic status. Comparative analysis of the 16S rRNA gene sequences revealed that they belong to the genus Lysobacter and to be highly related to the type strains of L. antibioticus DSM 2044(T), L. gummosus DSM 6980(T), and L. capsici DSM 19286(T), displaying 99.1-99.3%, 99.2-99.6% and 99.4-100% sequence similarities, respectively, to these species. The results of DNA-DNA hybridization studies unambigiously indicated that the four strains belonged to the species L. capsici. Nevertheless, DNA fingerprinting and phenotypic characterization indicated that there was a considerable diversification and niche differentiation among the strains belonging to L. capsici. The newly identified L. capsici strains strongly inhibit Rhizoctonia solani AG2 and originate from Rhizoctonia-suppressive soils where also populations of L. antibioticus and L. gummosus were present. This is the first report of the presence of combined populations of closely related Lysobacter spp. within agricultural soils.

Specific detection of Lysobacter enzymogenes (Christensen and Cook 1978) strain 3.1T8 with TaqMan PCR.

AIMS: To develop a strain-specific TaqMan PCR method for detecting and quantifying the biocontrol strain Lysobacter enzymogenes 3.1T8. METHODS AND RESULTS: A primer-probe combination was designed on the basis of a strain-specific sequence selected using REP-PCR (repetitive extragenic palindromic-polymerase chain reaction). The specificity of this combination was demonstrated by 14 other Lysobacter strains that did not react with the selected primer-probe combination. To quantify strain 3.1T8 in cucumber root samples, a calibration curve was prepared by spiking roots with a 10-fold dilution series of the strain. Detection of the biocontrol strain 3.1T8 with this method showed that the strain survived well for 22 days on root tips as well as on older cucumber roots. Survival was higher when the strain was inoculated to younger plants. In a cucumber production system with large volumes of substrate, strain 3.1T8 was detected in high numbers on cucumber roots 3 weeks after inoculation. CONCLUSIONS: The primer-probe combination developed was strain specific, because it did not react with other strains of the same species and genus. The TaqMan PCR method successfully quantified the inoculated biocontrol strain on cucumber roots grown in different cropping systems. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed TaqMan PCR method is a strain-specific real-time detection method that can be used to assess the population dynamics of L. enzymogenes strain 3.1T8 for further optimization of its biocontrol efficacy.

Microbial enrichment of a novel growing substrate and its effect on plant growth.

The quality of torrefied grass fibers (TGF) as a new potting soil ingredient was tested in a greenhouse experiment. TGF was colonized with previously selected microorganisms. Four colonization treatments were compared: (1) no inoculants, (2) the fungus Coniochaeta ligniaria F/TGF15 alone, (3) the fungus followed by inoculation with two selected bacteria, and (4) the fungus with seven selected bacteria. Cultivation-based and DNA-based methods, i.e., PCR-DGGE and BOX-PCR, were applied to assess the bacterial and fungal communities established in the TGF. Although colonization was not performed under sterile conditions, all inoculated strains were recovered from TGF up to 26 days incubation. Stable fungal and bacterial populations of 10(8) and 10(9) CFU/g TGF, respectively, were reached. As a side effect of the torrefaction process that aimed at the chemical stabilization of grass fibers, potentially phytotoxic compounds were generated. These phytotoxic compounds were cold-extracted from the fibers and analyzed by gas chromatography mass spectrometry. Four of 15 target compounds that had previously been found in the extract of TGF were encountered, namely phenol, 2-methoxyphenol, benzopyran-2-one, and tetrahydro-5,6,7,7a-benzofuranone. The concentration of these compounds decreased significantly during incubation. The colonized TGF was mixed with peat (P) in a range of 100%:0%, 50%:50%, 20%:80%, and 0%:100% TGF/P (w/w), respectively, to assess suitability for plant growth. Germination of tomato seeds was assessed three times, i.e., with inoculated TGF that had been incubated for 12, 21, and 26 days. In these tests, 90-100% of the seeds germinated in 50%:50% and 20%:80% TGF/P, whereas on average only 50% of the seeds germinated in pure TGF. Germination was not improved by the microbial inoculants. However, plant fresh weight as well as leaf area of 28-day-old tomato plants were significantly increased in all treatments where C. ligniaria F/TGF15 was inoculated compared to the control treatment without microbial inoculants. Colonization with C. ligniaria also protected the substrate from uncontrolled colonization by other fungi. The excellent colonization of TGF by the selected plant-health promoting bacteria in combination with the fungus C. ligniaria offers the possibility to create disease suppressive substrate, meanwhile replacing 20% to 50% of peat in potting soil by TGF.

Interactions of plant-beneficial bacteria with the ascomycete Coniochaeta ligniaria.

AIMS: To assess the interactions between Coniochaeta ligniaria F/TGF15 obtained from torrefied grass fibers (TGF) and selected bacteria from the same substrate. METHODS AND RESULTS: Upon coinoculation on potato dextrose agar, Pseudomonas putida 15/TGE5, Methylobacterium radiotolerans 56/TGF10, Serratia plymutica 23/TGE5, Pseudomonas corrugata 31/TGE5, Leifsonia xyli subsp. xyli 66/TGF10, Mycobacterium anthracenicum 70/TGF15 and Agromyces aurantiacus 95/TGF15 were translocated by C. ligniaria, but not in the absence of the fungus. Pseudomonas putida, P. corrugata, L. xyli subsp. xyli and A. aurantiacus were able to grow on compounds released by the fungus, but not M. radiotolerans. Antagonism towards C. ligniaria was observed for S. plymutica and P. corrugata. Pseudomonas putida was translocated by the fungus in TGF up to at least 45 mm. It also multiplied on the hyphae of C. ligniaria in TGF, reaching CFU densities of log 8.4 g(-1) dry TGF in 20 d, while the strain could not grow in nonfungal TGF. Methylobacterium radiotolerans was not translocated by the fungus in TGF. CONCLUSIONS: Several of the selected bacteria could grow on the compounds released by the fungus, whereas two bacteria inhibited or killed the fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: It is shown that C. ligniaria has a dual role in bacterial colonization of TGF, being crucial for the detoxification of TGF, meanwhile stimulating growth and translocation of a consortium of plant-growth-promoting bacteria.

Thermally treated grass fibers as colonizable substrate for beneficial bacterial inoculum.

This study investigates how thermally treated (i.e., torrefied) grass, a new prospective ingredient of potting soils, is colonized by microorganisms. Torrefied grass fibers (TGF) represent a specific colonizable niche, which is potentially useful to establish a beneficial microbial community that improves plant growth. TGF and torrefied grass extracts (TGE) were inoculated with a suspension of microorganisms obtained from soil. Sequential microbial enrichment steps were then performed in both substrates. The microbial communities developing in the substrates were assessed using cultivation-based and cultivation-independent approaches. Thus, bacterial isolates were obtained, and polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) analyses for bacterial communities were performed. Partial sequencing of the 16S ribosomal RNA gene from isolates and bands from DGGE gels showed diverse communities after enrichment in TGE and TGF. Bacterial isolates affiliated with representatives of the alpha-proteobacteria (Methylobacterium radiotolerans, Rhizobium radiobacter), gamma-proteobacteria (Serratia plymuthica, Pseudomonas putida), Cytophaga-Flavobacterium-Bacteroides (CFB) group (Flavobacterium denitrificans), beta-proteobacteria (Ralstonia campinensis), actinobacteria (Cellulomonas parahominis, Leifsonia poae, L. xyli subsp. xyli, and Mycobacterium anthracenicum), and the firmicutes (Bacillus megaterium) were found. In TGE, gamma-proteobacteria were dominant (61.5% of the culturable community), and 20% belonged to the CFB group, whereas actinobacteria (67.4%) and alpha-proteobacteria (21.7%) were prevalent in TGF. A germination assay with lettuce seeds showed that the phytotoxicity of TGF and TGE decreased due to the microbial enrichment.

Fragmentation of alpha- and beta-alanine molecules by ions at Bragg-peak energies.

The interaction of keV He(+), He(2+), and O(5+) ions with isolated alpha and beta isomers of the amino acid alanine was studied by means of high resolution coincidence time-of-flight mass spectrometry. We observed a strong isomer dependence of characteristic fragmentation channels which manifests in strongly altered branching ratios. Despite the ultrashort initial perturbation by the incoming ion, evidence for molecular rearrangement leading to the formation of H(3)(+) was found. The measured kinetic energies of ionic alanine fragments can be sufficient to induce secondary damage to DNA in a biological environment.

Effect of above-ground plant species on soil microbial community structure and its impact on suppression of Rhizoctonia solani AG3.

The extent of soil microbial diversity is seen to be critical to the maintenance of soil health and quality. Different agricultural practices are able to affect soil microbial diversity and thus the level of suppressiveness of plant diseases. In a 4-year field experiment, we investigated the microbial diversity of soil under different agricultural regimes. We studied permanent grassland, grassland turned into arable land, long-term arable land and arable land turned into grassland. The diversity of microbial communities was described by using cultivation-based and cultivation-independent methods. Both types of methods revealed differences in the diversities of soil microbial communities between different treatments. The treatments with higher above-ground biodiversity generally maintained higher levels of microbial diversity. Moreover, a positive correlation between suppression of Rhizoctonia solani AG3 and microbial diversity was observed. Permanent (species-rich) grassland and grassland turned into maize stimulated higher microbial diversities and higher levels of suppressiveness of R. solani AG3 compared with the long-term arable land. Effects of agricultural practices on Bacillus and Pseudomonas communities were also observed and clear correlations between the levels of suppressiveness and the diversities of these bacterial groups were found. This study highlighted the importance of agricultural management regime for soil microbial community structure and diversity as well as the level of soil suppressiveness.

Distribution of microcystins in a lake foodweb: no evidence for biomagnification.

Microcystins, toxins produced by cyanobacteria, may play a role in fish kills, although their specific contribution remains unclear. A better understanding of the eco-toxicological effects of microcystins is hampered by a lack of analyses at different trophic levels in lake foodwebs. We present 3 years of monitoring data, and directly compare the transfer of microcystin in the foodweb starting with the uptake of (toxic) cyanobacteria by two different filter feeders: the cladoceran Daphnia galeata and the zebra mussel Dreissena polymorpha. Furthermore foodwebs are compared in years in which the colonial cyanobacterium Microcystis aeruginosa or the filamentous cyanobacterium Planktothrix agardhii dominated; there are implications in terms of the types and amount of microcystins produced and in the ingestion of cyanobacteria. Microcystin concentrations in the seston commonly reached levels where harmful effects on zooplankton are to be expected. Likewise, concentrations in zooplankton reached levels where intoxication of fish is likely. The food chain starting with Dreissena (consumed by roach and diving ducks) remained relatively free from microcystins. Liver damage, typical for exposure to microcystins, was observed in a large fraction of the populations of different fish species, although no relation with the amount of microcystin could be established. Microcystin levels were especially high in the livers of planktivorous fish, mainly smelt. This puts piscivorous birds at risk. We found no evidence for biomagnification of microcystins. Concentrations in filter feeders were always much below those in the seston, and yet vectorial transport to higher trophic levels took place. Concentrations of microcystin in smelt liver exceeded those in the diet of these fish, but it is incorrect to compare levels in a selected organ to those in a whole organism (zooplankton). The discussion focuses on the implications of detoxication and covalent binding of microcystin for the transfer of the toxin in the foodweb. It seems likely that microcystins are one, but not the sole, factor involved in fish kills during blooms of cyanobacteria.

Environmental impact and recovery at two dumping sites for dredged material in the North Sea.

The environmental impact and recovery associated with the long and uninterrupted disposal of large volumes of moderately contaminated dredged material from the port of Rotterdam was studied at nearby dumping sites in the North Sea. Observations were made on sediment contamination, ecotoxicity, biomarker responses and benthic community changes shortly after dumping at the 'North' site had ceased and at the start of disposal at the new dumping site 'Northwest'. During the period of dumping, very few benthic invertebrates were found at the North site. Concentrations of cadmium, mercury, polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs) and tributyltin (TBT) in the fine sediment fraction (<63 microm) from this site were 2-3 times higher than at the reference site. In four different bioassays with marine invertebrates the sediments showed no acute toxic effects. In tissue (pyloric caeca) of resident starfish Asterias rubens, residual levels of mercury, zinc, PCBs and dioxin-like activity were never more than twice those at the reference site. Four different biomarkers (DNA integrity, cytochrome P450 content, benzo[a]pyrene hydroxylase activity and acetylcholinesterase inhibition) were used on the starfish tissues, but no significant differences were found between North and the reference site. Minor pathological effects were observed in resident dab Limanda limanda. One year after dumping had ceased at the North site, a significant increase in the species richness and abundance of benthic invertebrates and a concomitant decrease in the fine sediment fraction of the seabed were observed. After 8.2 million m3 of moderately contaminated dredged material had been dumped at the new dumping site Northwest, the species richness and abundance of benthic invertebrates declined over an area extending about 1-2 km eastwards. This correlated with a shift in sediment texture from sand to silt. The contamination of the fine sediment fraction at the Northwest location doubled. It is concluded that marine benthic resources at and around the dumping sites have been adversely affected by physical disturbance (burial, smothering). However, no causal link could be established with sediment-associated contaminants from the dredged spoils.

Inability to find consistent bacterial biocontrol agents of Pythium aphanidermatum in cucumber using screens based on ecophysiological traits.

A collection of 821 rhizobacteria from cucumber, originating from different root locations and stages of plant development, was screened for potential biocontrol agents of Pythium aphanidermatum (Edson) Fitzp. The screening procedure exploited carbon source utilization profiles and growth rates of bacteria as indicators of a partial niche overlap with the pathogen. The bacteria were tested for growth on nine carbon sources (glucose, fucose, sucrose, maltose, asparagine, alanine, galacturonic acid, succinic acid, and linoleic acid), most of which are reported to be used by the zoospores of P. aphanidermatum in the infection process. The isolates were classified as fast- or slow-growing, depending on their growth rate in 1/10 strength TSB. By nonhierarchical cluster analysis, 20 clusters were generated of bacteria with similar profiles of carbon source utilization. Redundancy analysis showed that the type of root sample explained 47% of the variance found in the relative abundance of bacteria from the clusters. Bacteria from clusters using none or few of the carbon sources, e.g., maltose and linoleic acid, with many slow-growing isolates, showed a preference for plants in the vegetative or generative stage, or for old root regions (root base). Bacteria from clusters with fast-growing isolates, using many carbon sources, were relatively abundant in the seedling stage. A selection of 127 bacteria from the different clusters was tested for disease suppressive capabilities in bioassays on young cucumber plants in nutrient solution, inoculated with zoospores of P. aphanidermatum. Nine of these bacteria produced biosurfactants, and 27 showed antibiosis against mycelial growth in plate assays. For 31 isolates, significant positive effects on plant biomass were shown, as analyzed with a general linear regression model. For most isolates, these effects occurred only in one of two replicate assays and no reductions in the degree of root and crown rot were found. Of the isolates that used many of the tested carbon sources, only four had positive effects on plant biomass. The majority of the isolates that positively affected plant biomass used few to moderate numbers of carbon sources and did not produce antibiotics or biosurfactants. In conclusion, competition for the tested carbon sources with the zoospores did not play a decisive role in disease suppression, and no clear relation was found between ecophysiological traits and disease suppression. Only isolate 3.1T8, isolated from root tips in the generative stage of plant growth, significantly increased plant biomass and suppressed root and crown rot symptoms in five out of six bioassays. The isolate produced an antifungal substance in plate assays and showed biosurfactant production in several (cucumber-derived) media.

Confounding factors in bioassays with freshwater and marine organisms.

The use of bioassays in ecological risk assessments often raises questions about the causative factors, and insight into the possibility that confounding factors, such as pH or increased ammonia concentrations, might be responsible for the observed toxicity is needed. It was decided to develop a practical approach for the Dutch situation, in which a first screening is carried out based on provisional criteria. In collecting the required data, dozens of experiments were performed, while the scientific literature was searched for additional information. It is concluded that the provisional criteria specified are at present useful tools in interpreting results of bioassays. Depending on the outcome and the aim of the research, it might be necessary to further reduce uncertainties in the interpretation. This might require some additional experiments, using alternative controls or test procedures or altering the composition of the original sample.

Bioaccumulation and biomarkers in the sea star Asterias rubens (Echinodermata: Asteroidea): a North Sea field study.

During two North Sea field trips in March 1995 and September 1996 sea stars, Asterias rubens, were collected at various stations along pollution gradients in order to study the relation between biochemical markers and levels of accumulated contaminants. Biomarkers measured were: cytochrome P450 level, benzo[a]pyrene hydroxylase (BPH) activity, acetylcholinesterase (AChE) activity and DNA integrity. Accumulation levels of heavy metals, polychlorinated biphenyls, organochlorine pesticides and polycyclic aromatic hydrocarbons (PAHs) in the pyloric caeca of sea stars indicate different pollution gradients, influenced by rivers in The Netherlands, UK and Germany. For some contaminants, especially PAHs, relatively high levels were found in the central part of the North Sea (Dogger Bank). On the basis of multivariate statistics, stations near the mouth of the Elbe and the Rhine/Meuse were shown to have different patterns of biomarker responses. Sea stars from stations in coastal zones showed relatively high levels of cytochrome P450 and 'P418', another haemoprotein that is present in most marine invertebrates. The station nearest to the Elbe Estuary showed the lowest BPH and AChE activity. DNA integrity was lower especially in stations near the Dutch coast and in a station near the Tees/Tyne estuaries. Using these biomarkers as early warning signals of exposure and/or adverse effects, this type of monitoring can be used also in the future to study the spatial and temporal trends in the quality of coastal waters.

Comparison of laboratory and in situ sediment bioassays using Corophium volutator.

Bioassays with the marine amphipod Corophium volutator were performed simultaneously in situ and in the laboratory using sediments sampled from the in situ locations. In most cases, the in situ response was significantly higher compared to the laboratory response. This difference was not caused by direct influence of the use of the field chamber on Corophium sp., nor was the difference caused by the overlying water used. Experiments showed homogenization can affect the toxicity of a sediment, but not in such a way that it can completely explain the difference between the response in situ and in the laboratory. Possible explanatory factors are harbor activity, storms, and temperature. To reduce the influence of some of these factors, the best period of the year to perform in situ bioassays with C. volutator is May, June, or September.

Population dynamics of Pythium aphanidermatum in cucumber grown in closed systems.

Foot and root rot in cucumber, caused by Pythium aphanidermatum (Edson) Fitzp., is an economically important disease in soilless culture systems. Nevertheless, very few data are available on the populations of this pathogen. Therefore, two detection methods, nested PCR (polymerase chain reaction) and plating on a selective medium after concentration of samples, were optimised and evaluated. With both methods very low concentrations of P. aphanidermatum could be detected; i.e. the detection limits were around 0.05 CFU/ml nutrient solution. In addition, real-time quantitative PCR using a Molecular Beacon probe was designed and tested. The potential and limitations of the different detection methods are discussed. With these different detection techniques, the population dynamics of P. aphanidermatum in a cucumber crop was followed. The impact of different disinfection treatments was studied in a greenhouse experiment with a cucumber crop growing on rockwool slabs in 12 independent closed systems. The nutrient solution was recirculated without disinfection (control), after UV-irradiation (250 mJ/cm2), or after slow sand filtration treatment. Part of the crop was inoculated with an isolate of P. aphanidermatum. The non-inoculated part could only become infected through the recirculated nutrient solution. Disease symptoms (stem rot, wilt, and root rot) and the yield loss were recorded in addition to the population dynamics of the pathogen. Very clear differences in the spread of the pathogen and in disease symptoms were measured between the systems with and without disinfection. UV-irradiation and slow sand filtration were both effective in removing the pathogen and protected the crop from disease symptoms. Correlation indices between the final yield and the different measurements during the experiment were calculated.

Ecophysiological Characterization of Rhizosphere Bacterial Communities at Different Root Locations and Plant Developmental Stages of Cucumber Grown on Rockwool.

Bacterial communities from the rhizosphere of cucumber were characterized with respect to growth rates and carbon source utilization, in order to develop a selection strategy for biocontrol agents against Pythium aphanidermatum. Rhizosphere samples were collected from different root regions (root tips, the root base, and the intermediate region where lateral roots emerge) and developmental stages (the seedling, vegetative, and generative stage) from plants cultivated on reused rockwool. By colony counts on 1/10 strength TSA on subsequent days after plating, percentages of fast- and slow-growing isolates (i.e., forming visible colonies within 2 days, or after 3 or more days, respectively) were determined for each rhizosphere sample. At all plant developmental stages, root tips had the highest percentages of fast growing isolates, and root bases the lowest. During plant growth, the relative amounts of slowly growing bacteria increased. Community-level carbon source utilization was determined for the different rhizosphere samples with Biolog GN plates. Principal component analysis showed that rhizosphere samples from different developmental stages and root locations had distinct carbon source utilization patterns. Communities from root tips of seedlings showed the highest utilization of several monosaccharides. Communities from tips and intermediate regions of plants in the vegetative stage utilized relatively many amino acids and several organic acids, and in the generative stage, more di- and polysaccharides were used. Root base samples scored low with respect to carbon source utilization, except for some disaccharides. From the different rhizosphere samples, 826 bacteria, randomly collected from 1/10 strength TSA plates, were screened on the utilization of 9 carbon sources. The 9 selected carbon sources were chosen because they are reported to occur in the rhizosphere, to be used by the zoospores of Pythium in the infection process, or appeared to be discriminant in the analysis of community-level carbon source utilization performed in this study. It appeared that monosaccharides (glucose and fucose), amino acids (alanine and asparagine), and organic acids (galacturonic, succinic, and linoleic acid) were used for growth mainly by bacteria from the root tips, and to a lesser extent from the intermediate region, of young plants. Disaccharides were predominantly utilized by isolates from plants in the vegetative stage. Overall, the results indicated that growth rates and carbon source utilization reflect the adaptation of bacteria to the rhizosphere environment. The possibility of using these characteristics to screen for rhizosphere competent biocontrol agents that compete for substrates with P. aphanidermatum is discussed.

Effect of the Indigenous Microflora on the Development of Root and Crown Rot Caused by Pythium aphanidermatum in Cucumber Grown on Rockwool.

ABSTRACT The capacity of the microflora in rockwool to suppress Pythium aphanidermatum, the causative agent of root and crown rot in cucumber, was assessed. Disease development of cucumber (Cucumis sativus) grown on rockwool was evaluated in an "ebb-and-flood" system with a recirculating nutrient solution after inoculation with P. aphanidermatum. In five independent experiments from 1995 to 1998, 11 batches of used rockwool were tested. All batches without P. aphanidermatum problems in the preceding cucumber crop had significantly lower numbers of diseased plants in nonautoclaved than in autoclaved used rockwool; the disease incidence was reduced by 52 to 100%. Suppressiveness also was present in rockwool previously used to grow other vegetable crops. Rockwool originating from a cucumber crop that was severely attacked by Pythium resulted in a high disease incidence. Previously unused (new) rockwool had higher or similar percentages of diseased plants than did nonsterilized used rockwool. Disease suppression in used rockwool could also be measured in a smaller test system. In both systems, autoclaved rockwool became suppressive to Pythium after recolonization with the indigenous microflora. Population sizes of total culturable aerobic bacteria as well as of fluorescent pseudomonads did not correlate with disease suppressiveness, as numbers of bacteria and pseudomonads were similar or lower in nonautoclaved (suppressive) than in autoclaved (nonsuppressive) rockwool. Differences in the structure of the bacterial populations could be visualized by using eubacterial polymerase chain reaction (PCR) followed by denaturing gradient gel electrophoresis (DGGE). Interestingly, the nonautoclaved and the recolonized used rockwool, which were both suppressive, showed different dominating bacterial groups as compared with the autoclaved rockwool. PCR-DGGE patterns obtained at different sampling times showed that the composition of the bacterial populations changed during plant growth. Fungal populations were present in the treatments that yielded suppressive rockwool, i.e., the nonautoclaved and the recolonized rockwool, but they were absent or present in low numbers in the autoclaved rockwool, which permitted a high disease incidence. Suppressiveness of rockwool to Pythium root and crown rot is a hitherto undescribed phenomenon, and knowledge of the mechanism and microorganisms involved will stimulate the development of microbially balanced soilless growing systems.