Soil Microcosm System Research Articles

Combining cyclic lipopeptides and cinnamon extract enhance antifungal activity against Fusarium oxysporum strains pathogenic to banana and delay Fusarium wilt under greenhouse conditions

AbstractFusarium wilt of banana (FWB) caused by the soil-borne fungus Fusarium oxysporum f. sp. cubense (Foc) is a widely distributed disease that generates devastating losses in banana production. Foc belongs to the Fusarium oxysporum species complex (FOSC) which includes several evolutionary lineages. Nine of them are pathogenic to banana such as F. phialophorum, F. grosmichelli, F. duoseptatum and the most aggressive F. odoratissimum tropical race 4 (TR4). No control method has been successfully implemented to manage FWB, then enhancing the potential of management approaches can avoid or delay disease epidemics and reduce disease severity. Here we determined the antifungal effect of different plant-based extracts against Foc in vitro, and whether the combination of cinnamon (Cinnamomum zeylanicum) extract and Bacillus tequilensis EA-CB0015 cyclic lipopeptides had an additive effect against different Foc lineages in vitro and against FWB in banana plants in greenhouse. We found, from 17 plant-based natural extracts, that cinnamon was highly active against Foc strain IB (race 1). Furthermore, cinnamon and cyclic lipopeptides inhibited different strains of various evolutionary lineages of Foc belonging to race 1 and TR4, and their combination increased in 1.4-fold the effect of the single extracts in vitro. Our results showed that soil concentration of F. odoratissimum TR4-II5 decreased by 1000-fold when treated with the combination of 488 mg L−1 cinnamon and 128 mg L−1 lipopeptides in a soil microcosm system after 5 days of incubation, followed by a partial population recovery after 21 days. In greenhouse experiments, the combination reduced external but not internal FWB symptoms, and cinnamon extract had a significant impact on internal plant symptoms. Taken together, the effect of cyclic lipopeptides with cinnamon extract on Foc supports their function towards delaying the effect of disease progression and suggests that the combination enhances the effect of the single extracts.

On the balance between practical relevance and standardization - Testing the effects of zinc and pyrene on native nematode communities in soil microcosms

Soils are among the most densely inhabited and biodiverse habitats on our planet, and many important soil ecosystem services depend on the health condition of the native soil fauna. Anthropogenic stress such as chemical pollution acting on the native soil fauna might jeopardize these functions. Laboratory microcosm tests are an appropriate tool for assessing the risk of chemicals on the native soil fauna and can be regarded as intermediate tier tests, bridging the gap between single species toxicity tests and field testing. Nematodes are one of the most abundant and divers soil invertebrates, and as such native nematode communities might be suitable for ecotoxicological assessments in laboratory microcosm set ups. In order to test such a small-scale (30 g soil) microcosm system, two different chemicals (zinc and pyrene) were assessed in various soil types for their effects on the respective native nematode communities. Various community parameters such as total nematode density, genus richness and genus composition, as well as trait-related indices (e.g. maturity index) were monitored over a period of 8–10 weeks. The response of the nematode communities strongly varied between soil types, and these differences were more pronounced for Zn than for pyrene. Interestingly, the structure of the respective native nematode communities was shown to play a larger role for explaining the varying toxic effects than soil properties governing the bioavailability of the spiked chemicals. We demonstrated that exposure of natural nematode communities in their original soil matrix to the metal zinc and to pyrene under climatically highly controlled conditions resulted in quantitatively and qualitatively distinct responses. Upon comparison of various community indices, the maturity index was shown to be the most sensitive toxicity endpoint for all tested soils and chemicals.

Mycelial network-mediated rhizobial dispersal enhances legume nodulation.

The access of rhizobia to legume host is a prerequisite for nodulation. Rhizobia are poorly motile in soil, while filamentous fungi are known to grow extensively across soil pores. Since root exudates-driven bacterial chemotaxis cannot explain rhizobial long-distance dispersal, mycelia could constitute ideal dispersal networks to help rhizobial enrichment in the legume rhizosphere from bulk soil. Thus, we hypothesized that mycelia networks act as vectors that enable contact between rhizobia and legume and influence subsequent nodulation. By developing a soil microcosm system, we found that a facultatively biotrophic fungus, Phomopsis liquidambaris, helps rhizobial migration from bulk soil to the peanut (Arachis hypogaea) rhizosphere and, hence, triggers peanut-rhizobium nodulation but not seen in the absence of mycelia. Assays of dispersal modes suggested that cell proliferation and motility mediated rhizobial dispersal along mycelia, and fungal exudates might contribute to this process. Furthermore, transcriptomic analysis indicated that genes associated with the cell division, chemosensory system, flagellum biosynthesis, and motility were regulated by Ph. liquidambaris, thus accounting for the detected rhizobial dispersal along hyphae. Our results indicate that rhizobia use mycelia as dispersal networks that migrate to legume rhizosphere and trigger nodulation. This work highlights the importance of mycelial network-based bacterial dispersal in legume-rhizobium symbiosis.

Effects of functionalized and raw multi-walled carbon nanotubes on soil bacterial community composition.

Carbon nanotubes (CNTs) are widely used in industry, but their environmental impacts on soil microbial communities are poorly known. In this paper, we compare the effect of both raw and acid treated or functionalized (fCNTs) multi-walled carbon nanotubes (MWCNTs) on soil bacterial communities, applying different concentrations of MWCNTs (0 µg/g, 50 µg/g, 500 µg/g and 5000 µg/g) to a soil microcosm system. Soil DNA was extracted at 0, 2 and 8 weeks and the V3 region of the 16S rRNA gene was PCR-amplified and sequenced using paired-end Illumina bar-coded sequencing. The results show that bacterial diversity was not affected by either type of MWCNT. However, overall soil bacterial community composition, as illustrated by NMDS, was affected only by fMWCNT at high concentrations. This effect, detectable at 2 weeks, remained equally strong by 8 weeks. In the case of fMWCNTs, overall changes in relative abundance of the dominant phyla were also found. The stronger effect of fMWCNTs could be explained by their intrinsically acidic nature, as the soil pH was lower at higher concentrations of fMWCNTs. Overall, this study suggests that fMWCNTs may at least temporarily alter microbial community composition on the timescale of at least weeks to months. It appears, by contrast, that raw MWCNTs do not affect soil microbial community composition.

Identification of Active Denitrifiers in Rice Paddy Soil by DNA- and RNA-Based Analyses

Denitrification occurs markedly in rice paddy fields; however, few microbes that are actively involved in denitrification in these environments have been identified. In this study, we used a laboratory soil microcosm system in which denitrification activity was enhanced. DNA and RNA were extracted from soil at six time points after enhancing denitrification activity, and quantitative PCR and clone library analyses were performed targeting the 16S rRNA gene and denitrification functional genes (nirS, nirK and nosZ) to clarify which microbes are actively involved in denitrification in rice paddy soil. Based on the quantitative PCR results, transcription levels of the functional genes agreed with the denitrification activity, although gene abundance did not change at the DNA level. Diverse denitrifiers were detected in clone library analysis, but comparative analysis suggested that only some of the putative denitrifiers, especially those belonging to the orders Neisseriales, Rhodocyclales and Burkholderiales, were actively involved in denitrification in rice paddy soil.

Invertebrate grazing affects nitrogen partitioning in the saprotrophic fungus Phanerochaete velutina

The heterogeneity of nutrients in forest soils is governed by many biotic and abiotic factors. The significance of nutrient patchiness in determining soil processes remains poorly understood. Some saprotrophic basidiomycete fungi influence nutrient heterogeneity by forming large mycelial networks that enable translocation of nutrients between colonized patches of dead organic matter. The effect of mycophagous soil fauna on these networks and subsequent nutrient redistribution has, however, been little studied. We used a soil microcosm system to investigate the potential effects of a mycophagous collembola, Protaphorura armata, on nutrient transfer within, and nutrient loss from, the mycelium of a saprotrophic basidiomycete fungus, Phanerochaete velutina. A 15N label, added to central mycelium, was used to track nitrogen movement within the microcosms across 32 days. Although collembola grazing had little impact on δ 15N values, it did alter the partitioning of 15N between different regions of mycelia. Less 15N was transferred to new mycelial growth in grazed systems than in ungrazed systems, presumably because collembola reduced fungal growth rate and altered mycelial morphology. Surprisingly, collembola grazing did not increase the mineralization of N from mycelium into the bulk soil. Overall, our results suggest that mycophagous soil fauna can alter nutrient flux and partitioning within fungal mycelium; this has the potential to affect the dynamics and spatial heterogeneity of forest floor nutrients.

Recovery of 15N-labeled urea and soil nitrogen dynamics as affected by irrigation management and nitrogen application rate in a double rice cropping system

A sophisticated soil microcosm system and 15N-labeled urea were used to investigate nitrogen (N) use efficiency and soil N dynamics in a rice monoculture system in two successive seasons. Topsoil (0 cm–20 cm) and subsoil (20 cm–50 cm) samples were collected from a traditional double rice cropping field in the Jiangxi Province, China, and these soil samples were derived from Quaternary red clay. Treatments were randomly assigned with two irrigation regimes and three N application rates (no application control, 80% traditional rate and 100% traditional rate noted as N0, N1 and N2, respectively). The levels of 15N recovery of plants, 15N and N remaining in soil were determined. Moreover, the N dynamic of soil solution from different layers of the soil profile was surveyed. The results showed that the effects of irrigation management and N application rate varied in different rice growing seasons. Irrigation regimes had remarkable effects on grain yield and chemical 15N fertilizers (CF-15N) uptake. When compared to flood irrigation (FI), the shallow water depth with wetting and drying (WD) increased grain yield up to 5.7%–20%. Although the highest grain yield was obtained with reduced N application level, both N apparent recovery (NAR) and 15N use efficiency (the percentage of plant N uptake derived from applied N, %Ndfan) significantly decreased with increasing N inputs. However, the interaction between irrigation management and N application rate on grain yield and N use efficiency (NUE) of CF-15N were not significant. A survey of soil solutions every 5 days indicated that NH4+-N was the main residual form of N, and high NH4+-N leaching was observed. When compared to FI, WD decreased vertical NH4+-N and TN leaching, especially at 10–50 cm depths of soil profile in the second season. NH4+-N was the main N residual form in the soil profile. Therefore, in this study, the WD irrigation regime and reduced rate (N1) was the optimal irrigation and fertilizer management strategy to increase the NUE of CF-N, increase the after effects of CF-15N, decrease leaching loss of CF-15N and minimize the shallow groundwater pollution risk, which were all beneficial for the ecological environment.

Dynamics of microbial community during bioremediation of phenanthrene and chromium(VI)-contaminated soil microcosms

The combined effect of phenanthrene and Cr(VI) on soil microbial activity, community composition and on the efficiency of bioremediation processes has been studied. Biometer flask systems and soil microcosm systems contaminated with 2,000 mg of phenanthrene per kg of dry soil and different Cr(VI) concentrations were investigated. Temperature, soil moisture and oxygen availability were controlled to support bioremediation. Cr(VI) inhibited the phenanthrene mineralization (CO(2) production) and cultivable PAH degrading bacteria at levels of 500-2,600 mg kg(-1). In the bioremediation experiments in soil microcosms the degradation of phenanthrene, the dehydrogenase activity and the increase in PAH degrading bacteria counts were retarded by the presence of Cr(VI) at all studied concentrations (25, 50 and 100 mg kg(-1)). These negative effects did not show a correlation with Cr(VI) concentration. Whereas the presence of Cr(VI) had a negative effect on the phenanthrene elimination rate, co-contamination with phenanthrene reduced the residual Cr(VI) concentration in the water exchangeable Cr(VI) fraction (WEF) in comparison with the soil microcosm contaminated only with Cr(VI). Clear differences were found between the denaturing gradient gel electrophoresis (DGGE) patterns of each soil microcosm, showing that the presence of different Cr(VI) concentrations did modulate the community response to phenanthrene and caused perdurable changes in the structure of the microbial soil community.

A microcosm system and an analytical protocol to assess PAH degradation and metabolite formation in soils

During bioremediation of polycyclic aromatic hydrocarbon (PAH)-polluted soils accumulation of polar metabolites resulting from the biological activity may occur. Since these polar metabolites are potentially more toxic than the parental products, a better understanding of the processes involved in the production and fate of these oxidation products in soil is needed. In the present work we describe the design and set-up of a static soil microcosm system and an analytical methodology for detection of PAHs and their oxidation products in soils. When applied to a soil contaminated with phenanthrene, as a model PAH, and 1-hydroxy-2-naphthoic acid, diphenic acid, and phthalic acid as putative metabolites, the extraction and fractionation procedures resulted in recoveries of 93%, 89%, 100%, and 89%, respectively. The application of the standardized system to study the biodegradation of phenanthrene in an agricultural soil with and without inoculation of the high molecular weight PAH-degrading strain Mycobacterium sp. AP1, demonstrates its suitability for determining the environmental fate of PAHs in polluted soils and for evaluating the effect of bioremediative treatments. In inoculated microcosms 35% of the added phenanthrene was depleted, 19% being recovered as CO(2) and 3% as diphenic acid. The latter, together with other two unidentified metabolites, accumulated in soil.

The effect of mixing ground leaf litters to soil on the development of pitch pine ectomycorrhizal and soil arthropod communities in natural soil microcosm systems

The addition of leaf litter to soil influences both the nutrients and polyphenols of soil. It is likely that contrasting nutrient and polyphenolic composition of different plant litters may affect plant growth, mycorrhizal and soil arthropod communities. We report results from a microcosm experiment of effects of incorporation of three single leaf litter species and a mixture of all three on pitch pine seedling growth, their ectomycorrhizal community and soil arthropod community. The three litter species (pine, oak and huckleberry) represent co-dominant species within the New Jersey pine barrens ecosystem. We show that the leaf litters have different composition of nutrients and polyphenols, with rooting matrix containing pine litter having lower inorganic nitrogen content (1.6 μg g −1) than oak (19.9 μg g −1) and huckleberry (4.4 μg g −1), but oak litter having the highest extractable phosphorus (13.3 cf. 0–0.08 μg g −1) and total phenol content and lowest condensed tannin content. These differences were imparted to rooting matrix of homogenized humic (Oa) layer of pine barrens soil to which milled leaf litter was added and used in the microcosms. Pitch pine seedlings grew significantly better in un-amended rooting matrix (0.33±0.02 g) than any of the litter treatments (0.15±0.02–0.17±0.01 g) and tissue P concentrations tracked phosphate concentrations in the rooting matrix. Total P accumulation into plant tissue was higher in oak than control, attributable to a significantly higher ( P<0.05) accumulation in roots (3.3±0.19 mg g −1) compared to other species (1.1±0.04–2.3±0.08 mg g −1). No relationship was seen between tissue N concentration and soil N, but seedlings growing in huckleberry litter amended soil accumulated less N than control. The effect of leaf litters on the ectomycorrhizal community composition were determined by PCA (first two axes accounted for 81% of the variance) and stepwise multiple regression analysis. These analyses showed that huckleberry leaf litter had a significant impact on mycorrhizal community composition with morphotypes Cg and DB being more abundant in the presence of huckleberry litter (178±13 cf. 68±15–106±15 for Cg and 141±11 cf. 88±23–111±18 for DB) and its influence of elevating nitrate nitrogen, organic nitrogen, total phenols and protein precipitation content of the rooting matrix. Mycorrhizal morphotypes BS and SB were significantly more abundant in the community where these soil factors were low in the absence of leaf litter addition. Total ectomycorrhizal abundance was negatively related to hydrolysable tannin concentration in the rooting matrix ( r 2=0.132, P<0.05). There was no influence of leaf litter type on mite density (dominated by non-burrowing phthiracarids), but collembolan density (dominated by Folsomia spp) showed a greater than threefold reduction in population density in the presence of leaf litter ( F=6.47, P<0.05). Collembolan density was positively correlated with mycorrhizal morphotypes GS and SB ( P<0.05) and negatively related to morphotypes DB ( P<0.05) and soil extractable NH 4–N ( P<0.05), suggesting a possible selection of fungal species in their diet and a relationship between collembola and nitrification.

Fate of 14C from glucose and the herbicide metsulfuron–methyl in a plant–soil microcosm system

The effects of weeds and soil nutrient additions on the fate of 14C-labelled glucose and metsulfuron–methyl (metsulfuron) herbicide were determined in a growth-chamber soil–plant microcosm study. Nodding thistle (Carduus nutans) was used as a test weed species to which these compounds were applied. The experiment consisted of a full factorial design of N addition, P addition, planting with C. nutans, and addition of 14C-labelled compounds (glucose and herbicide metsulfuron). The experiment ran for 131 days following addition of the 14C-labelled compounds. There was a significant positive effect of C. nutans plants on soil microbial biomass-C and basal respiration. The addition of nutrients did not consistently affect any of the parameters measured in this study. The fate of 14C-labelled compounds was determined by quantifying the relative amounts of 14C absorbed by plants, incorporated into soil, taken up into microbial biomass and respired by soil organisms. Two days after application of the 14C compounds, 7–8% of the 14C was absorbed by the C. nutans (weed) canopy. By day 131, approximately 2–3% of the applied 14C was still detected in the tissues of dead thistle plants. The amounts and pattern of decomposition differed considerably between metsulfuron and glucose. Almost all (99%) of the applied 14C glucose was either respired by the microbes or incorporated into microbial biomass by day 131 while only 38–42% of the applied metsulfuron was respired or incorporated into microbes by this time. Greater amounts of metsulfuron pesticide were degraded in microcosms containing C. nutans plants than in those without plants. By day 131, 27.4% of 14C added from metsulfuron was respired in the planted treatment and only 22.8% from the non-planted treatment. This would suggest that the presence of decomposing weed litter (dying weed plants) has the potential to enhance the decomposition of added xenobiotics such as herbicides.

Sulfide profile determination in a waterlogged soil microcosm by in situ filtration

Abstract An experimental soil microcosm system was built and soil was added and flooded to study the vertical distribution of dissolved sulfide. We compared the interstitial dissolved sulfide profiles among three experimental units by fitting them to the empirical model: sulfide = exp (b0 + b1 / (1-depth)). The 95% confidence intervals for b0 and b1 of the three experimental units overlapped for the same time period for the eight different sampling times during a 95-day experiment, thus suggesting that depth-sulfide profiles were not statistically different for the three experimental units. The profile determination in this study, which used an extraction method through porous polyethylene filters, was less invasive than methods where sediment samples have been removed since it involved a minimum sediment alteration. Our system could be used to study chemical transformation in flooded soils over short intervals.

Laboratory Estimates of Trace Gas Emissions following Surface Application and Injection of Cattle Slurry

AbstractApplying cattle slurry to soil may induce emissions of the greenhouse gases N2O and CH4. Our objective was to determine the effects of different application techniques (surface application and slit injection) of cattle (Bostaurus) slurry on the decomposition of slurry organic matter and the emissions of N2O and CH2. The effects of slurry application (43.6 m3 ha−1) were studied for 9 wk under controlled laboratory conditions using a soil microcosm system with automated monitoring of the CO2, N2O, and CH4 fluxes. The soil used was a silty loam (Ap horizon of a cambisol) with a constant water‐filled pore space of 67% during the experiment. About 38% of the organic matter applied with the slurry was decomposed within 9 wk. Production of CO2 was not affected by the application technique. Emissions of N2O and CH4 from the injected slurry were significantly higher than from the surface‐applied slurry, probably because of restricted aeration at the injected‐slurry treatment. Total N2O‐N emissions were 0.2% (surface application) and 3.3% (slit injection) of the slurry N added. Methane emission occurred only during the first few days following application. The total net flux of CH4‐C for 2 wk was −12 g ha−1 for the control (CH4 uptake), 2 g ha−1 for the surface‐applied slurry, and 39 g ha−1 for the injected slurry. Slurry injection, which is recommended to reduce NH3 volatilization, appears to increase emissions of the greenhouse gases N2O and CH4 from the fertilized fields.

Soil microcosm experiments to study the effects of waste material application on nitrogen and carbon turnover of lignite mine spoils in Lusatia (Germany)

The lignite and pyrite containing spoil substrates of the Lusatian mining district are marked by very high acidity and salt concentrations due to pyrite oxidation and by a very low content of pedogenic organic matter and nutrients. The effects of fly ash application to neutralize the produced acid and of organic waste material application to improve the ecological soil functions were studied considering the carbon and nitrogen cycling. Nineteen, 38 and 57 t ha−1 sewage sludge and 22, 44 and 66 t ha−1 compost were applied to ameliorated lignite and pyrite containing substrate. An automated soil microcosm system was used to analyse the solid, gaseous and liquid phases. Almost 9% of total N applied with sewage sludge (620, 1240 and 1860 t N ha−1 applied) were lost over a period of 150 days mainly as NO3-N. The total N losses from compost treatments were three times lower (2.8–3.1% of applied Nt) and occurred in similar quantities as NH4-N and NO3-N. Only sewage sludge treatments showed slightly increased N2O emissions at the beginning of the experiment. CO2 emissions determined the carbon losses of all treatments. The C losses amounted to 3.2–4.7% and 1.5–2.7% of Ct applied with sewage sludge and with compost, respectively.

Soil enzyme activities in the rhizosphere of field-grown sugar beet inoculated with the biocontrol agent Pseudomonas fluorescens F113

Pseudomonas fluorescens F113, which produces the antimicrobial compound 2,4-diacetylphloroglucinol, is a prospective biocontrol agent. Soil enzyme activities were used to investigate the ecological impact of strain F113 in the rhizosphere of field-grown sugar beet. There were distinct trends in rhizosphere enzyme activities in relation to soil chemistry [determined by electro-ultrafiltration (EUF)]. The activities of enzymes from the P cycle (acid phosphatase, alkaline phosphatase and phosphodiesterase) and of arylsulphatase were negatively correlated with the amount of readily available P, whereas urease activity was positively correlated with the latter. Significant correlations between nutrient levels determined by EUF and enzyme activities in the rhizosphere were obtained, highlighting the usefulness of enzyme assays to document variations in soil nutrient cycling. Contrary to previous microcosm studies, which did not investigate plants grown to maturity, the biocontrol inoculant had no effect on enzyme activities or on soil chemistry in the rhizosphere. The results showed the importance of using homogenous soil microcosm systems, also employed in previous work, for risk assessment studies, whereby the effects of inherent soil variability were minimised, and effects of the pseudomonad on soil enzymology could be detected.

Survival, metabolic activity and conjugative interactions of indigenous and introduced streptomycete strains in soil microcosms.

The growth and activity of introduced (S. lividans TK24 pIJ673 and S. lividans TK23) and indigenous (S. griseus CAG17) streptomycete strains in soil was studied, under controlled conditions. The effect of environmental parameters such as temperature, soil water content and nutrient availability on the growth and activity of these strains, was studied using a highly dynamic fed-batch soil microcosm system. Using this new system, repeated cycles of active streptomycete growth were achieved, allowing long-term investigation of metabolic activity, plasmid stability and conjugative plasmid transfer. In long-term experiments, respiration rates and enzyme activity patterns matched the pattern of germination/sporulation cycles of the inoculants. In situ hybridisation, using fluorescently labelled oligonucleotides, also proved the presence of metabolically active streptomycete mycelia in sterile soil. Plasmid stability under varying temperatures and selective pressure was studied using the above system. In both sterile and non sterile amended antibiotic containing soil, no intraspecific transfer of plasmid pIJ673 from S. lividans TK24 to S. griseus CAG17 was detected. The soil microcosm system used, though, permitted detection of intraspecific conjugative transfer of this plasmid from S. lividans TK24 to S. lividans TK23 in soil.

The influence of nitrate and ammonium fertilization on N2O release and CH4 uptake of a well‐drained topsoil demonstrated by a soil microcosm experiment

AbstractThe effects of the application of KNO3 and NH4Cl (100 kg N ha−1) on N2O release and CH4 uptake by a well‐aerated topsoil (porosity: 55%, water‐filled pore space: 67% of the total pore space) were studied in a laboratory incubation experiment over 50 days using a soil microcosm system with an automated registration of N2O and CH4 fluxes. The total N2O‐N losses over 50 days were low for all treatments and amounted to 0.9 mg m−2 for the control, 1.2 mg m−2 for the soil columns fertilized with KNO3, and 7.3 mg m−2 for the soil columns fertilized with NH4Cl. The slightly elevated N2O release after the application Of NH4Cl was associated with the nitrification of NH4+ added. Only −0.06% of the fertilized NH4−N was lost as N2O. This nitrogen fertilization reduced the CH4 uptake of the soil columns by 43% (NH4Cl) and 21% (KNO3), respectively.

Effect of a genetically modified Rhizobium meliloti inoculant on the development of arbuscular mycorrhizas, root morphology, nutrient uptake and biomass accumulation in Medicago sativa

summaryA soil microcosm system was used to evaluate the impact of a genetically modified (GM) Rhizobium meliloti strain on development and function of arbuscular mycorrhiza in alfalfa plants (Medicago sativa L.). There was no indication that this GM Rhizobium strain, which had an enhanced nodulation competitiveness ability, interfered with mycorrhiza formation by Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe. Indeed, inoculation with the GM Rhizobium strain greatly increased the number of mycorrhizal entry points in the alfalfa root system in comparison to the wild‐type strain. Mycorrhizal development and quality of nodulation increased with time and coincided with increased biomass of and nutrient (N, P) uptake by the host plant. The establishment of the symbiotic interactions also induced changes in root morphology; in particular, the degree of branching increased and the number of lateral roots was greater in plants inoculated with the GM Rhizobium strain. These results demonstrate that the GM Rhizobium strain does affect adversely the performance of arbuscular mycorrhizal symbiosis, a biosafety model system based on a functional rhizosphere.

Production and processing of a 59-kilodalton exochitinase during growth of Streptomyces lividans carrying pCHIO12 in soil microcosms amended with crab or fungal chitin.

Streptomyces lividans (pCHIO12), which carries the previously cloned Streptomyces olivaceoviridis exo-chiO1 gene on a multicopy vector, secretes a 59-kDa exochitinase, consisting of a catalytic domain (40 kDa), a central fibronectin type III-like module, and a chitin-binding domain (12 kDa). The propagation rate of S. lividans (pCHIO12) was higher in soil microcosms amended with fungal mycelia than in those containing crab chitin. Comparative biochemical and immunological studies allowed the following conclusions to be drawn. Within soil microcosm systems amended with crab shell chitin or chitin-containing Aspergillus proliferans mycelia, the strain expressed the clones exo-chiO1 gene and produced high quantities of a 59-kDa exochitinase. The enzyme was preferentially attached via its binding domain to the pellet from soil or liquid cultures. In contrast, truncated forms of 47, 40, and 25 kDa could be easily extracted from soil. The relative proportions of the 59-kDa enzyme and its truncated forms varied depending on the source of chitin and differed in soil and in liquid cultures.

Establishment and Meloidogyne-capture of an adhesive hyphaeforming isolate of the fungus Arthrobotrys oligospora in soil

In situ observations of an isolate of Arthrobotyrs oligospora (CBS 289.82) in sterilized and untreated soil revealed attachment of second-stage juveniles of the root-knot nematode Meloidogyne hapla to hyphae, development of rings around the nematode body and nematode colonization by trophic hyphae. Abundant growth of rings from infected nematodes was never observed. Establishment and nematode capture by this isolate was compared with those of several other fungi from the Dactylaria-complex in a simple soil microcosm system. Application of hyphal fragments of A. oligospora (CBS 289.82), about 30 mm g −1 oven-dry soil, resulted in hyphal lengths of 100–170 m g −1 oven-dry soil, reduction of 90% of living juveniles of M. hapla within 1 day and extermination of the nematodes after 10 days. Arthrobotrys oligospora (CBS 289.82) performed far better than any of the other fungi. Establishment in non-sterilized soil was not as good, but still total hyphal length of about 10 g −1 oven-dry soil resulted in 70% reduction of living nematodes at Day 10. At 13°C results were similar to those at 20°C. The results support the conclusion that adhesive hyphae-forming fungi may be useful for the control of nematodes in soil.