Diversity and structure of AMF communities as affected by tillage in a temperate soil

Arbuscular mycorrhizal fungi (AMF) were studied in differently tilled soils from a long-term field experiment in Switzerland. Diversity and structure of AMF communities were surveyed either directly on spores isolated from the field soil or on spores isolated from trap cultures, planted with different host plants. Single-spore cultures were established from the AMF spores obtained from trap cultures. Identification of the AMF was made by observation of spore morphology and confirmed by sequencing of ITS rDNA. At least 17 recognised AMF species were identified in samples from field and/or trap cultures, belonging to five genera of AMF--Glomus, Gigaspora, Scutellospora, Acaulospora, and Entrophospora. Tillage had a significant influence on the sporulation of some species and non- Glomus AMF tended to be more abundant in the no-tilled soil. The community structure of AMF in the field soil was significantly affected by tillage treatment. However, no significant differences in AMF diversity were detected among different soil tillage treatments. AMF community composition in trap cultures was affected much more by the species of the trap plant than by the original tillage treatment of the field soil. The use of trap cultures for fungal diversity estimation in comparison with direct observation of field samples is discussed.     

Isolation and characterization of Arthrobacter bacteriophages and their application to phage typing of soil arthrobacters.

Seventeen bacteriophages, active against 19 Arthrobacter soil isolates, were isolated from concentrated samples of river water and sewage. Attempts to isolate Arthrobacter bacteriophages from filtrates of broth cultures of the soil isolates or from ultraviolet light-irradiated cultures were unsuccessful. Bacteriophages were not detected in either concentrated or unconcentrated soil extracts. Electron microscopic studies of 11 phages showed morphologies characteristic of Bradley's groups B (exhibited by 9 phages) and C (exhibited by 2 phages). Moles percent guanine plus cytosine, calculated from the deoxyribonucleic acid density of three phages, ranged from 60.2 to 65.3. The phages were characterized by their plague and virion morphology, host range, and serological specificity.     

Isolation and characterization of Arthrobacter bacteriophages and their application to phage typing of soil arthrobacters.

Seventeen bacteriophages, active against 19 Arthrobacter soil isolates, were isolated from concentrated samples of river water and sewage. Attempts to isolate Arthrobacter bacteriophages from filtrates of broth cultures of the soil isolates or from ultraviolet light-irradiated cultures were unsuccessful. Bacteriophages were not detected in either concentrated or unconcentrated soil extracts. Electron microscopic studies of 11 phages showed morphologies characteristic of Bradley's groups B (exhibited by 9 phages) and C (exhibited by 2 phages). Moles percent guanine plus cytosine, calculated from the deoxyribonucleic acid density of three phages, ranged from 60.2 to 65.3. The phages were characterized by their plague and virion morphology, host range, and serological specificity.     

Isolation and partial characterization of cellulose-degrading strain of Streptomyces sp. LX from soil

X. LI AND P. GAO. 1996. A new bacterium, Streptomyces sp. LX, was isolated from soil, which was aerobic Gram-positive and could decompose crystalline cellulose completely. Endo-cellulase with CMC-liquefying activity was detected when α-cellulose, Avicel, Whatman CF₁₁ or CMC was used as carbon source, and its production varied with nature of the carbon source. Only traces of reducing sugar were found in cultures during incubation. This strain could produce FPase, β-glucanase and short fibre generating activity. Exo- and endo-cellulase were detected in cultures by measuring formation of total sugar but were not detected by determining release of reducing sugar.     

Prevalence of Clostridium botulinum type E and coexistence of C. botulinum nonproteolytic type B in the river soil of Japan.

Soil samples from 98 sites in the whole systems of four rivers in Japan were examined for the presence of Clostridium botulinum. Type E organism was prevalently shown throughout the whole river systems including upper part; detection rates of type E toxin in soil culture ranged from 33 to 82%. This type was also detected in soil of adjacent mountainous district. Type B and C toxins were detected at 7% and 9% of the sites examined, respectively. C. botulinum type E and nonproteolytic type B strains were isolated from enrichment cultures of soil samples. These results suggest that the terrestrial origin of type E organism would be considered as one of the reasons for the high incidence of this organism in the sea areas, and prove that C. botulinum nonproteolytic type B exists in the soil of Japan.     

Denitration of 2,4,6-trinitrotoluene by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> ESA-5 in the presence of ferrihydrite

Denitration of 2,4,6-trinitrotoluene (TNT) was evaluated in oxygen-depleted enrichment cultures. These cultures were established starting with an uncontaminated or a TNT-contaminated soil inoculum and contained TNT as sole nitrogen source. Incubations were carried out in the presence or absence of ferrihydrite. A significant release of nitrite was observed in the liquid culture containing TNT, ferrihydrite, and inoculum from a TNT-contaminated soil. Under these conditions, Pseudomonas aeruginosa was the predominant bacterium in the enrichment, leading to the isolation of P. aeruginosa ESA-5 as a pure strain. The isolate had TNT denitration capabilities as confirmed by nitrite release in oxygen-depleted cultures containing TNT and ferrihydrite. In addition to reduced derivatives of TNT, several unidentified metabolites were detected. Concomitant to a decrease of TNT concentration, a release of nitrite was observed. The concentration of nitrite peaked and then it slowly decreased. In the absence of TNT, the drop in the concentration of nitrite in oxygen-depleted cultures was lower when ferrihydrite was provided, suggesting that ferrihydrite inhibited the utilization of nitrite by P. aeruginosa ESA-5.     

Decomposition of the herbicide bromoxynil in soil and in bacterial cultures

It was found in field, and laboratory experiments that of 50 ppm of the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile added to grey forest soil 20-80% were still detected after three months). Bromoxynil did not influence (except for a short-termed stimulation of the number of bacteria) the amount and composition of the basic groups of soil microorganisms. In enrichment cultures of soil microorganisms metabolie products of bromoxynil decomposition (3,5-dibromo-4-hydroxybenzamide and 3,5-dibromo-4-hydroxybenzoic acid) were detected and a stimulating effect of cosubstratos on its decomposition was demonstrated. Bromoxynil concentration, aeration conditions and the presence of cosubstrates (ribose in particular) influenced the rate and degree of the decomposition process inPsevdomonas putida. In addition to the degradation products mentioned above, production of methoxylated and partially dehalogenated aromatic compounds was detected.     

Degradability of polychlorinated phenols by bacterial populations in soil

The biodegradabilities of polychlorinated phenols including 5 isomers of trichlorophenols, 3 isomers of tetrachlorophenols and pentachlorophenol, were tested with 170 samples of soil collected from various environments. After the samples were inoculated into a succinate-containing mineral medium and incubated, the cultures were acclimatized to phenol concentrations from 10 to 100 ppm. Twenty six samples (15%) were observed to degrade 2, 4, 6-trichlorophenol (246TrCP) and a mixed sample of soil degraded 2, 3, 4, 6-tetrachlorophenol, but no degradation was seen with other chlorophenols. All of the mixed cultures acclimatized to and degrading 246TrCP also degraded phenol. For the degradation of 246TrCP, the NO₃′ ion was preferred to the NH₄⁺ ion as a nitrogen source. At concentrations below 500 ppm, 246TrCP was degraded completely within 8 d and the chloride ion was detected in the culture broth at an amount corresponding to that of the chlorinated phenol, although cell growth was inhibited at a 246TrCP concentration of 1,000 ppm. No possible intermediate product of 246TrCP was detected in the cultures.     

Toxicities of DDE on Wheat and Bioremediation of DDE by Fungus Pleurotus pulmonarius

An in vitro study was performed to determine the acute phytotoxicities and genotoxicity of DDE either spiked to soil or added to hydrophonic cultures on wheat Triticum aestivum. A 24-well plate was first used to determine toxicity on individual grains using conventional seed germination/seedling growth toxicity tests whereas a single cell electrophoresis system was applied to measure genotoxicity at single cell level for wheat. Hydrophonic cultures provide a simplified environment to screen for toxicities with high sensitivity. Inverse dose-response relationships were detected between exogenous DDE levels and one of the following parameters: seed germination, seedling growth, and genotoxicity. In contrast, soil reduced the stress on T. aestivum by lowering bioavailability leading to less DDE distributed in radicle and coleoptile, modulated growth, and enhanced tolerance. At all DDE doses spiked to soil including the reference safety level of 0.5 mg/kg, DNA breakage was detected in both radicle and coleoptile but their magnitudes did not correlate with the organ nor the soil DDE contents. Thus, although wheat is highly sensitive to the genotoxic effect of DDE, first demonstrated here, the seed germination test offers a simple quantitative measure of DDE's phytotoxicity in soil and hydrophonic cultures. This study also found that fungus Pleurotus pulmonarius, which secretes extracellular ligninolytic enzymes causing non-specific cleavage of lignin and organopollutants, remediated DDE spiked to soil. In 5 weeks, 78% of 10 mg/kg DDE was biodegraded, and the fungal-treated soil reduced acute toxicity on T. aestivum using the seed germination test.     

Selection of appropriate host plants used in trap culture of arbuscular mycorrhizal fungi

Arbuscular mycorrhizal (AM) fungi in coalmine spoil, island forest and saline soils were enriched in pot culture with maize (Zea mays L.), tobacco (Nicotiana tabacum L.), white clover (Trifolium repens Linn.) and silverweed cinquefoil (Potentilla anserina L.). Based on spores, there were more species of AM fungi in the coalmine spoil (15 species, 3 genera), than in the forest soil (11 species, 4 genera) and the saline soil (5 species, 2 genera). In the trap cultures, the total of 28 species in Acaulospora, Gigaspora, Glomus, and Sclerocystis detected in the original soils were all recovered with at least one of the four trap plants. The highest spore and species numbers were recovered in trap cultures of T. repens inoculated with coalmine spoil. Glomus constrictum and Glomus multicaule were the dominant species associated with N. tabacum grown in saline soil and forest soil. The dominant species of AM fungi on the four hosts was Acaulospora mellea, which had over 90% of the spore incidence in pot trap culture in coalmine spoil. It is suggested that there be selectivity between host plants and AM fungi. The number of species of AM fungi detected was influenced by host plants under certain conditions and white clover was generally the optimal host plant to detect diversity of AM fungi.     

Immunological Detection of Nitrospira-like Bacteria in Various Soils

Chemolithotrophic nitrite oxidizers were enriched from five different soils including freshwater marsh, permafrost, garden, agricultural, and desert soils and monitored during the cultivation procedure. Immunoblot analysis was used to identify the nitrite oxidizing organisms with monoclonal antibodies, which recognize the key enzyme of nitrite oxidation in a genus-specific reaction [Bartosch et al. (1999) Appl Environ Microbiol 65:4126-4133]. The morphological characteristics of the enriched nitrite oxidizers were additionally studied using transmission electron microscopy (TEM) and fluorescence microscopy. By means of the antibodies and TEM analysis Nitrospira could be clearly identified in enrichment cultures derived from freshwater marsh and from permafrost soil. Nitrospira cells were enriched simultaneously with cells of the genus Nitrobacter when nitrite concentrations of 0.2 g of NaNO2 L(-1) were used. However, in enrichment cultures containing 2 g of NaNO2 L(-1) Nitrobacter was exclusively detected. During fluorescence microscopic observations of DAPI stained samples microcolonies were found in enrichment cultures from freshwater marsh, permafrost, garden, and agricultural soil. They had a similar morphology to Nitrospira-like microcolonies from activated sludge. In conclusion, Nitrospira seems to be not only a common aquatic but also a usual soil bacterium.     

Involvement of fungi and bacteria in enhanced and nonenhanced biodegradation of carbendazim and other benzimidazole compounds in soil

The relationship between chemical structure and the enhancement of microbial degradation of three benzimidazole compounds in soil was determined. Preapplication of methyl benzimidazole-2-ylcarbamate (carbendazim or MBC), 2-aminobenzimidazole (2AB), and benzimidazole enhanced their degradation upon repeated application (self-enhanced degradation). MBC and 2AB cross-enhanced the degradation of each of these two compounds, whereas benzimidazole did not enhance the degradation of MBC. Thiabendazole (TBZ) did not enhance its own degradation or cross-enhance the degradation of MBC. No increase in the number of MBC-degrading fungi or in the capacity of soilborne fungi to degrade MBC was detected in soil exhibiting enhanced MBC degradation (MBC-history). A sharp increase in esterolytic activity in the microsomal fraction of Alternaria alternata capable of degrading MBC in culture was induced by the presence of MBC in the growth medium. 2AB was the main metabolite of MBC that accumulated in A. alternata cultures and in cell-free preparations. MBC was degraded much faster by mixed bacterial cultures that originated from MBC-history soil than in cultures from MBC-nonhistory soil. Fluctuations in the MBC degrading capacity of mixed bacterial cultures occurred during repeated subculturing of the mixed culture. Inoculation of nonhistory soil with mixed bacterial cultures resulted in enhanced MBC degradation, whereas inoculation with A. alternata did not enhance MBC degradation. It is suggested that while fungi contribute to MBC dissipation in soil, bacteria have a greater role in enhanced biodegradation of MBC in soil.     

Isolation and partial properties of cellulose-decomposing strain of Cytophaga sp. LX-7 from soil

A new facultatively anaerobic, Gram-negative bacterium, Cytophaga sp. LX-7, degrading crystalline cellulose completely, was isolated from soil by dilution plating on cellodextrin agarose plates. This strain could excrete extracellularly all three types of cellulase and cellulosic substrates were the strongest inducer of endocellulase with CMC-liquefying activity production. No reducing sugar was found in cultures of cellulose during incubation. An enzyme which degrades crystalline cellulose was detected in cultures of cellulose by measuring the formation of soluble carbohydrate but was not detected by determining the reducing sugar released. This strain also synthesized cell-bound cellobiose oxidizing enzyme which was previously noted only in fungi. Both cellulose and soluble sugars could promote the production of cellobiose oxidizing enzyme.     

Molecular analyses of methyl-coenzyme M reductase alpha-subunit (mcrA) genes in rice field soil and enrichment cultures reveal the methanogenic phenotype of a novel archaeal lineage

The diversity of methanogen-specific methyl-coenzyme M reductase alpha-subunit (mcrA/mrtA) genes in Italian rice field soil was analysed using a combination of molecular techniques and enrichment cultures. From 75 mcrA/mrtA clones retrieved from rice field soil, 52 were related to members of the Methanosarcinaceae, Methanosaetaceae and Methanobacteriaceae. However, 19 and four clones formed two novel clusters of deeply branching mcrA sequences, respectively, which could not be affiliated to known methanogens. A new methanogen-specific fingerprinting assay based on terminal restriction fragment length polymorphism (T-RFLP) analysis of fluorescently labelled polymerase chain reaction (PCR) products allowed us to distinguish all environmental mcrA/mrtA sequences via group-specific Sau96I restriction sites. Even genes for the isoenzyme methyl-coenzyme M reductase two (mrtA) of Methanobacteriaceae present in rice field soil were represented by a unique 470 bp terminal restriction fragment (T-RF). Both cloning and T-RFLP analysis indicated a significant representation of novel environmental mcrA sequences in rice field soil (238 bp T-RF). To identify these mcrA sequences, methanogenic enrichment cultures with rice field soil as inoculum were established with H2/CO2 as substrates at a temperature of 50 degrees C, and these were monitored using molecular tools. In subsequent transfers of these enrichment cultures, cloning and T-RFLP analysis detected predominantly SSU rRNA genes of rice cluster I (RC-I), an uncultivated euryarchaeotal lineage discovered previously in anoxic rice field soil. In parallel, both mcrA cloning and T-RFLP analyses of the enrichment culture identified the more frequent cluster of novel environmental mcrA sequences as belonging to members of RC-I. Thus, we could demonstrate the genotype and phenotype of RC-I Archaea by the presence of a catabolic gene in a methanogenic enrichment culture before the isolation of pure cultures.     

Occurrence of collagen-degrading microorganisms in associations of mesophilic heterotrophic bacteria from various soils

A total of 437 bacterial cultures was isolated from various soils and sewage water that were tested for the ability to decompose reconstituted collagen. This activity was found in 6.6% of the cultures isolated from sewage water, 15% of the cultures from organic horizons of the spruce growth soil, 30% of the cultures from the meadow soil, 29% of the cultures from the vegetable field soil and in 37% of those isolated from garden soil. The capability to produce collagenolytic enzymes does not appear to be rare among soil bacteria.     

Association of the Plant-beneficial Fungus Verticillium lecanii with Soybean Roots and Rhizosphere

Colonization of soybean roots by the biocontrol fungus Verticillium lecanii was studied in vitro and in situ. For in vitro experiments, V. lecanii was applied to soybean root tip explant cultures. Four weeks after inoculation, the fungus grew externally on at least half of the roots (all treatments combined), colonizing 31% to 71% of root length (treatment means). However, when a potato dextrose agar plug was available as a nutrient source for the fungus, root tips inoculated soon after transfer were not colonized by V. lecanii unless Heterodera glycines was present. Scanning electron microscopy of colonized roots from in vitro cultures revealed a close fungus-root association, including fungal penetration of root cells in some specimens. In the greenhouse, soybeans in sandy soil and in loamy sand soil were treated with V. lecanii applied in alginate prills. The fungus was detected at greater depths from the sandy soil than from the loamy sand soil treatment, but fungus population numbers were small and variable in both soils. Root box studies coupled with image processing analysis of the spatial distribution of V. lecanii in sandy soil supported these findings. Verticillium lecanii was detected randomly in the rhizosphere and soil of root boxes, and was rarely extensively distributed. These in vitro and in situ experiments indicate that V. lecanii can grow in association with soybean roots but is a poor colonizer of soybean rhizosphere in the soil environment.     

Genetic exchange in Bacillus subtilis in soil.

Summary Genetically labelled strains of Bacillus subtilis have been shown to exchange blocks of linked genes while growing together in soil. After eight days of incubation, 79% of unselected colony-forming units exhibited a phenotype containing markers from both parents; the parental strains were not detected after the first day of incubation. High frequencies of trans-formation were also obtained by adding genetically labelled deoxyribonucleic acid to single-strain soil cultures. Observed linkage of genetic markers was greater in soil transformation than in standard laboratory procedures. The results indicate that transformation may play an important role in the adaptation of the Bacilli to their natural habitat.     

Biomineralization of an organophosphorus pesticide,Monocrotophos, by soil bacteria

AIMS: To study biomineralization of Monocrotophos (MCP) and identify the metabolites formed during biodegradation. METHODS AND RESULTS: Two cultures, namely Arthrobacter atrocyaneus MCM B-425 and Bacillus megaterium MCM B-423, were isolated by enrichment and adaptation culture technique from soil exposed to MCP. The isolates were able to degrade MCP to the extent of 93% and 83%, respectively, from synthetic medium containing MCP at the concentration of 1000 mg x l(-1), within 8 d, under shake culture condition at 30 degrees C. The cultures degraded MCP to carbon dioxide, ammonia and phosphates through formation of one unknown compound--Metabolite I, valeric or acetic acid and methylamine, as intermediate metabolites. The enzymes phosphatase and esterase, reported to be involved in biodegradation of organophosphorus compounds, were detected in both the organisms. CONCLUSIONS:Arthrobacter atrocyaneus MCM B-425 and B. megaterium MCM B-423 isolated from soil exposed to MCP were able to mineralize MCP to carbon dioxide, ammonia and phosphates. SIGNIFICANCE AND IMPACT OF THE STUDY: Pathway for biodegradation of MCP in plants and animals has been reported. A microbial metabolic pathway of degradation involving phosphatase and esterase enzymes has been proposed. The microbial cultures could be used for bioremediation of wastewater or soil contaminated with Monocrotophos.     

Degradation of 2,4,6-trichlorophenol by a specialized organism and by indigenous soil microflora: bioaugmentation and self-remediability for soil restoration

A selected mixed culture and a strain of Alcaligenes eutrophus TCP were able to totally degrade 2,4,6-TCP with stoichiometric release of Cl⁻. In cultures of Alc. eutrophus TCP, a dioxygenated dichlorinated metabolite was detected after 48 h of incubation. Experiments conducted with soil microcosms gave evidence that : the degradative process had a biotic nature and was accompanied by microbial growth ; the soil used presented an intrinsic degradative capacity versus 2,4,6-TCP ; the specialized organism used as inoculum was effective in degrading 2,4,6-TCP in a short time. These results could be utilized for the adoption of appropriate remediation techniques for contaminated soil.