Anna Mårtensson

Where's the limit? Changes in the microbiological properties of agricultural soils at low levels of metal contamination

A number of microbial properties previously shown to be sensitive to heavy metal toxicity were determined in soils from field experiments at Brunnby and Robacksdalen in Sweden. The properties investigated were the acetylene reduction activity (ARA) potential of blue-green algae and heterotrophic soil bacteria, population size of Rhizobium leguminosarum bv. trifolii, size of the microbial biomass, basal respiration, specific respiration rate of the biomass, bacterial community metal tolerance, phospholipid fatty acid pattern of the soil microbial community and lag period and specific microbial growth rate determined after glucose addition to soil. In the Brunnby soils, sewage sludge applications between 1966 and 1989 had increased the soil C content from 2.3 to 2.6% and reduced soil pH from 6.1 to 5.8. Concentrations of Cd, Cr, Cu, Pb and Zn in the soil had increased by up to 76%, but had not reached the current lower EC limits for soils. Most of the measured microbial properties were affected by the sludge additions, although effects were generally moderate. We observed reductions of between 15 and 80% in autotrophic and heterotrophic ARA potential, in numbers of rhizobia and in the biomass C-to-organic C ratio, and increases between 25 and 76% in the specific microbial respiration rate, and in the lag time and the specific microbial growth rate upon glucose addition. There were significant differences in the community structure determined by phospholipid fatty acid (PLFA) patterns between the high sludge treatment and the control and low sludge treatment, and in the bacterial community Cu tolerance between the high sludge treatment and the control. Basal respiration was not significantly affected by past sludge additions. At Robacksdalen, additions of metal salt solutions between 1979 and 1991 had increased soil concentrations of Cd, Cu and Pb by up to 23%, but soil concentrations were nevertheless below the background concentrations at Brunnby. In spite of the low metal concentrations, small, but statistically significant effects of metal addition on the specific respiration rate, lag time before the onset of microbial growth upon glucose addition and on potential autotrophic and heterotrophic ARA were found. The findings are discussed in relation to current legislation for soil protection.

Size of the soil microbial biomass in a long-term field experiment as affected by different n-fertilizers and organic manures

The size of the soil microbial biomass was measured in a more than 30 yr old field experiment, whose treatments included different N fertilizers and organic manures. The size of the microbial biomass was measured as biomass C and N by the chloroform fumigation-incubation technique, as K2SO4 extractable ninhydrin-reactive N released upon fumigation and as the soils ATP content. There was a high degree of correlation (r > 0.88) between the fumigation-based methods and the ATP determinations. Compared with the biomass estimate by ATP, biomass C was underestimated in the ammonium sulphate fertilized soil (pH 4.4), the peat-amended soils, and the sewage sludge amended soil. Biomass N was only underestimated in the ammonium sulphate and peat-amended soil, whereas there was a good correlation between the ninhydrin assay and the ATP assay for all soils. Between three successive years biomass C showed larger, statistically significant, variations than the size of the biomass measured by the ninhydrin assay. There was a high degree of correlation (r > 0.90) between both the rate of base respiration and the size of the microbial biomass and the soils carbon content. These relationships generally held independent of whether carbon was derived from stabilized soil organic matter (in the fallow soil), from crop residues, or from organic manures such as straw, green manure, farmyard manure, or sawdust. Relative to the soils carbon content the microbial biomass was smaller than expected in the peat amended-soils, the ammonium sulphate fertilized, and the sewage sludge-amended soil. The rate of base respiration was only lower than expected in the sewage sludge treated soil. The size of the biomass was negatively affected by a low soil pH, but the rate of base respiration was not. Liming some of the soils indicated that other factors than low pH restricted the size of the biomass in the peat and sewage-sludge amended soils, but not in the ammonium sulphate fertilized soils.

Potential for improving pea production by co-inoculation with fluorescent Pseudomonas and Rhizobium

Seed bacterization with five plant growth promoting fluorescent Pseudomonas strains isolated from Indian and Swedish soils and three Rhizobium leguminosarumbiovar viceae strains isolated from Swedish soils were shown to promote plant growth in Pisum sativum L. cv. Capella. Co-inoculation of the fluorescent pseudomonads and Rhizobium improved plant growth in terms of shoot height, root length and dry weight. Both the fluorescent pseudomonads and Rhizobium were shown to exhibit a wide range of antifungal activity against pathogens specific to pea. Seed bacterization with plant growth promoting strains alone and together with a rhizobial isolate, R 361-27 reduced the number of infected peas grown in Fusarium oxysporum infested soils. We found that the introduced organisms were able to colonize the roots, which was confirmed using immunofluorescence staining and drug resistant mutant strains. In a synthetic culture medium, all the plant growth promoting fluorescent pseudomonads strains produced siderophores, which shown to express antifungal and antibacterial activity. Our results suggest the potential use of these bacteria to induce plant growth and disease suppression in sustainable agriculture production systems.

Influence of various soil amendments on nitrogen-fixing soil microorganisms in a long-term field experiment, with special reference to sewage sludge

The effects of additions of various organic materials and nitrogen fertilizers on legume bacteria, blue-green algal populations and free-living nitrogen-fixing soil microorganisms in a 30 yr old field experiment have been investigated. Soil pH was found to be an important regulating factor for the occurrence and activity of nitrogen-fixing microorganisms. Nitrogen-fixing processes were retarded in the soil treated with sewage sludge compared to soils with a similar pH, indicating that other unfavourable conditions had build up in the soil through the sewage sludge additions, likely elevated metal concentrations. Relatively high numbers of Rhizobium leguminosarum biovar trifolii were found in all treatments although no legumes have been grown since the start of the experiment. Numbers of rhizobia in the soils were related to soil pH and all isolates were equally effective. Delayed nodulation was observed in plants inoculated with bacteria isolated from plots treated with sewage sludge. No increase in heavy-metal resistance was found in these bacteria when studied on agar plates. The occurrence and nitrogen-fixing activity of blue-green algal populations were correlated with soil pH. Algae were present in all treatments, except for the ammonium sulphate fertilized treatment (pH 4.4). Maximum nitrogen-fixing activity of the blue-green algae when grown under optimum conditions occurred after 1.5 months, except for the soil treated with sewage sludge, where maximum activity was found after 5 months, when it was ca 100 times lower than in the other treatments. There was no indication that this slowly established population was better adapted to growth in the presence of elevated heavy-metal concentrations when grown in liquid media. Nitrogen-fixation activity of the free-living aerobic nitrogen-fixing bacteria was correlated with soil pH and was reduced in the sewage sludge treated soil, compared to soils with similar pH. The implications of the results for control of metal contamination of soils is discussed.

Potential to improve transfer of N in intercropped systems by optimising host-endophyte combinations

Possibilities for improving N transfer from N2-fixing plants to non-N2-fixing plants by mycorrhiza have been investigated. Initially, the genetic variability with respect to N uptake was assessed by screening five varieties of chicory (Cichorium intybus L.), four of peas (Pisum sativum L.) and three of red clover (Trifolium pratense L.) in combination with eight isolates of arbuscular mycorrhizal fungi. The most promising plant - fungi combinations identified through the cultivar screening were used to optimise conditions for N transfer between intercropped N2-fixing plants (peas and clover) and non-N2-fixing chicory. In the first experiment, the recovery of fixed legume N was investigated using three cultivars, of chicory intercropped with pea variety, and inoculated with one of four mycorrhizal isolates. Roots of the N2-fixing pea and the non-N2-fixing chicory were separated by a root-free soil layer in a three-compartment container. A section of the legume roots was forced to grow into a separate compartment which received four split applications of 15N. The percentage of N in the chicory derived from transfer ranged between 3% and 50%. In a second experiment one chicory variety was intercropped with one red clover variety and inoculated with four mycorrhizal isolates respecetively. A harvest regime was chosen in which the shoots were harvested from intercropped plants at 3,4.5 and 6 months of age. At three months the percentage of N in the chicory derived from transfer ranged between 15% and 18% and at a plant age of 4.5 months from 46 to 77%. At six months the percentage of N in the chicory roots derived from transfer of legume N ranged from 20 to 34% and varied with fungal isolate. Our results show that there is potential for improving N transfer in intercropped plant systems through the methodological selection of suitable plant and mycorrhizal partners.

Organically produced plant foods: Evidence of health benefits

If in plant secondary metabolites, the presence of pesticides and nutritional balances are the most important determinants of nutritional value of food plants, then organically produced plant foods could be expected to bemore health-promoting than conventional foods. Differences arise in management practices between organic and conventional farming where the former support (a) an activation of the plant defence mechanisms by excluding synthetic plant protection agents, (b) an active soil life where plants and microbes interact, exchanging certain metabolic compounds and (c) a balanced mineral nutrient uptake where excesses of easily available nutrients are avoided.

Nitrogen fertilization does not affect micronutrient uptake in grain maize (Zea mays L.)

Abstract Due to continuous single nitrogen fertilization, we hypothesized a built-up deficiency of micronutrients in crops that would limit plant growth and crop quality. In 2-year field experiments using urea-N fertilized grain maize (Zea mays L.), hybrid KWS 2376 at 0, 120 and 240 kg N ha−1 crop uptake of Zn, Mn, Cu and Fe was studied at DC 32, DC 61 and in the grain harvested. Micronutrient contents at DC 32 stage – 1st node (aboveground phytomass) and DC 61 – flowering (ear leaf) were all at levels indicative of adequate micronutrient supply to the crop. At both sampling occasions the Fe:Zn and Fe:Mn ratios were adequate implying that Fe did not inhibit the uptake of Zn and Mn. The application of nitrogen increased the Fe content at the 1st sampling in both years; in the second year the same was also the case for the Zn content. Nitrogen nutrition increased the contents of Mn and Fe at the 2nd sampling only in year 2; in the other treatments no changes were observed in the micronutrient contents. Micronutrient correlations in the grain were discovered between Zn and Mn contents and between Fe and Mn contents. In the second year the highest N-rate significantly increased the Fe and Zn content of the grain compared with the lower rates of nitrogen fertilization. Grain yields were not affected by the rate of nitrogen and ranged between 13.65 and 14.34 t ha−1 (1st year) and between 13.68 and 14.18 t ha−1 (2nd year). Nitrogen fertilization did not reduce the content of micronutrients in the plant or grain of maize. It is evident that the continuous single use of N fertilization so far has not resulted in a micronutrient deficiency of the plants limiting the nutrient density of the grain or reducing its quality.

A Modified, Highly Sensitive Enzyme-linked Immunosorbent Assay (ELISA) for Rhizobium meliloti Strain Identification

SUMMARY: An evaluation of the ELISA technique was made in order to obtain a very specific and sensitive method for strain identification of Rhizobium meliloti. Antisera against an R. meliloti strain were produced by using as antigen either whole cells or purified lipopolysaccharides from the cell wall. The use of whole cells as antigen gave rise to antibodies which were more sensitive and strain specific than those produced from purified lipopolysaccharides. A further evaluation of the ELISA method was the use of a new type of conjugate consisting of a purified antibody linked to β-galactosidase by using as coupling reagent a hetero-bifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), which contains two reactive groups directed towards different functional groups. Substrate for this type of conjugate consisted of o-nitrophenyl-β-D-galactopyranoside. This improved technique makes it possible to detect bacteria in samples containing 1 × 103 cells ml−1 whilst retaining strain specificity. Furthermore, preparations of nodules formed by different R. meliloti strains containing 1 × 105 cells ml−1 could be analysed whilst retaining strain specificity. The technique presented thus offers advantages in higher sensitivity and reliability than conventional ELISA techniques.

Occurrence of Glomeromycota spores and some arbuscular mycorrhiza fungal species in arable fields in Sweden

Fungi within the phylum Glomeromycota were investigated in arable fields throughout Sweden. Sweden is located between 55° and 69° North. The fungi within this phylum form arbuscular mycorrhizal symbiosis with plant roots. Sampling of soil was carried out to a depth of 30 cm in the rhizosphere. Arbuscular mycorrhizal fungi were found at all 45 sampling sites, at densities between 3 and 44 spores per g dry weight of soil. No significant differences in spore densities were found between different agro-climatic zones or between semi-natural grassland and ploughed fields. Our study revealed that the upper half (0–15 cm) of the soil profiles had significantly more spores than the lower half (15–30 cm). Spores from eight sampling sites were identified from the indigenous soils. Almost 90% were shown to belong to the genus Glomus. The other genera found were Gigaspora and Scutellospora.

Monitoring sewage sludge using heterotrophic nitrogen fixing microorganisms

Abstract Sewage sludge was studied using free-living N 2 -fixing bacteria in two types of soil amended with six types of municipal sewage sludges and cow and pig manures, respectively. Sludge and manure treatments were as follows: no addition, Swedish recommended rates of 5 t dry wt ha −1 , twice the standard rate of addition (2RR), and 10 times the standard rate (10RR). The N 2 -fixing activities of the soils were unaffected by additions of sludge with elevated concentrations of Cd, Co, Pb, Zn and naphthalene, corresponding to the recommended rate and 2RR and 10RR. Addition of a sludge with low contents of heavy metals and organic pollutants reduced N 2 -fixation activities at all concentrations in the Galbo soil. Additions of a sludge with an elevated content of Pb and PCBs reduced the N 2 -fixation when added at 10RR. The Ulleraker soil, which had higher contents of organic carbon and clay than the Galbo soil, showed reduced N 2 -fixation when treated with these sludges at 10RR. Addition of a sludge with elevated concentrations of Ag, Cr, Hg and naphthalene reduced N 2 -fixation when applied at 10RR in both soils. Sludge with the highest concentration of Cu together with elevated concentrations of Ag, nonylphenol and PCBs reduced the N 2 -fixation when applied at the standard rate and 10RR in both soils. The sludge with a moderate concentration of all studied heavy metals except Ag, but with elevated concentrations of nonylphenol and toluene, reduced N 2 -fixation when applied at 2RR in both soils. Additions of cow manure with high concentrations of ammonium and nonylphenol did not affect N 2 -fixation in the Galbo soil at the recommended rate, but reduced it at all concentrations in the Ulleraker soil. Additions of pig manure, with elevated concentrations of ammonium, phenols and toluene, strongly reduced N 2 -fixation at all rates in both soils. The adverse effects of sludges or manures on potential N 2 -fixing activities could partly be explained by the increasing concentrations of ammonium present in soil. Above a certain concentration of added ammonium, which varied between the soils, N 2 -fixation was inhibited. In some sludge-soil combinations, however, adverse effects on N 2 -fixation were observed that could not be explained by elevated ammonium rates. In these combinations, elevated concentrations of Ag, Cu and nonylphenol were observed. Adverse effects of these substances on heterotrophic N 2 -fixation were also observed in pure culture studies where Azotobacter sp. isolated from the Galbo soil were found to be sensitive to Ag, Cd, Co, Cu, Hg, Ni, Zn and nonylphenol at maximum concentrations present in the soils after additions of the sludges. Our findings indicate that particular elements or substances, such as Ag, Cu and nonylphenol present in the sludges or manures, are particularly toxic and deserve attention and that measurement of N 2 -fixation in sludge or manure treated soils is a useful method for predicting the biological quality of a particular organic slurry.