Elevation of atmospheric CO2 and N-nutritional status modify nodulation, nodule-carbon supply, and root exudation of Phaseolus vulgaris L.

Increased root exudation and a related stimulation of rhizosphere-microbial growth have been hypothesised as possible explanations for a lower nitrogen- (N-) nutritional status of plants grown under elevated atmospheric CO₂ concentrations, due to enhanced plant-microbial N competition in the rhizosphere. Leguminous plants may be able to counterbalance the enhanced N requirement by increased symbiotic N₂ fixation. Only limited information is available about the factors determining the stimulation of symbiotic N₂ fixation in response to elevated CO₂.In this study, short-term effects of elevated CO₂ on quality and quantity of root exudation, and on carbon supply to the nodules were assessed in Phaseolus vulgaris, grown in soil culture with limited (30 mg N kg⁻¹ soil) and sufficient N supply (200 mg N kg⁻¹ soil), at ambient (400 μmol mol⁻¹) and elevated (800 μmol mol⁻¹) atmospheric CO₂ concentrations.Elevated CO₂ reduced N tissue concentrations in both N treatments, accelerated the expression of N deficiency symptoms in the N-limited variant, but did not affect plant biomass production. ¹⁴CO₂ pulse-chase labelling revealed no indication for a general increase in root exudation with subsequent stimulation of rhizosphere microbial growth, resulting in increased N-competition in the rhizosphere at elevated CO₂. However, a CO₂-induced stimulation in root exudation of sugars and malate as a chemo-attractant for rhizobia was detected in 0.5-1.5 cm apical root zones as potential infection sites. Particularly in nodules, elevated CO₂ increased the accumulation of malate as a major carbon source for the microsymbiont and of malonate with essential functions for nodule development. Nodule number, biomass and the proportion of leghaemoglobin-producing nodules were also enhanced. The release of nod-gene-inducing flavonoids (genistein, daidzein and coumestrol) was stimulated under elevated CO₂, independent of the N supply, and was already detectable at early stages of seedling development at 6 days after sowing.     

Soil organic matter dynamics and land use change at a grassland/forest ecotone

In the grassland/forest ecotone of North America, many areas are experiencing afforestation and subsequent shifts in ecosystem carbon (C) stocks. Ecosystem scientists commonly employ a suite of techniques to examine how such land use changes can impact soil organic matter (SOM) forms and dynamics. This study employs four such techniques to compare SOM in grassland (Bromus inermis) and recently forested (∼35 year, Ulmus spp. and Quercus spp.) sites with similar soil types and long-term histories in Kansas, USA. The work examines C and nitrogen (N) parameters in labile and recalcitrant SOM fractions isolated via size and density fractionation, acid hydrolysis, and long-term incubations. Size fractionation highlighted differences between grassland and forested areas. N concentration of forested soils’ 63-212 μm fraction was higher than corresponding grassland soils’ values (3.0±0.3 vs. 2.3±0.3 mg g_fraction⁻¹, P<0.05), and N concentration of grassland soils’ 212-2000 μm fraction was higher than forested soils (3.0±0.4 vs. 2.3±0.2 mg g_fraction⁻¹, P<0.05). Similar trends were observed for these same fractions for C concentration; forested soils exhibited 1.3 times the C concentration in the 63-212 μm fraction compared to this fraction in grassland soils. Fractions separated via density separation and acid hydrolysis exhibited no differences in [C], [N], δ¹⁵N, or δ¹³C when compared across land use types. Plant litterfall from forested sites possessed significantly greater N concentrations than that from grassland sites (12.41±0.10 vs. 11.62±0.19 mg g_litter⁻¹). Long-term incubations revealed no differences in C or N dynamics between grassland and forested soils. δ¹³C and δ¹⁵N values of the smallest size and the heavier density fractions, likely representing older and more recalcitrant SOM, were enriched compared to younger and more labile SOM fractions; δ¹⁵N of forested soils’ 212-2000 μm fraction were higher than corresponding grassland soils (1.7±0.3‰ vs. 0.5±0.4‰). δ¹³C values of acid hydrolysis fractions likely reflect preferential losses of ¹³C-depleted compounds during hydrolysis. Though C and N data from size fractions were most effective at exhibiting differences between grassland and forested soils, no technique conclusively indicates consistent changes in SOM dynamics with forest growth on these soils. The study also highlights some of the challenges associated with describing SOM parameters, particularly δ¹³C, in SOM fractions isolated by acid hydrolysis.     

Sampling effects on the assessment of genetic diversity of rhizobia associated with soybean and common bean

Biological nitrogen fixation plays a key role in agriculture sustainability, and assessment of rhizobial diversity contributes to worldwide knowledge of biodiversity of soil microorganisms, to the usefulness of rhizobial collections and to the establishment of long-term strategies aimed at increasing contributions of legume-fixed N to agriculture. Although in recent decades the use of molecular techniques has contributed greatly to enhancing knowledge of rhizobial diversity, concerns remain over simple issues such as the effects of sampling on estimates of diversity. In this study, rhizobia were isolated from nodules of plants grown under field conditions, in pots containing soil, or in Leonard jars receiving a 10⁻² or a 10⁻⁴ serially-diluted soil inoculum, using one exotic (soybean, Glycine max) and one indigenous (common bean, Phaseolus vulgaris) legume species. The experiments were performed using an oxisol with a high population (10⁵ cells g⁻¹ soil) of both soybean rhizobia, composed of naturalized strains introduced in inoculants and of indigenous common-bean rhizobia. BOX-PCR was used to evaluate strain diversity, while RFLP-PCR of the ITS (internally transcribed spacer) region with five restriction enzymes aimed at discriminating rhizobial species. In both analyses the genetic diversity of common-bean rhizobia was greater than that of soybean. For the common bean, diversity was greatly enhanced at the 10⁻⁴ dilution, while for the soybean dilution decreased diversity. Qualitative differences were also observed, as the DNA profiles differed for each treatment in both host plants. Differences obtained can be attributed to dissimilarity in the history of the introduction of both the host plant and the rhizobia (exotic vs. indigenous), to host-plant specificity, rhizobial competitiveness, and population structure, including ease with which some types are released from microcolonies in soil. Therefore, sampling method should be considered both in the interpretation and comparison of the results obtained in different studies, and in the setting of the goals of any study, e.g. selection of competitive strains, or collection of a larger spectrum of rhizobia. Furthermore, effects of sampling should be investigated for each symbiosis.     

Influence of selenium on oxidoreductive enzymes activity in soil and in plants

The aim of this greenhouse experiment was the assessment of the influence of H₂SeO₃ at soil concentrations of 0.05, 0.15 and 0.45 mmol kg⁻¹, on the activity of selected oxidoreductive enzymes in wheat (Triticum aestivum). The wheat plants were grown in 2 dm³ pots filled with dust-silt black soil of pH 7.7. Applied H₂SeO₃ caused activation of plant nitrate reductase at all concentrations, but activation of plant polyphenol oxidase at only two lower concentrations. The highest concentration caused inhibition of polyphenol oxidase and peroxidase. Plant catalase activity decreased under the influence of 0.15 and 0.45 mmol kg⁻¹ concentration. After the final analysis Se was quantified in plants and soil. The amounts in plants were: control (unamended soil) 1.95 mg kg⁻¹; I dose (0.05 mmol kg⁻¹) 18.27 mg kg⁻¹; II dose (0.15 mmol kg⁻¹) 33.20 mg kg⁻¹ and III dose (0.45 mmol kg⁻¹) 38.37 mg kg⁻¹, in soil: 0.265 mg kg⁻¹; 3.61 mg kg⁻¹; 10.53 mg kg⁻¹; 30.53 mg kg⁻¹; respectively. Simultaneously, a laboratory experiment was performed, where the activity of soil catalase and peroxidase were tested after 1, 3, 7, 14, 28, 56, and 112 days after Se treatment. Peroxidase activity in soil decreased with increasing Se content, over the whole experiment. The lowest dose of Se caused activation a significant 10% increase in catalase activity, but the influence of others doses was unclear.     

Specific flavonoids as interconnecting signals in the tripartite symbiosis formed by arbuscular mycorrhizal fungi, Bradyrhizobium japonicum (Kirchner) Jordan and soybean (Glycine max (L.) Merr.)

Many legume plants benefit from the tripartite symbiosis of arbuscular mycorrhizal fungi (AMF) and rhizobia. Beneficial effects for the plant have been assumed to rely on increased P supply through the mycorrhizas. Recently, we demonstrated that P does not regulate the establishment of the tripartite symbiosis. Flavonoids appear to play a role as early signals for both rhizobia and AMF. Four soybean lines known to express different concentrations of the isoflavones genistein, daidzein and glycitein in the seed were used to test three hypotheses: (i) The establishment of the tripartite symbiosis is not dependent of a nutrient mediated effect; (ii) There is a positive correlation between seed isoflavone concentrations of different soybean lines and the progress of the tripartite symbiosis; (iii) Specific flavonoids control the establishment of the tripartite symbiosis in that a change in flavonoid root accumulation resulting from the development of one microbial partner can stimulate colonization of soybean roots by the other. Disturbed versus undisturbed soil treatments were produced to vary the potential for indigenous AMF colonization of soybean. In contrast, the potential for Bradyrhizobium was kept identical in both soil disturbance treatments. The uptake of P and Zn and the concentration of flavonoids in mycorrhizal soybean roots at 10 d after emergence were analysed either separately of Bradyrhizobium or in context of the tripartite symbiosis. Zinc nutrition did not differ between AMF treatments which supports the first hypothesis. The concentration of daidzein was at least four times greater in the root than in the seed reaching 3958±249 μg g⁻¹ dry across soybean lines. Coumestrol, which was absent in the seed, was synthesized to reach 2154±64 μg g⁻¹ dry. Conversely, the concentration of genistein was approximately three times smaller in the root that in the seed (301±15 μg g⁻¹ dry), while glycitein and formononetin were never detected. The establishment of the tripartite symbiosis was identical across soybean lines which does not support the second hypothesis. Concentrations of flavonoids were significantly greater in roots under disturbed soil, for which both symbioses were not as developed as in plants from undisturbed soil. This clearly supports the third hypothesis. This research provides the first data linking the function of different flavonoids to the establishment of the tripartite symbiosis, and suggests that these compounds are produced and released into the rhizosphere as a function of the colonization process.     

Isolation and characterisation of symbiotically effective Rhizobium resistant to arsenic and heavy metals after the toxic spill at the Aznalcóllar pyrite mine

After the toxic spill occurred at Aznalcóllar pyrite mine (Southern Spain), a wide area of croplands near the Doñana Wild Park was contaminated with 4.5 million m³ of slurries composed of acidic waters loaded with toxic metals and metalloids such as As, Sb, Zn, Pb, Cu, Co, Tl, Bi, Cd, Ag, Hg and Se. Today, 6 years after the spill, the concentration of toxic elements in these soils is still very high, in spite of the efforts to clean the zone. However, some plant species have colonised this contaminated area. Legumes possessing N₂-fixing nodules on their roots represented a significant proportion of these plants. Our objective was to use the Rhizobium-legume symbiosis as a new tool for bioremediate the affected area. We have isolated about 100 Rhizobium strains, 41 of them being resistant to high concentrations of As (300 mg l⁻¹), Cu (100 mg l⁻¹) and Pb (500 mg l⁻¹). Their phenotypes and bioaccumulation potentials have been characterised by their growth rates in media supplemented with As and heavy metals. The presence of the resistance genes in some strains has been confirmed by PCR and Southern blot hybridisation. Several Rhizobium were symbiotically effective in the contaminated soils. On the other hand, the first steps in nodule establishment seemed to be more affected by heavy metals than N₂-fixation.     

Volatile organic compounds in the roots and rhizosphere of Pinus spp.

Plant roots normally release a complex mixture of chemicals which have important effects in the rhizosphere. Among these different root-emitted compounds, volatile isoprenoids have received very little attention, yet they may play important and diverse roles in the rhizosphere, contributing to the regulation of microbial activity and nutrient availability. It is therefore important to estimate their abundance in the rhizosphere, but so far, there is no reliable sampling method that can be used to measure realistic rates of root emissions from plants growing in field conditions, or even in pots. Here, we measured root content of volatile isoprenoids (specifically monoterpenes) for Pinus pinea, and explored the feasibility of using a dynamic bag enclosure method to measure emissions from roots of intact pot-grown plants with different degrees of root cleaning. We also investigated a passive diffusion method for exploring monoterpenes in soil at incremental distances from mature Pinus sylvestris trees growing in field conditions. Total monoterpene content of P. pinea roots was 415±50 μg g⁻¹ fresh wt in an initial screening study, and between 688±103 and 1144±208 μg g⁻¹ dry wt in subsequent investigations. Emissions from shaken-clean roots of intact plants and roots of intact plants washed to remove remaining soil after shaken-clean experiments were 119±14 and 26±5 μg g⁻¹ dry wt h⁻¹, respectively. Emissions from intact roots in soil-balls were an order of magnitude lower than from shaken-clean roots, and probably reflected the amount of emitted compounds taken up by physical, chemical or biological processes in the soil matrix surrounding the roots. Although monoterpene content was not significantly different in droughted roots, emission rates from droughted roots were generally significantly lower than from well-watered roots. Finally, passive sampling of monoterpenes in the soil at different distances from mature P. sylvestris trees in field conditions showed significantly decreasing sampling rates with increasing distance from the trunk. We conclude that it is feasible to measure volatile isoprenoid emissions from roots but the method of root preparation affects magnitude of measured emissions and therefore must be decided according to the application. We also conclude that the rhizosphere of Pinus species is a strong and previously un-characterized source of volatile isoprenoid emissions and these are likely to impact significantly on rhizosphere function.     

Enzyme activities and microbial biomass in coastal soils of India

Soil salinity is a serious problem for agriculture in coastal regions, wherein salinity is temporal in nature. We studied the effect of salinity, in summer, monsoon and winter seasons, on microbial biomass carbon (MBC) and enzyme activities (EAs) of the salt-affected soils of the coastal region of the Bay of Bengal, Sundarbans, India. The average pH of soils collected from different sites, during different seasons varied from 4.8 to 7.8. The average organic C (OC) and total N (TN) content of the soils ranged between 5.2-14.1 and 0.6-1.4 g kg⁻¹, respectively. The electrical conductivity of the saturation extract (ECe) of soils, averaged over season, varied from 2.2 to 16.3 dSm⁻¹. The ECe of the soils increased five fold during the summer season (13.8 dSm⁻¹) than the monsoon season (2.7 dSm⁻¹). The major cation and anion detected were Na⁺ and Cl⁻, respectively. Seasonality exerted considerable effects on MBC and soil EAs, with the lowest values recorded during the summer season. The activities of β-glucosidase, urease, acid phosphatase and alkaline phosphatase were similar during the winter and monsoon season. The dehydrogenase activity of soils was higher in monsoon than in winter. Average MBC, dehydrogenase, β-glucosidase, urease, acid phosphatase and alkaline phosphatase activities of the saline soils ranged from 125 to 346 mg kg⁻¹ oven dry soil, 6-9.9 mg triphenyl formazan (TPF) kg⁻¹ oven dry soil h⁻¹, 18-53 mg p-nitro phenol (PNP) kg⁻¹ oven dry soil h⁻¹, 38-86 mg urea hydrolyzed kg⁻¹ oven dry soil h⁻¹, 213-584 mg PNP kg⁻¹ oven dry soil h⁻¹ and 176-362 mg PNP g⁻¹ oven dry soil h⁻¹, respectively. The same for the non-saline soils were 274-446 mg kg⁻¹ oven dry soil, 8.8-14.4 mg TPF kg⁻¹ oven dry soil h⁻¹, 41-80 mg PNP kg⁻¹ oven dry soil h⁻¹, 89-134 mg urea hydrolyzed kg⁻¹ oven dry soil h⁻¹, 219-287 mg PNP kg⁻¹ oven dry soil h⁻¹ and 407-417 mg PNP kg⁻¹ oven dry soil h⁻¹, respectively. About 48%, 82%, 48%, 63%, 40% and 48% variation in MBC, dehydrogenase activity, β-glucosidase activity, urease activity, acid phosphatase activity and alkaline phosphatase activity, respectively, could be explained by the variation in ECe of saline soils. Suppression of EAs of the coastal soils during summer due to salinity rise is of immense agronomic significance and needs suitable interventions for sustainable crop production.     

Lead tolerance in Aporrectodea rosea earthworms from a clay pigeon shooting site

Lead tolerance in individuals of the earthworm species Aporrectodea rosea collected from a clay pigeon shooting site was investigated. Lead concentrations in the shooting site soil and the un-shot control site were 6410±2250 and 296±98 mg_Pb kg⁻¹ dry weight, respectively. Of these concentrations 1050±240 and 12±9 mg_Pb kg⁻¹ dry weight were suggested to be available, using ammonium acetate (1 M), respectively. With respect to earthworm body burdens of lead the shooting site earthworms had a body burden of 6.1±1.2 mg_Pb g⁻¹ dry weight while the uncontaminated site earthworms had almost a 1000-times lower body burden of 7.1±9.0 μg_Pb g⁻¹ dry weight. Lead tolerance was assessed in uncontaminated soil that had been augmented with lead, using lead nitrate solutions, to obtain lead concentrations in soil of 0.5, 5 and 50 mg_Pb kg⁻¹ dry weight. Earthworms were exposed for 28 days during which time a semi-qualitative assessment was made of their condition. Results showed no decrease in condition in the shooting site earthworms with increasing exposure time or concentration. In contrast, earthworms collected from an uncontaminated site showed a significant (p<0.05) decrease in condition when exposed to lead concentrations above, and including, a concentration of 5 mg kg⁻¹ dry weight soil. These results suggested lead tolerance in the shooting site earthworms.     

Seasonal variation in trehalose contents of roots and nodules of leguminous trees in a tropical deciduous forest in Mexico

Seasonal variations of trehalose contents in roots and root-nodules of five legumes in a tropical deciduous forest in Jalisco, México, were determined. The tree species were: Lonchocarpus eriocarinalis and Erythrina lanata (sub-family Papilionoideae) and Piptadenia constricta, Albizia occidentalis and Lysiloma microphylum (sub-family Mimosoideae). Trehalose accumulation in nodules and roots varied seasonally and among species. For example, the Papilionoid-species retained nodules longer than those in the Mimosoideae (5 and 4 months, respectively), and accumulated the highest amounts of trehalose (average values of 178 vs. 2.88 mg g⁻¹ nodule (d. wt), respectively). Generally, maximum trehalose contents in nodules and roots were observed in November, at the beginning of the dry season.     

Variation in NH4 mineralization and microbial communities with stand age in lodgepole pine (Pinus contorta) forests, Yellowstone National Park (USA)

Soils and vegetation were analyzed in 20 lodgepole pine (Pinus contorta) forest stands, varying in age from 50 to 350 years, that had initiated following stand-replacing fire. Our goal was to determine how nitrogen availability (NH₄⁺-N) and microbial community composition varied with stand age-class and to determine whether differences could be explained by canopy, soil, or understory characteristics. Gross NH₄⁺ mineralization was measured using laboratory isotopic pool dilution, and microbial community composition was evaluated using microbial membrane lipids. The microbial community composition of stands in the 300-350 age class was distinct from stands in younger age classes. Microbial community composition among sites varied with pH, % organic matter, and phosphorus. Gross NH₄⁺ mineralization rates averaged 1.45±0.07 mg NH₄⁺ kg soil⁻¹ d⁻¹ while consumption averaged 1.37±0.20 mg NH₄⁺ kg soil⁻¹ d⁻¹, resulting in low net NH₄⁺ mineralization rates (0.08±0.18 mg NH₄⁺ kg soil⁻¹ d⁻¹), but rates were not significantly different with stand age-class at p<0.05. At p<0.10, net NH₄⁺ mineralization was significantly higher in the 300-350 age class compared to the 125-175 age class. None of the measured variables significantly explained NH₄⁺ consumption and net mineralization patterns. However, gross NH₄⁺ mineralization rates were best explained by information on microbial community structure (i.e. lipids). Variation among stands within a given age-classes was high, indicating that patterns of N cycling across landscapes reflect substantial heterogeneity among mature stands.     

Transfer and loss of naturally-occurring plasmids among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils

Plasmid transfer among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils from a long-term experiment in Braunschweig, Germany, was investigated under laboratory conditions. Three replicate samples each of four sterilized soils with total Zn contents of 54, 104, 208 and 340 mg kg⁻¹ were inoculated with an equal number (1×10⁵ cells g⁻¹ soil) of seven different, well-characterized isolates of R. leguminosarum bv. viciae. Four of the isolates were from an uncontaminated control plot (total Zn 54 mg kg⁻¹) and three were from a metal-contaminated plot (total Zn 340 mg kg⁻¹).After 1 year the population size was between 10⁶ and 10⁷ g⁻¹ soil, and remained at this level in all but the most contaminated soil. In the soil from the most contaminated plot no initial increase in rhizobial numbers was seen, and the population declined after 1 year to <30 cells g⁻¹ soil after 4 years. One isolate originally from uncontaminated soil that had five large plasmids (no. 2-8-27) was the most abundant type re-isolated from all of the soils. Isolates originally from the metal-contaminated soils were only recovered in the most contaminated soil. After 1 year, four isolates with plasmid profiles distinct from those inoculated into the soils were recovered. One isolate in the control soil appeared to have lost a plasmid. Three isolates from heavy metal contaminated soils (one isolate from the soil with total Zn 208 mg kg⁻¹ and two isolates from the soil with total Zn 340 mg kg⁻¹) had all acquired one plasmid. Plasmid transfer was confirmed using the distinct ITS-RFLP types of the isolates and DNA hybridization using probes specific to the transferred plasmid. The transconjugant of 2-8-27 which had gained a plasmid was found in one replicate after 2 years of the most contaminated soil but comprised more than 50% of the isolates. A similar type appeared in a separate replicate of the most contaminated soil after 3 years and persisted in both of these soils until the final sampling after 4 years. After 2 years isolates were recovered from four of the soil replicates with the chromosomal type of 2-8-27 which appeared to have lost one plasmid, but these were not recovered subsequently.Isolate 2-8-27 was among the isolates most sensitive to Zn in laboratory assays, whereas isolate 7-13-1 showed greater zinc tolerance. Acquisition of the plasmid conferred enhanced Zn tolerance to the recipients, but transconjugant isolates were not as metal tolerant as 7-13-1, the putative donor. Laboratory matings between 2-8-27 and 7-13-1 in the presence of Zn resulted in the conjugal transfer of the same small plasmid from 7-13-1 to isolate 2-8-27 and the transconjugant had enhanced metal tolerance. Our results show that transfer of naturally-occurring plasmids among rhizobial strains is stimulated by increased metal concentrations in soil. We further demonstrate that the transfer of naturally-occurring plasmids is important in conferring enhanced tolerance to elevated zinc concentrations in rhizobia.     

Community composition and activities of denitrifying bacteria from adjacent agricultural soil, riparian soil, and creek sediment in Oregon, USA

We examined denitrifying bacteria from wet soils and creek sediment in an agroecosystem in Oregon, USA that received inputs of nitrogen (N) fertilizer. Our objective was to determine the variation in denitrifying community composition and activities across three adjacent habitats: a fertilized agricultural field planted to perennial ryegrass, a naturally vegetated riparian area, and creek sediment. Using C₂H₂ inhibition, denitrifying enzyme and N₂O-reductase activities were determined in short-term incubations of anaerobic slurries. A key gene in the denitrification pathway, N₂O reductase (nosZ), served as a marker for denitrifiers. Mean denitrifying enzyme activity (DEA) was similar among habitats, ranging from 0.5 to 1.8 μg N g⁻¹ dry soil h⁻¹. However, the ratio of N₂O production, without C₂H₂, to DEA was substantially higher in riparian soil (0.64±0.02; mean±standard error, n=12) than in agricultural soil (0.19±0.02) or creek sediment (0.32±0.03). Mean N₂O-reductase activity ranged from 0.5 to 3.2 μg N g⁻¹ dry soil h⁻¹, with greater activity in agricultural soil than in riparian soil. Denitrifying community composition differed significantly among habitats based on nosZ terminal-restriction fragment length polymorphisms. The creek sediment community was unique. Communities in the agricultural and riparian soil were more closely related but distinct. A number of unique nosZ genotypes were detected in creek sediment. Sequences of nosZ obtained from riparian soil were closely related to nosZ from Bradyrhizobium japonicum. Although nosZ distribution and N₂O-reductase activity differed among habitats, relationships between activity and community composition appeared uncoupled across the agroecosystem.     

Competition between Rhizobium strain NGR234 and Bradyrhizobium strain CP283 for nodulation in Siratro investigated with the GUS reporter gene

The use of the GUS reporter gene was found to be very suitable for studying the competitiveness of the Rhizobium strain NGR234 and Bradyrhizobium strain CP283 for nodulation in siratro. However, the expression from the transposon mTn5SSgusA20 declined in the nodules of old plants, particularly the nodules at 35 d after inoculation and onward. Siratro inoculated with both gusA-marked NGR234 and CP283 strains showed a similar nodulation and acetylene reduction activity (ARA) to those of their parental strains. No major changes in nodulation and symbiotic properties in these marked strains were observed. When the Rhizobium strain NGR234 and Bradyrhizobium strain CP283 were inoculated separately to siratro, both of them were found equally effective for nodulation in the plant. But when the Rhizobium strain NGR234, and gusAmarked Bradyrhizobium strain CP283 were co-inoculated to the plants in a ratio of 1:1, the strain NGR234 occupied 75% of the nodules, while, the strain CP283 occupied only 25%, irrespective of the growth stages and inoculum concentrations. Similar results were also observed in the plants in which the Bradyrhizobium strain CP283 and gusA-marked Rhizobium strain NGR234 had been co-inoculated. Thus, the Rhizobium strain NGR234 was more competitive for nodulation in siratro.     

Size, symbiotic effectiveness and genetic diversity of field pea rhizobia (Rhizobium leguminosarum bv. viciae) populations in South Australian soils

Field pea (Pisum sativum L.) is widely grown in South Australia (SA), often without inoculation with commercial rhizobia. To establish if symbiotic factors are limiting the growth of field pea we examined the size, symbiotic effectiveness and diversity of populations of field pea rhizobia (Rhizobium leguminosarum bv. viciae) that have become naturalised in South Australian soils and nodulate many pea crops. Most probable number plant infection tests on 33 soils showed that R. l. bv. viciae populations ranged from undetectable (six soils) to 32×10³ rhizobia g⁻¹ of dry soil. Twenty-four of the 33 soils contained more than 100 rhizobia g⁻¹ soil. Three of the six soils in which no R. l. bv. viciae were detected had not grown a host legume (field pea, faba bean, vetch or lentil). For soils that had grown a host legume, there was no correlation between the size of R. l. bv. viciae populations and either the time since a host legume had been grown or any measured soil factor (pH, inorganic N and organic C). In glasshouse experiments, inoculation of the field pea cultivar Parafield with the commercial Rhizobium strain SU303 resulted in a highly effective symbiosis. The SU303 treatment produced as much shoot dry weight as the mineral N treatment and more than 2.9 times the shoot dry weight of the uninoculated treatment. Twenty-two of the 33 naturalised populations of rhizobia (applied to pea plants as soil suspensions) produced prompt and abundant nodulation. These symbioses were generally effective at N₂ fixation, with shoot dry weight ranging from 98% (soil 21) down to 61% (soil 30) of the SU303 treatment, the least effective population of rhizobia still producing nearly double the growth of the uninoculated treatment. Low shoot dry weights resulting from most of the remaining soil treatments were associated with delayed or erratic nodulation caused by low numbers of rhizobia. Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) fingerprinting of 70 rhizobial isolates recovered from five of the 33 soils (14 isolates from each soil) showed that naturalised populations were composed of multiple (5-9) strain types. There was little evidence of strain dominance, with a single strain type occupying more than 30% of trap host nodules in only two of the five populations. Cluster analysis of RAPD PCR banding patterns showed that strain types in naturalised populations were not closely related to the current commercial inoculant strain for field pea (SU303, ≥75% dissimilarity), six previous field pea inoculant strains (≥55% dissimilarity) or a former commercial inoculant strain for faba bean (WSM1274, ≥66% dissimilarity). Two of the most closely related strain types (≤15% dissimilarity) were found at widely separate locations in SA and may have potential as commercial inoculant strains. Given the size and diversity of the naturalised pea rhizobia populations in SA soils and their relative effectiveness, it is unlikely that inoculation with a commercial strain of rhizobia will improve N₂ fixation in field pea crops, unless the number of rhizobia in the soil is very low or absent (e.g. where a legume host has not been previously grown and for three soils from western Eyre Peninsula). The general effectiveness of the pea rhizobia populations also indicates that reduced N₂ fixation is unlikely to be the major cause of the declining field pea yields observed in recent times.     

Heavy metal accumulation by two earthworm species and its relationship to total and DTPA-extractable metals in soils

The concentrations of Zn, Cd, Pb and Cu in earthworm tissues were compared with the total and DTPA-extractable contents of these heavy metals in contaminated soils. Samples were taken from a pasture polluted by waste from a metallurgic industry over 70 y ago. Three individuals of Aporrectodea caliginosa and Lumbricus rubellus and soil samples were collected at six points along a gradient of increasing pollution. Total metal contents of earthworms, soil, and metals extracted by DTPA from the soil were measured. Total heavy metal contents of the soils ranged from 165.7 to 1231.7 mg Zn kg⁻¹, 2.7 to 5.2 mg Cd kg⁻¹, 45.8 to 465.5 mg Pb kg⁻¹ and 30.0 to 107.5 mg Cu kg⁻¹. Their correlations with metals extracted by DTPA were highly significant. Contents of the metals in earthworm tissues were higher in A. caliginosa than in L. rubellus, with values ranging from 556 to 3381 mg Zn kg⁻¹, 11.6 to 102.9 mg Cd kg⁻¹, 1.9 to 182.8 mg Pb kg⁻¹ and 17.9 to 35.9 mg Cu kg⁻¹ in A. caliginosa, and from 667.9 to 2645 mg Zn kg⁻¹, 7.7 to 26.3 mg Cd kg⁻¹, 0.5 to 37.9 mg Pb kg⁻¹ and 16.0 to 37.6 mg Cu kg⁻¹ in L. rubellus, respectively. Correlations between body loads in earthworms with either total or DTPA-extractable contents of soil metals were significant, except for Cd in L. rubellus and Cu in A. caliginosa. Considering its simple analytical procedure, DTPA-extractable fraction may be preferable to total metal content as a predictor of bio-concentrations of heavy metals in earthworms. Biota-to-Soil Accumulation Factor (BSAF) of these four metals are Cd>Zn>Cu>Pb, with range of mean values between: Cd (6.18-17.02), Zn (1.95-7.91), Cu (0.27-0.89) and Pb (0.08-0.38) in A. caliginosa, and Cd (3.64-6.34), Zn (1.5-6.35), Cu (0.29-0.87) and Pb (0.04-0.13) in L. rubellus. The BSAF of Ca, Fe and Mn are Ca>Mn>Fe, with mean values of: Ca (0.46-1.31), Mn (0.041-0.111), Fe (0.017-0.07) in A. caliginosa and Ca (0.98-2.13), Mn (0.14-0.23), Fe (0.019-0.048) in L. rubellus, respectively. Results of principal component analysis showed that the two earthworm species differ in the pattern of metal bioaccumulation which is related to their ecological roles in contaminated soils.     

Cyanobacterial inoculation of heated soils: effect on microorganisms of C and N cycles and on chemical composition in soil surface

Physiological groups of soil microorganisms, total C and N and available nutrients were investigated in four heated (350 °C, 1 h) soils (one Ortic Podsol over sandstone and three Humic Cambisol over granite, schist or limestone) inoculated (1.5 μg chlorophyll a g⁻¹ soil or 3.0 μg chlorophyll a g⁻¹ soil) with four cyanobacterial strains of the genus Oscillatoria, Nostoc or Scytonema and a mixture of them.Cyanobacterial inoculation promoted the formation of microbiotic crusts which contained a relatively high number of NH₄⁺-producers (7.4×10⁹ g⁻¹ crust), starch-mineralizing microbes (1.7×10⁸ g⁻¹ crust), cellulose-mineralizing microbes (1.4×10⁶ g⁻¹ crust) and NO₂⁻ and NO₃⁻ producers (6.9×10⁴ and 7.3×10³ g⁻¹ crust, respectively). These crusts showed a wide range of C and N contents with an average of 293 g C kg⁻¹ crust and 50 g N kg⁻¹ crust, respectively. In general, Ca was the most abundant available nutrient (804 mg kg⁻¹ crust), followed by Mg (269 mg kg⁻¹ crust), K (173 mg kg⁻¹ crust), Na (164 mg kg⁻¹ crust) and P (129 mg kg⁻¹ crust). There were close positive correlations among all the biotic and abiotic components of the crusts.Biofertilization with cyanobacteria induced great microbial proliferation as well as high increases in organic matter and nutrients in the surface of the heated soils. In general, cellulolytics were increased by four logarithmic units, amylolytics and ammonifiers by three logarithmic units and nitrifiers by more than two logarithmic units. C and N contents rose an average of 275 g C kg⁻¹ soil and 50 g N kg⁻¹ soil while the C:N ratio decreased up to 7 units. Among the available nutrients the highest increase was for Ca (315 mg kg⁻¹ soil) followed by Mg (189 mg kg⁻¹ soil), K (111 mg kg⁻¹ soil), Na (109 mg kg⁻¹ soil) and P (89 mg kg⁻¹ soil). Fluctuations of the microbial groups as well as those of organic matter and nutrients were positively correlated.The efficacy of inoculation depended on both the type of soil and the class of inoculum. The best treatment was the mixture of the four strains and, whatever the inoculum used, the soil over lime showed the most developed crust followed by the soils over schist, granite and sandstone. In the medium term there were not significant differences between the two inocula amounts tested.These results showed that inoculation of burned soils with alien N₂-fixing cyanobacteria may be a biotechnological means of promoting microbiotic crust formation, enhancing C and N cycling microorganisms and increasing organic matter and nutrient contents in heated soils.     

Chemical composition, or quality, of agroforestry residues influences N2O emissions after their addition to soil

Emissions of N₂O were measured following addition of ¹⁵N-labelled (2.6-4.7 atom% excess ¹⁵N) agroforestry residues (Sesbania sesban, mixed Sesbania/Macroptilium atropurpureum, Crotalaria grahamiana and Calliandra calothyrsus) to a Kenyan oxisol at a rate of 100 mg N kg soil⁻¹ under controlled environment conditions. Emissions were increased following addition of residues, with 22.6 mg N m⁻² (124.4 mg N m⁻² kg biomass⁻¹; 1.1 mg ¹⁵N m⁻²; 1.03% of ¹⁵N applied) emitted as N₂O over 29 d after addition of both Sesbania and Macroptilium residues in the mixed treatment. Fluxes of N₂O were positively correlated with CO₂ fluxes, and N₂O emissions and available soil N were negatively correlated with residue lignin content (r=−0.49;P<0.05), polyphenol content (r=−0.94;P<0.05), protein binding capacity (r=−0.92;P<0.05) and with (lignin+polyphenol)-to-N ratio (r=−0.55;P<0.05). Lower emission (13.6 mg N m⁻² over 29 d; 94.5 mg N m⁻² kg biomass⁻¹; 0.6 mg ¹⁵N m⁻²; 0.29% of ¹⁵N applied) after addition of Calliandra residue was attributed to the high polyphenol content (7.4%) and high polyphenol protein binding capacity (383 μg BSA mg plant⁻¹) of this residue binding to plant protein and reducing its availability for microbial attack, despite the residue having a N content of 2.9%. Our results indicate that residue chemical composition, or quality, needs to be considered when proposing mitigation strategies to reduce N₂O emissions from systems relying on incorporation of plant biomass, e.g. improved-fallow agroforestry systems, and that this consideration should extend beyond the C-to-N ratio of the residue to include polyphenol content and their protein binding capacity.     

Laccase and peroxidase isoenzymes during leaf litter decomposition of Quercus ilex in a Mediterranean ecosystem

The dynamics of leaf litter decomposition of Quercus ilex (L.) were investigated over a 2 year period by determining the activities and isoenzyme distribution of laccases and peroxidases. The analysis of isoenzymes was performed by isoelectric focusing on high stability pH gradients with high resolving power. The preparation of zymograms was carried out using the leaf litter extract without previous concentration. During litter decomposition, laccase and peroxidase activities changed as well as the type and number of enzyme isoforms. The activities of both enzymes were low (≤0.017 and ≤0.031 mmol o-tolidine oxidized h⁻¹ g⁻¹ d.w. for laccase and peroxidase, respectively) in first year and increased in October-January of the second year of litter decay. The highest activities measured after 15-18 months of litter exposure (0.37±0.03 and 0.19±0.02 mmol o-tolidine oxidized h⁻¹ g⁻¹ d.w. for laccase and peroxidase, respectively), showed that litter chemical composition affected the growth of ligninolytic microbial community. The activation energy for laccase and peroxidase reactions also changed during decomposition: the highest values (55±6 kJ mol⁻¹ for laccase and 60±6 kJ mol⁻¹ for peroxidase) occurred in autumn-winter, even if spatial changes were evidenced. Some enzyme isoforms (pI=5.3 and 5.5 for laccase and pI=5.0 and 5.1 for peroxidase, respectively), contributed more than others to the overall laccase and peroxidase activity, suggesting that some ligninolytic species bloomed in particular seasons of the year, even if other species with similar functional activities colonized the litter.     

Mycorrhizae activity and diversity in conventional and organic apple orchards from Brazil

The aim of this study was to characterize the impacts of a conventional, an organic apple orchard and a native grassland on the activity and diversity of arbuscular mycorrhizal fungi (AMF) located at the south of Brazil during winter and summer. AMF activity was measured by the mycorrhizal inoculum potential (MIP), mycorrhizal fungal hyphal length (HL), easily extractable and total Bradford-reactive soil protein (BRSP). AMF diversity was represented by richness, Shannon diversity index and number of spores. Orchards reduced the MIP of the soil and increased the HL when compared to the grassland site. The amount of easily extractable BRSP was not different among orchards and between seasons evaluated, with overall mean value of 1.23 mg g⁻¹. However, the amount of total-BRSP was smaller in the conventional orchard (4.55 mg g⁻¹) than in the organic orchard (4.91 mg g⁻¹) and in the native grassland (5.12 mg g⁻¹). T-BRSP and total organic carbon were strongly correlated in the grassland during the winter, suggesting the contribution of this protein for carbon stocks in the native soil of this region. The organic orchard presented the highest AMF richness, but sporulation and Shannon diversity index were larger in the conventional orchard. Our data suggest that the conventional orchard promoted higher impacts on the natural condition of AMF activity, being considered an unadvisable practice to soil conservation.