Potential gene exchange between Bacillus thuringiensis subsp. kurstaki and Bacillus spp. in soil in situ

The possible transfer of genes from Bacillus thuringiensis subsp. kurstaki (Btk) to indigenous Bacillus spp. was investigated in soil samples from stands of cork oak in Orotelli (Sardinia, Italy) collected 5 years after spraying of the stands with a commercial insecticidal preparation (FORAY 48B) of Btk. Two colonies with a morphology different from that of Btk were isolated and identified as Bacillus mycoides by morphological and physiological characteristics and by 16S rDNA analysis. Amplification by the polymerase chain reaction (PCR) of the DNA of the two isolated B. mycoides colonies with primers used for the identification of the Btk cry genes showed the presence of a fragment of 238 bp of the cry1Ab9 gene that had a similarity of 100% with the sequence of the cry1Ab9 gene present in GenBank, indicating that the isolates of B. mycoides acquired part of the sequence of this gene from Btk. No cells of Btk or B. mycoides carrying the 238-bp fragment of the cry1Ab9 gene were isolated from samples of unsprayed control soil. However, the isolates of B. mycoides were not able to express the partial Cry1Ab protein. Hybridization with probes for IS231 and the cry1Ab9 gene suggested that the inverted repeated sequence, IS231, was probably involved in the transfer of the 238-bp fragment from Btk to B. mycoides. These results indicate that transfer of genes between introduced Btk and indigenous Bacillus spp. can occur in soil under field conditions.     

Soil organic N dynamics and stand quality in Pinus radiata pinewoods of the temperate humid region

Soil organic-N dynamics, its controlling factors and its relationships with stand quality were studied in the 0-15 cm soil layer of 24 pinewoods with contrasting age, productivity and parent material (granite; acid schists), searching for N variables useful to predict stand growth and site quality. No significant differences were found between young and old stands for any of the N variables considered, nor two- or three-order interactions among stand age, site quality and parent material. The soil total-N content, which was correlated positively with the Al oxides content (a soil organic matter (SOM) stabilizing agent), did not vary significantly according to parent material, but it was lower (P≤0.02) in stands with high than with low site index (2.68±1.11 and 3.97±1.13 g N kg⁻¹ soil, respectively). The soil δ¹⁵N ranged from +3.5 to +6.5 δ, without significant differences among stand groups, and it was negatively correlated with water holding capacity, exchangeable bases, Al oxides and N content, suggesting that: (i) N losses by NO₃⁻ leaching are the most important controlling factor of δ¹⁵N in these temperate humid region soils; and (ii) soil N richness is related with limited N losses, which discriminate against ¹⁵N. At any incubation time, no significant differences were found in soil inorganic-N content among stand groups (7.78±4.57, 39.33±16.20 and 67.80±26.50 mg N kg⁻¹ soil at 0, 42 and 84 d, respectively). During the incubation, the relative importance of ammonification decreased and that of the nitrification increased. The net N mineralization rate (NNMR, in percentage of organic N) was significantly higher in granite than in schists soils at both 42 d (1.24±0.34 and 0.75±0.37%, respectively) and 84 d (2.18±0.56 and 1.53±0.66%, respectively). In high quality pinewoods, the NNMR at 42 and 84 d (1.16±0.45 and 2.12±0.79%, respectively) were significantly higher than in low quality stands (0.83±0.35 and 1.59±0.45%, respectively). This result, together with those on soil total-N and inorganic-N supply, suggests that soil N dynamics in low and high quality stands are different: in the former there is a bigger N pool with a slower turnover, whereas in the latter there is a smaller N pool with a faster turnover, both factors being nearly compensated, making the soil available N supply in both types of stand similar. After 42 and 84 d of incubation, the NNMR and the nitrification rates were higher in the coarse textured soils, likely due to the low physical and chemical protection of their SOM; both rates were positively correlated with available P, exchangeable K⁺ and CEC base saturation, suggesting strong relationships among the availabilities of the main plant nutrients, and they increased with SOM quality (low C-to-N ratio). The strong negative correlation of site index with soil total-N (r=−0.707; P≤0.005), and its positive correlations with NNMR after 42 and 84 d of incubation, suggested that site quality and potential productivity are closely related to soil organic-N dynamics. Half of the site index variation in the stands studied could be predicted with a cheap and easy analysis of soil N content, the prediction being slightly improved if soil δ¹⁵N is included and, more significantly, by including N mineralization measurements.     

Pre-symbiotic and symbiotic interactions between Glomus intraradices and two Paenibacillus species isolated from AM propagules. In vitro and in vivo assays with soybean (AG043RG) as plant host

Two indole-producing Paenibacillus species, known to be associated with propagules of arbuscular mycorrhizal (AM) fungi, were examined for their mycorrhization helper bacteria activity at pre-symbiotic and symbiotic stages of the AM association. The effects were tested under in vitro and in vivo conditions using an axenically propagated strain of the AM fungus Glomus intraradices and Glycine max (soybean) as the plant host. The rates of spore germination and re-growth of intraradical mycelium were not affected by inoculation with Paenibacillus strains in spite of the variation of indole production measured in the bacterial supernatants. However, a significant promotion in pre-symbiotic mycelium development occurred after inoculation of both bacteria under in vitro conditions. The Paenibacillus rhizosphaerae strain TGX5E significantly increased the extraradical mycelium network, the rates of sporulation, and root colonization in the in vitro symbiotic association. These results were also observed in the rhizosphere of soybean plants grown under greenhouse conditions, when P. rhizosphaerae was co-inoculated with G. intraradices. However, soybean dry biomass production was not associated with the increased development and infectivity values of G. intraradices. Paenibacillus favisporus strain TG1R2 caused suppression of the parameters evaluated for G. intraradices during in vitro symbiotic stages, but not under in vivo conditions. The extraradical mycelium network produced and the colonization of soybean roots by G. intraradices were promoted compared to the control treatments. In addition, dual inoculation had a promoting effect on soybean biomass production. In summary, species of Paenibacillus associated with AM fungus structures in the soil, may have a promoting effect on short term pre-symbiotic mycelium development, and little impact on AM propagule germination. These findings could explain the associations found between some bacterial strains and AM fungus propagules.     

Diversity and antagonistic potential of Pseudomonas spp. associated to the rhizosphere of maize grown in a subtropical organic farm

Pseudomonas spp. are one of the most important bacteria inhabiting the rhizosphere of diverse crop plants and have been frequently reported as biological control agents (BCAs). In this work, the diversity and antagonistic potential of Pseudomonas spp. in the rhizosphere of maize cultivars Nitroflint and Nitrodent grown at an organic farm in Brazil was studied by means of culture-dependent and -independent methods, respectively. Sampling of rhizosphere soil took place at three different stages of plant development: 20, 40 and 106 days after sowing. A PCR-DGGE strategy was used to generate specific Pseudomonas spp. fingerprints of 16S rRNA genes amplified from total community rhizosphere DNA. Shifts in the relative abundance of dominant populations (i.e. PCR-DGGE ribotypes) along plant development were detected. A few PCR-DGGE ribotypes were shown to display cultivar-dependent relative abundance. No significant differences in diversity measures of DGGE fingerprints were observed for different maize cultivars and sampling times. The characterisation and assessment of the antagonistic potential of a group of 142 fluorescent Pseudomonas isolated from the rhizosphere of both maize cultivars were carried out. Isolates were phenotypically and genotypically characterised and screened for in vitro antagonism towards three phytopathogenic fungi and the phytopathogenic bacterium Ralstonia solanacearum. Anti-fungal activity was displayed by 13 fluorescent isolates while 40 isolates were antagonistic towards R. solanacearum. High genotypic and phenotypic diversity was estimated for antagonistic fluorescent Pseudomonas spp. PCR-DGGE ribotypes displayed by antagonists matched dominant ribotypes of Pseudomonas DGGE fingerprints, suggesting that antagonists may belong to major Pseudomonas populations in the maize rhizosphere. Antagonists differing in their genotypic and phenotypic characteristics shared the same DGGE electrophoretic mobility, indicating that an enormous genotypic and functional diversity might be hidden behind one single DGGE band. Cloning and sequencing was performed for a DGGE double-band which had no corresponding PCR-DGGE ribotypes among the antagonists. Sequences derived from this band were affiliated to Pseudomonas stutzeri and P. alcaligenes 16S rRNA gene sequences. As used in this study, the combination of culture-dependent and -independent methods has proven to be a powerful tool to relate functional and structural diversity of Pseudomonas spp. in the rhizosphere.     

Molecular markers and ex situ conservation of the European elms (Ulmus spp.)

The enormous losses suffered by the European elms during recent Dutch elm disease outbreaks led to concern over the conservation of elm genetic resources, and the subsequent establishment of a series of ex situ collections. However, as ex situ collections are inevitably finite in size, some consideration needs to be given to selecting which samples to include in them. To contribute towards this process for European ex situ elm collections we have undertaken genetic studies on a Europe-wide sample of 535 individuals. A major aim has been to use genetic markers to clarify the identification of samples to ensure that the ex situ collections contain a representative spread of taxonomic diversity. This is important given the paucity of mature elms in the landscape due to Dutch elm disease. The lack of mature material (critical for identification) compounds identification problems in what was already a taxonomically difficult group. Our data (derived from random amplified polymorphic DNA and inter-simple sequence repeats) have provided a useful supplement to morphology in undertaking such sample identifications. The molecular data served to highlight mis-identified samples and led to extensive revisions of sample identities within individual countries. Our results were less useful in detecting regional intra-specific genetic structure, and do not provide sufficient information for prioritising within-species sample selections.     

Non-pathogenic Fusarium strains protect the seedlings of Lepidium sativum from Pythium ultimum

Two root-colonizing Fusarium strains, Ls-F-in-4-1 and Rs-F-in-11, isolated from roots of Brassicaceae plants, induced the resistance in Lepidium sativum seedlings against Pythium ultimum. These strains caused an increase in the content of benzyl isothiocyanate, and of its precursor glucotropaeolin, in the roots of the host plants. The increased isothiocyanate content is one of the factors contributing to the resistance of L. sativum against P. ultimum. To be transformed into the fungitoxic compound benzyl isothiocyanate, glucotropaeolin has to be hydrolyzed by myrosinase, which can be produced either by plants or microorganisms. The Fusarium strain Ls-F-in-4-1 has a myrosinase activity but the strain Rs-F-in-11 has not. These results suggest that both strains are able to trigger the metabolic pathway leading to benzyl isothiocyanate production in the plant. In the case of the myrosinase-negative strain Rs-F-in-11, hydrolyzation into isothiocyanate is only due to the myrosinase activity of the plant, and in the other case, the myrosinase produced by the strain Ls-F-in-11 also would contribute to the production of isothiocyanate. This paper reports a new mode of action of non-pathogenic Fusarium strains in controlling P. ultimum.     

Quantification of Wautersia [Ralstonia] basilensis in the mycorrhizosphere of Pinus thunbergii Parl. and its effect on mycorrhizal formation

The bacterium Wautersia [Ralstonia] basilensis has been shown to enhance the mycorrhizal symbiosis between Suillus granulatus and Pinus thunbergii (Japanese black pine). However, no information is available about this bacterium under field conditions. The objectives of this study were to detect W. basilensis in bulk and mycorhizosphere soils in a Japanese pine plantation in the Tottori Sand Dunes, determine the density of W. basilensis in soil, and determine the optimal cell density of W. basilensis for mycorrhizal formation in pine seedlings. We designed and validated 16S rRNA gene-targeted specific primers for detection and quantification of W. basilensis. SYBR Green I real-time PCR assay was used. A standard curve relating cultured W. basilensis cell density (10³-10⁸ cells ml⁻¹) to amplification of DNA showed a strong linear relationship (R = 0.9968). The specificity of the reaction was confirmed by analyzing DNA melting curves and sequencing of the amplicon. The average cell density of W. basilensis was >4.8 × 10⁷ cells g⁻¹ of soil in the mycorrhizosphere and 7.0 × 10⁶ cells g⁻¹ in the bulk soil. We evaluated the W. basilensis cell density required for mycorrhizal formation using an in vitro microcosm with various inoculum densities ranging from 10² to 10⁷ cells g⁻¹ soil (10⁴-10⁹ cells ml⁻¹). Cell densities of W. basilensis of >10⁶ cells g⁻¹ of soil were required to stimulate mycorrhizal formation. In vivo and in vitro experiments showed that W. basilensis was sufficiently abundant to enhance mycorrhizal formation in the mycorrhizosphere of Japanese black pine sampled from the Tottori Sand Dunes.     

Litter- and ecosystem-driven decomposition under elevated CO2 and enhanced N deposition in a Sphagnum peatland

Peatlands represent massive global C pools and sinks. Carbon accumulation depends on the ratio between net primary production and decomposition, both of which can change under projected increases of atmospheric CO₂ and N deposition. The decomposition of litter is influenced by 1) the quality of the litter, and 2) the microenvironmental conditions in which the litter decomposes. This study aims at experimentally testing the effects of these two drivers in the context of global change. We studied the in situ litter decomposition from three common peatland species (Eriophorum vaginatum, Polytrichum strictum and Sphagnum fallax) collected after one year of litter production under pre-treatment conditions (elevated CO₂: 560 ppm or enhanced N: 3 g m⁻² y⁻¹ NH₄NO₃) and decomposed the following year under treatment conditions (same as pre-treatment). By considering the cross-effects between pre-treatments and treatments, we distinguished between the effects on mass loss of 1) the pre-treatment-induced litter quality and 2) the treatment conditions under which the litters were decomposing. The combination between CO₂ pre-treatment and CO₂ treatment reduced Polytrichum decomposition by −24% and this can be explained by litter quality-driven decomposition changes brought by the pre-treatment. CO₂ pre-treatment reduced Eriophorum litter quality, although this was not sufficient to predict decomposition. The N addition pre-treatment reduced the decomposition of Eriophorum, due to enhanced lignin and soluble phenols concentrations in the initial litter, and reduced litter-driven losses of starch and enhanced litter-driven losses of soluble phenols. While decomposition indices based on initial litter quality provide a broad explanation of quantitative and qualitative decomposition, they can only be taken as first approximations. Indeed, the microbial ATP activity, the litter N loss and resulting litter quality, were strongly altered irrespective of the compounds' initial concentration and by means of processes that occurred independently of the initial litter-qualitative changes. The experimental design was valuable to assess litter- and ecosystem-driven decomposition pathways simultaneously or independently. The ability to separate these two drivers makes it possible to attest the presence of litter-qualitative changes even without any litter biochemical determinations, and shows the screening potential of this approach for future experiments dealing with multiple plant species.     

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.     

The abundance of nitrogen cycle genes amoA and nifH depends on land-uses and soil types in South-Eastern Australia

Nitrogen is a critical nutrient in plant-based primary production systems, therefore measurements of N cycling by microorganisms may add value to agricultural soil monitoring programs. Bacterial-mediated nitrogen cycling was investigated in soils from two broad land-uses (managed and remnant vegetation) across different Soil Orders from three geomorphic zones in Victoria, Australia, by examining the abundance of the genes amoA and nifH using quantitative polymerase chain reaction (qPCR). The aim of the study was to identify parameters influencing bacterial populations possessing the genes nifH and amoA, and examine their distribution at a regional scale across different management treatments. The gene amoA was most abundant in the neutral to slightly alkaline surface soils from Calcarosols in North-West Victoria. There was a highly significant (P < 0.001) interaction between land-use and geomorphic zones in terms of the abundance of amoA. Detection of the gene nifH was site specific with low copy number (less than 100 copies per nanogram of DNA) observed for some strongly acidic surface soil sites in North-East Victoria (Dermosols) and South-West Victoria (Sodosols/Chromosols), while nifH was more abundant in selected Calcarosols of North-West Victoria. The gene amoA was detected across more sites than nifH and was strongly influenced by land-use, with almost consistently greater abundance in managed compared to remnant sites, particularly for North-West and South-West Victoria. The abundance of nifH was not related to land-use, with similar copy numbers observed for both managed and remnant sites at some locations. For the gene nifH, there was no significant interaction between land-use and geomorphic zones, between managed and remnant sites or between the three geomorphic zones. Regression tree analysis revealed a number of likely soil chemical and microbial variables which may act as drivers of gene abundance of amoA and nifH. Variables identified as drivers for amoA included pH, Olsen P, microbial biomass carbon, nitrate and total nitrogen while for nifH the variables were microbial biomass carbon, electrical conductivity, microbial biomass nitrogen, total nitrogen and total potassium. Measures of N cycling genes could be used as an additional indicator of soil health to assess potential ecosystem functions. The spatial scale of the current study demonstrates that a landscape approach may assist soil health monitoring programs by evaluating N cycle gene abundance in the context of the different microbial and chemical conditions related to Soil Order and land-use management.     

Five years of simulated atmospheric nitrogen deposition have only subtle effects on the fate of newly synthesized carbon in Calluna vulgaris and Eriophorum vaginatum

To understand the implications of atmospheric nitrogen deposition on carbon turnover in peatlands, we conducted a ¹³C pulse labeling experiment on Calluna vulgaris and Eriophorum vaginatum already receiving long-term (5 years) amendments of 56 kg N ha⁻¹ y⁻¹ as ammonium or nitrate. We examined shoot tissue retention, net ecosystem respiration returns of the ¹³C pulse, and soil porewater DOC content under the two species. ¹³C fixation in Eriophorum leaves was enhanced with nitrogen addition and doubled with nitrate supply. This newly fixed C appeared to be relocated below-ground faster with nitrogen fertilization as respiration returns were unaffected by N inputs. By contrast, increases in ¹³C fixation were not observed in Calluna. Instead, net ecosystem respiration rates over Calluna increased with N fertilization. There was no significant label incorporation into DOC, suggesting a conservative strategy of peatland vegetation regarding allocation of C through root exudation. Greater concentrations of total DOC were identified with nitrate addition in Calluna. Given the long-term nature of the experiment and the high N inputs, the overall impacts of nitrogen amendments on the fate of recently synthesized C in Eriophorum and Calluna in this ombrotrophic peatland were surprisingly more moderate than originally hypothesized. This may be due to N being effectively retained within the bryophyte layer, thus limiting, and delaying the onset of, below-ground effects.     

Effects of nitrogen deposition on growth and survival of montane Racomitrium lanuginosum heath

Montane heaths dominated by the moss Racomitrium lanuginosum are in decline, for which increased atmospheric nitrogen (N) deposition may be partially responsible. To test this, field plots in northeast Scotland were treated with either low or high (10 or 40 kg N ha⁻¹year⁻¹) doses of nitrogen (as NO₃⁻ or NH₄⁺) for 2 years. Although Racomitrium tissue N increased after treatment, with greater response for low than high N application, activity of the enzyme nitrate reductase and Racomitrium growth were severely inhibited by increasing N addition. Racomitrium cover declined following N addition and graminoid cover increased, also with greatest effect at high doses. Of all measurements, only nitrate reductase showed a distinction between NO₃⁻ and NH₄⁺ application. The results demonstrate the detrimental effects of even low increases in nitrogen deposition on the moss heath, suggesting that loss of Racomitrium and its replacement by graminoids is strongly linked to increased levels of anthropogenic N pollution.     

The abundance and efficacy of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

For optimum production, the use of commercial rhizobial inoculant on pea (Pisum sativum L.) at seeding is necessary in the absence of compatible rhizobial strains or when rhizobial soil populations are low or symbiotically ineffective. Multiple site experiments were conducted to characterize the abundance and effectiveness of resident populations of Rhizobium leguminosarum bv. viciae (Rlv) in eastern Canadian prairie soils. A survey of 20 sites across a broad geographical range of southern Manitoba was carried out in 1998 and was followed by more intensive study of five of the sites in 1999 and 2000. Appreciable nodulation of uninoculated pea was observed at all sites which had previously grown inoculated pea. However, uninoculated pea grown at two sites, which had not previously grown pea, had negligible nodulation. Likewise, wild Lathyrus sp. and Vicia sp. plants collected from uncultivated areas adjacent to agricultural sites were poorly nodulated. In the more intensively studied sites, there was a tendency towards higher nodulation in pea plants receiving commercial inoculant containing Rlv strain PBC108 across all site-years (e.g., 4.7% in nodulation and 22% in nodule mass), but the effect was significant at only 2 of 10 site-years. Despite a relatively high range of soil pH (6-8), regression analysis indicated that decreasing soil pH resulted in lower nodulation rates. Likewise, electrical conductivity (EC) was correlated to nodulation levels, however the effect of EC was likely more indicative of the influence of soil texture and organic matter than salinity. As with nodulation, commercial inoculation tended to increase above-ground dry matter (DM) and fixed-N (estimated by the difference method) at the early pod-filling stage, but again the effects were significant at only 2 of 10 site-years. Specifically, above-ground DM and fixed-N levels were up to 29 and 51% greater, respectively, in inoculated compared to non-inoculated treatments at these sites. Addition of N-fertilizer at a rate of 100 kg N ha⁻¹ decreased nodulation at almost all site-years (by as much as 70% at one site), but rarely resulted in increases in above-ground DM compared to inoculated plots. The study indicates for the first time that populations of infective, and generally effective strains of Rlv occur broadly in agricultural soils across the eastern Canadian prairie, but that there is a tendency for increased symbiotic efficiency with the use of commercial inoculant.     

Lignin and cellulose degradation and nitrogen dynamics during decomposition of three leaf litter species in a Mediterranean ecosystem

Cellulose and lignin degradation dynamics was monitored during the leaf litter decomposition of three typical species of the Mediterranean area, Cistus incanus L., Myrtus communis L. and Quercus ilex L., using the litter bag method. Total N and its distribution among lignin, cellulose and acid-detergent-soluble fractions were measured and related to the overall decay process. The litter organic substance of Cistus and Myrtus decomposed more rapidly than that of Quercus. The decay constants were 0.47 year⁻¹, 0.75 year⁻¹ and 0.30 year⁻¹ for Cistus, Myrtus and Quercus, respectively. Lignin and cellulose contents were different as were their relative amounts (34 and 18%, 15 and 37%, 37 and 39% of the overall litter organic matter before exposure, for Cistus, Myrtus and Quercus, respectively). Lignin began to decrease after 6 and 8 months of exposure in Cistus and Myrtus, respectively, while it did not change significantly during the entire study period in Quercus. The holocellulose, in contrast, began to decompose in Cistus after 1 year, while in Quercus and Myrtus immediately. Nitrogen was strongly immobilized in all the litters in the early period of decay. Its release began after the first year in Cistus and Myrtus and after 2 years of decomposition in Quercus. These litters still contained about 60, 20 and 90% of the initial nitrogen at the end of the experiment (3 years). Prior to litter exposure nitrogen associated with the lignin fraction was 65, 54 and 37% in Cistus, Myrtus and Quercus, while that associated with the cellulose fraction was 30, 24 and 28%. Although most of the nitrogen was not lost from litters, its distribution among the litter components changed significantly during decomposition. In Cistus and Myrtus the nitrogen associated with lignin began to decrease just 4 months after exposure. In Quercus this process was slowed and after 3 years of decomposition 8% of the nitrogen remained associated with lignin or lignin-like substances. The nitrogen associated with cellulose or cellulose-like substances, in contrast, began to decrease from the beginning of cellulose decomposition in all three species. At the end of the study period most of the nitrogen was not associated to the lignocellulose fraction but to the acid-detergent-soluble substance (87, 88 and 84% of the remaining litter nitrogen).     

The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

Soil populations of Rhizobium leguminosarum bv. viciae (Rlv) that are infective and symbiotically effective on pea (Pisum sativum L.) have recently been shown to be quite widespread in agricultural soils of the eastern Canadian prairie. Here we report on studies carried out to assess the genetic diversity amongst these endemic Rlv strains and to attempt to determine if the endemic strains arose from previously used commercial rhizobial inoculants. Isolates of Rlv were collected from nodules of uninoculated pea plants from 20 sites across southern Manitoba and analyzed by plasmid profiling and PCR-RFLP of the 16S-23S rDNA internally transcribed spacer (ITS) region. Of 214 field isolates analyzed, 67 different plasmid profiles were identified, indicating a relatively high degree of variability among the isolates. Plasmid profiling of isolates from proximal nodules (near the base of the stem) and distal nodules (on lateral roots further from the root crown) from individual plants from one site suggested that the endemic strains were quite competitive relative to a commercial inoculant, occupying 78% of the proximal nodules and 96% of the distal nodules. PCR-RFLP of the 16S-23S rDNA ITS also suggested a relatively high degree of genetic variability among the field isolates. Analysis of the PCR-RFLP patterns of 15 selected isolates by UPGMA indicated two clusters of three field isolates each, with simple matching coefficients (SMCs) ≥0.95. However, to group all field isolates together, the SMC has to be reduced to 0.70. Regarding the origin of the endemic Rlv strains, there were few occurrences of the plasmid profiles of field isolates being identical to the profiles of inoculant Rlv strains commonly used in the region. Likewise, the plasmid profiles of isolates from nodules of wild Lathyrus plants located near some of the sites were all different from those of the field isolates. However, comparison of PCR-RFLP patterns suggested an influence of some inoculant strains on the chromosomal composition of some of the field isolates with SMCs of ≥0.92. Overall, plasmid profiles and PCR-RFLP patterns of the isolates from endemic Rlv populations from across southern Manitoba indicate a relatively high degree of genetic diversity among both plasmid and chromosomal components of endemic strains, but also suggest some influence of chromosomal information from previously used inoculant strains on the endemic soil strains.     

Effects of Medicago sativa, Taraxacum officinale and Bromus sterilis on the density and diversity of Collembola in grassy arable fallows of different ages

The density and diversity of Collembola of nine grassy arable fallows of different ages were investigated in a factorial design with the factors ‘plant species’ (legume: Medicago sativa, herb: Taraxacum officinale, grass: Bromus sterilis) and ‘age class’ (2-3, 6-8 and 12-15 years) including the random effect ‘site’ (1-9). In May 2008, four plots were selected randomly at each fallow. Within each plot five M. sativa, T. officinale and B. sterilis plants were extracted with their associated soil using steel cylinders. The material from each plant species was used for extraction of Collembola and for determination of environmental parameters. Thus, the new aspect of the present study compared to other field studies investigating the relationships between plant and Collembolan communities is the focus on the “micro-scale”, investigating the Collembolan communities of the soil associated with single plants.We found that species richness and density of total and euedaphic Collembola were significantly higher in B. sterilis than in T. officinale samples with the M. sativa samples being intermediate. Fine-root feeding euedaphic Collembola particularly benefited from the higher amount of fine roots in B. sterilis samples. We also discovered that the age of the fallows had no significant influence on the number of Collembolan species and the density of the Collembolan groups. Notably, however, species of the epedaphic genera Lepidocyrtus and Sminthurinus were associated with the 12-15 year-old fallows and presumably benefited from the high number of plant species in the old fallows. Finally, canonical correspondence analysis (CCA) indicated that the soil organic matter content and the microbial biomass, both potential food sources for many Collembolan species, were important structuring forces for the Collembolan communities.     

Conserving marginal populations of the food plant (Impatiens noli-tangere) of an endangered moth (Eustroma reticulatum) in a changing climate

Impatiens noli-tangere is scarce in the UK and probably only native to the Lake District and Wales. It is the sole food plant for the endangered moth Eustroma reticulatum. Significant annual fluctuations in the size of I. noli-tangere populations endanger the continued presence of E. reticulatum in the UK. In this study, variation in population size was monitored across native populations of I. noli-tangere in the English Lake District and Wales. In 1998, there was a crash in the population size of all metapopulations in the Lake District but not of those found in Wales. A molecular survey of the genetic affinities of samples in 1999 from both regions and a reference population from Switzerland was performed using AFLP and ISSR analyses. The consensus UPGMA dendrogram and a PCO scatter plot revealed clear differentiation between the populations of I. noli-tangere in Wales and those in the Lake District. Most of the genetic variation in the UK (H_T=0.064) was partitioned between (G_ST=0.455) rather than within (H_S=0.034) regions, inferring little gene flow occurs between regions. There was similar bias towards differentiation between metapopulations in Wales, again consistent with low levels of interpopulation gene flow. This contrasts with far lower levels of differentiation in the Lake District which suggests modest rates of gene flow may occur between populations. It is concluded that in the event of local extinction of sites or populations, reintroductions should be restricted to samples collected from the same region. We then surveyed climatic variables to identify those most likely to cause local extinctions. Climatic correlates of population size were sought from two Lake District metapopulations situated close to a meteorological station. A combination of three climatic variables common to both sites explained 81-84% of the variation in plant number between 1990 and 2001. Projected trends for these climatic variables were used in a Monte Carlo simulation which suggested an increased risk of I. noli-tangere population crashes by 2050 at Coniston Water, but not at Derwentwater. Implications of these findings for practical conservation strategies are explored.     

Screening of bacterial isolates from various European soils for in vitro antagonistic activity towards Rhizoctonia solani and Fusarium oxysporum: Site-dependent composition and diversity revealed

A cultivation-based approach was used to determine the in vitro antagonistic potential of soil bacteria towards Rhizoctonia solani AG3 and Fusarium oxysporum f. sp. lini (Foln3). Four composite soil samples were collected from four agricultural sites with previous documentation of disease suppression, located in France (FR), the Netherlands (NL), Sweden (SE) and the United Kingdom (UK). Similarly, two sites from Germany (Berlin, G-BR; and Braunschweig, G-BS) without documentation of disease suppression were sampled. Total bacterial counts were determined by plating serial dilutions from the composite soil samples onto R2A, AGS and King's B media. A total of 1,788 isolates (approximately 100 isolates per medium and site) was screened for antifungal activity, and in vitro antagonists (327 isolates) were found amongst the dominant culturable bacteria isolated from all six soils. The overall proportion of antagonists and the number of isolates with inhibitory activity against F. oxysporum were highest in three of the suppressive soils (FR, NL and SE). Characterization of antagonistic bacteria revealed a high phenotypic and genotypic diversity. Siderophore and protease activity were the most prominent phenotypic traits amongst the antagonists. The composition and diversity of antagonists in each soil was site-specific. Nevertheless, none of the antimicrobial traits of bacteria potentially contributing to soil suppressiveness analyzed in this study could be regarded as specific to a given site.     

Rapid identification and discrimination among Egyptian genotypes of Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti nodulating faba bean (Vicia faba L.) by analysis of nodC, ARDRA, and rDNA sequence analysis

Twenty-eight Rhizobium strains were isolated from the root nodules of faba bean (Vicia faba L.) collected from 11 governorates in Egypt. A majority of these strains (57%) were identified as Rhizobium leguminosarum bv. viciae (Rlv) based on analysis of a nodC gene fragment amplified using specific primers for these faba bean symbionts. The strains were characterized using a polyphasic approach, including nodulation pattern, tolerance to environmental stresses, and genetic diversity based on amplified ribosomal DNA-restriction analysis (ARDRA) of both 16S and 23S rDNA. Analysis of tolerance to environmental stresses revealed that some of these strains can survive in the presence of 1% NaCl and a majority of them survived well at 37 °C. ARDRA indicated that the strains could be divided into six 16S rDNA genotypes and five 23S rDNA genotypes. Sequence analysis of 16S rDNA indicated that 57% were Rlv, two strains were Rhizobium etli, one strain was taxonomically related to Rhizobium rubi, and a group of strains were most closely related to Sinorhizobium meliloti. Results of these studies indicate that genetically diverse rhizobial strains are capable of forming N₂-fixing symbiotic associations with faba bean and PCR done using nodC primers allows for the rapid identification of V. faba symbionts.     

Changes in mineral nitrogen, phosphorus availability and salt-extractable aluminium following the application of green manure residues in two weathered soils of South Vietnam

Phosphorus deficiency and aluminium toxicity in weathered soils can be amended by applying organic residues. Nitrogen mineralization, changes in P-availability and changes in salt-extractable Al following the incorporation of residues of various green manures (Flemingia congesta, Mucuna pruriens, Pueraria phaseoloides, Tithonia diversifolia) were quantified in a field core incubation experiment. Dried residues were added at an application rate of 45 kg P ha⁻¹ to two soils representative for the main soil groups of the South Vietnamese uplands, set up in incubation cores in an experimental field near Ho Chi Minh City, Vietnam.Decomposition of the residues proceeded at high rates. Mineralized nitrogen from the residues was recovered mainly as ammonium during the first 2 weeks of incubation. Nitrogen release from Tithonia residues with the highest lignin content and lignin:N ratio occurred more gradually compared to the three legumes. Resin-extractable P was significantly increased by residue treatments. Largest and sustained increases in resin-extractable P (0.67 and 2.06 mg P kg⁻¹ in the two soils) were observed in samples amended with Tithonia, which was related to the large P-content (0.37%) and small C:P ratio (110) of the residues. The P-concentration in the residues, rather than the total amount of P applied through the residues, affected the increase in P-availability. The increase in resin-extractable P was correlated to the P-content (R=0.64) and C:P ratio (R=−0.65) of the residues. Salt-extractable Al-concentrations were considerably reduced by the organic amendments, up to 70 and 50% in the two soils. At the rate of 45 kg P ha⁻¹, no significant differences between the residue treatments to reduce soil acidity were observed.As such, the application of high quality residues that are rich in P, in particular T. diversifolia, may enhance crop production by creating favourable soil conditions during the initial stages of plant development of the main crop.