Diverse Mesorhizobium bacteria nodulate native Astragalus and Oxytropis in arctic and subarctic areas in Eurasia.

Rhizobia nodulating native Astragalus and Oxytropis spp. in Northern Europe are not well-studied. In this study, we isolated bacteria from nodules of four Astragalus spp. and two Oxytropis spp. from the arctic and subarctic regions of Sweden and Russia. The phylogenetic analyses were performed by using sequences of three housekeeping genes (16S rRNA, rpoB and recA) and two accessory genes (nodC and nifH). The results of our multilocus sequence analysis (MLSA) of the three housekeeping genes tree showed that all the 13 isolates belonged to the genus Mesorhizobium and were positioned in six clades. Our concatenated housekeeping gene tree also suggested that the isolates nodulating Astragalus inopinatus, Astragalus frigidus, Astragalus alpinus ssp. alpinus and Oxytropis revoluta might be designated as four new Mesorhizobium species. The 13 isolates were grouped in three clades in the nodC and nifH trees. 15N analysis suggested that the legumes in association with these isolates were actively fixing nitrogen.

Genetic diversity of rhizobia nodulating native Vicia spp. in Sweden.

Despite the recognition that Rhizobium leguminosarum sv. viciae is the most common symbiont of Vicia species worldwide, there is no available information on rhizobia nodulating native Vicia species in Sweden. We have therefore studied the genetic diversity and phylogeny of root nodule bacteria isolated from V. cracca, V. hirsuta, V. sepium, V. tetrasperma and V. sylvatica growing in different locations in Sweden as well as an isolate each from V. cracca in Tromsø, Norway, and V. multicaulis in Siberia, Russia. Out of 25 isolates sampled from the six Vicia species in 12 different locations, there were 14 different genotypes based on the atpD, recA and nodA gene phylogenies. All isolates were classified into Rhizobium leguminosarum sv. viciae group based on the concatenated atpD and recA phylogeny and the nodA phylogeny.

Ecological consequences of the expansion of N2-fixing plants in cold biomes.

Research in warm-climate biomes has shown that invasion by symbiotic dinitrogen (N2)-fixing plants can transform ecosystems in ways analogous to the transformations observed as a consequence of anthropogenic, atmospheric nitrogen (N) deposition: declines in biodiversity, soil acidification, and alterations to carbon and nutrient cycling, including increased N losses through nitrate leaching and emissions of the powerful greenhouse gas nitrous oxide (N2O). Here, we used literature review and case study approaches to assess the evidence for similar transformations in cold-climate ecosystems of the boreal, subarctic and upper montane-temperate life zones. Our assessment focuses on the plant genera Lupinus and Alnus, which have become invasive largely as a consequence of deliberate introductions and/or reduced land management. These cold biomes are commonly located in remote areas with low anthropogenic N inputs, and the environmental impacts of N2-fixer invasion appear to be as severe as those from anthropogenic N deposition in highly N polluted areas. Hence, inputs of N from N2 fixation can affect ecosystems as dramatically or even more strongly than N inputs from atmospheric deposition, and biomes in cold climates represent no exception with regard to the risk of being invaded by N2-fixing species. In particular, the cold biomes studied here show both a strong potential to be transformed by N2-fixing plants and a rapid subsequent saturation in the ecosystem's capacity to retain N. Therefore, analogous to increases in N deposition, N2-fixing plant invasions must be deemed significant threats to biodiversity and to environmental quality.

Nodulation of Thermopsis lupinoides by a Mesorhizobium huakuii strain with a unique nodA gene in Kamtchatka, Russia.

Very little is known about rhizobia that form nodules on Thermopsis spp. We report the isolation of a Mesorhizobium huakuii strain with a unique nodA gene that form nodules on Thermopsis lupinoides in Kamtchatka, Russia. The isolate did not form nodules on Thermopsis chinensis or Thermopsis caroliniana, which suggests it may be host specific.

Genetic diversity of root nodule bacteria nodulating Lotus corniculatus and Anthyllis vulneraria in Sweden.

Very little is known about the genetic diversity and phylogeny of rhizobia nodulating Lotus species in northern temperate regions. We have therefore studied the genetic diversity among a total of 61 root nodule bacteria isolated from Lotus corniculatus and Anthyllis vulneraria from different geographic sites and habitats in Sweden by restriction fragment length polymorphism (RFLP) of the internal transcribed spacer between their 16S rRNA and 23S rRNA (IGS) region. A high diversity consisting of 26 IGS types from 54 L. corniculatus isolates and five IGS types from seven A. vulneraria isolates was found. The 16S rRNA sequences and phylogeny of representatives of the different IGS types showed four interesting exceptions from the majority of the isolates belonging to the genus Mesorhizobium: Two isolates were both found to be closely related to Rhodococcus spp., and two other isolates showed close relationship with Geobacillus spp. and Paenibacillus spp., respectively. The nodA sequences and phylogeny showed that all the isolates, including those not belonging to the traditional rhizobia genera, harbored nodA sequences which were typical of Mesorhizobium loti. Generally, the 16S rRNA and nodA phylogenetic trees were not congruent in that isolates with similar 16S rRNA sequences were associated with isolates harboring different nodA sequences. All the isolates were confirmed to nodulate L. corniculatus in an inoculation test. This is the first report of members of these non-rhizobia genera being able to nodulate legumes, and we suggest that they may have acquired their nodulating properties through lateral gene transfer.

Transporters in Arabidopsis roots mediating uptake of amino acids at naturally occurring concentrations.

Recent studies of Arabidopsis have identified several transporters as being important for amino acid uptake. We used Arabidopsis plants with altered expression of lysine histidine transporter 1 (LHT1), amino acid permease 1 (AAP1) and amino acid permease 5 (AAP5) with the aim of disentangling the roles of each transporter in the uptake of different amino acids at naturally occurring concentrations (2-50 µM). LHT1 mutants displayed reduced uptake rates of L-Gln, L-Ala, L-Glu and L-Asp but not of L-Arg or L-Lys, while AAP5 mutants were affected in the uptake of L-Arg and L-Lys only. Double mutants (lht1aap5) exhibited reduced uptake of all tested amino acids. In the concentration range tested, AAP1 mutants did not display altered uptake rates for any of the studied amino acids. Expression analysis of amino acid transporter genes with important root functions revealed no major differences in the individual mutants other than for genes targeted for mutation. We conclude that LHT1 and AAP5, but not AAP1, are crucial for amino acid uptake at concentrations typically found in soils. LHT1 and AAP5 displayed complementary affinity spectra, and no redundancy with respect to gene expression was found between the two transporters, suggesting these two transporters have separate roles in amino acid uptake.

Long-term effects of plant diversity and composition on soil nematode communities in model grasslands.

An important component of plant-soil feedbacks is how plant species identity anddiversity influence soil organism communities. We examine the effects of grassland plant species growing alone and together up to a richness of 12 species on nematode diversity and feeding group composition, eight years after the establishment of experimental grassland plots at the BIODEPTH site in northern Sweden. This is a substantially longer time than most other experimental studies of plant effects on soil fauna. We address the hypotheses that (la) higher species or functional diversity of plants increases nematode diversity, as well as influences nematode community composition. Alternatively, (1b) individual plant species traits are most important for nematode diversity and community composition. (2) Plant effects on soil organisms will decrease with increasing number of trophic links between plants and soil fauna. Plant species identity was often more important than plant diversity for nematode community composition, supporting hypothesis 1b. There was a weak positive relation between plant and nematode richness;which could be attributed to the presence of the legume Trifolium pratense, but also to some other plant species, suggesting a selection or sampling effect. Several plant species in different functional groups affected nematode community composition. For example, we found that legumes increased bacterial-feeding nematodes, most notably r-selected Rhabditida, while fungal-feeding nematodes were enhanced by forbs. Other bacterial feeders and obligate root feeders were positively related to grasses. Plant effects were usually stronger on plant-, bacterial- and fungal-feeding nematodes than on omnivores/predators, which supports hypothesis 2. Our study suggests that plant identity has stronger effects than plant diversity on nematode community composition, but when comparing our results with similar previous studies the effects of particular plant species appear to vary. We also found that more productive plant species affected bacterial-feeding nematodes more than fungal feeders. Moreover, we observed stronger effects the fewer the number of trophic links there were between a nematode feeding group and plants. Although we found clear effects of plants on soil nematodes, these were probably not large enough to result in strong and persistent plant-soil-organism-plant feedback loops.

Effects of phosphorus and nitrogen on nodulation are seen already at the stage of early cortical cell divisions in Alnus incana.

BACKGROUND AND AIMS: The present work aimed to study early stages of nodulation in a chronological sequence and to study phosphorus and nitrogen effects on early stages of nodulation in Alnus incana infected by Frankia. A method was developed to quantify early nodulation stages in intact root systems in the root hair-infected actinorhizal plant A. incana. Plant tissue responses were followed every 2 d until 14 d after inoculation. Cortical cell divisions were already seen 2 d after inoculation with Frankia. Cortical cell division areas, prenodules, nodule primordia and emerging nodules were quantified as host responses to infection. METHODS: Seedlings were grown in pouches and received different levels of phosphorus and nitrogen. Four levels of phosphorus (from 0.03 to 1 mM P) and two levels of nitrogen (0.71 and 6.45 mM N) were used to study P and N effects on these early stages of nodule development. KEY RESULTS: P at a medium concentration (0.1 mM) stimulated cell divisions in the cortex and a number of prenodules, nodule primordia and emerging nodules as compared with higher or lower P levels. A high N level inhibited early cell divisions in the cortex, and this was particularly evident when the length of cell division areas and presence of the nodulation stages were related to root length. CONCLUSIONS: Extended cortical cell division areas were found that have not been previously shown in A. incana. The results show that effects of P and N are already expressed at the stage when the first cortical cell divisions are induced by Frankia.

Local and systemic effects of phosphorus and nitrogen on nodulation and nodule function in Alnus incana.

Phosphorus (P) and nitrogen (N) effects on nodulation, nitrogenase activity and plant growth were studied in the root-hair-infected actinorhizal plant Alnus incana (L.) Moench. A split-root experiment, as well as a short-term experiment with entire root systems and a broader range of P concentrations, showed that P effects were specific on nodulation and not a general stimulation via a plant growth effect. These results indicate that nodule initiation and nodule growth have a high P demand. The split-root assay, comprising seven combinations of two N and two P levels, showed that P could counteract systemic N inhibition of nodulation, but did not counteract N inhibition of nitrogenase activity.

Regulation of nodulation in the absence of N2 is different in actinorhizal plants with different infection pathways.

Root nodulation in actinorhizal plants, like Discaria trinervis and Alnus incana, is subject to feedback regulatory mechanisms that control infection by Frankia and nodule development. Nodule pattern in the root system is controlled by an autoregulatory process that is induced soon after inoculation with Frankia. The final number of nodules, as well as nodule biomass in relation to plant biomass, are both modulated by a second mechanism which seems to be related to the N status of the plant. Mature nodules are, in part, involved in the latter process, since nodule excision from the root system releases the inhibition of infection and nodule development. To study the effect of N(2) fixation in this process, nodulated D. trinervis and A. incana plants were incubated under a N(2)-free atmosphere. Discaria trinervis is an intercellularly infected species while A. incana is infected intracellularly, via root hairs. Both symbioses responded with an increment in nodule biomass, but with different strategies. Discaria trinervis increased the biomass of existing nodules without significant development of new nodules, while in A. incana nodule biomass increased due to the development of nodules from new infections, but also from the release of arrested infections. It appears that in D. trinervis nodules there is an additional source for inhibition of new infections and nodule development that is independent of N(2) fixation and nitrogen assimilation. It is proposed here that the intercellular Frankia filaments commonly present in the D. trinervis nodule apex, is the origin for the autoregulatory signals that sustain the blockage of initiated nodule primordia and prevent new roots from infections. When turning to A. incana plants, it seems likely that this signal is related to the early autoregulation of nodulation in A. incana seedlings and is no longer present in mature nodules. Thus, actinorhizal symbioses belonging to relatively distant phylogenetic groups and displaying different infection pathways, show different feedback regulatory processes that control root nodulation by Frankia.

Arabinogalactan proteins are expressed at the symbiotic interface in root nodules of Alnus spp.

• We have characterized the origin and distribution of arabinogalactan proteins (AGPs) at the symbiotic interface of dinitrogen (N 2 )-fixing root nodules of Alnus spp. The interface between the host plant cell and the microsymbiont is an important zone for signaling and growth regulation during nodulation. Arabinogalactan proteins are glycoproteins that have adhesive properties, and, potentially, participate in cell wall assembly, direction of growth, and signaling cascades. These glycoproteins are expressed in several symbiotic systems in an infection-specific pattern, but their occurrence has not been examined in actinorhizal nodules. • To characterize AGP epitopes in Alnus root nodules, we have used immunogold localization with anti-AGP antibodies, correlated with other techniques. • Arabinogalactan proteins are abundant in the nodule-infected tissue. One AGP epitope (JIM4) is localized in pectin-rich cell walls, while another (JIM13) is found at the membrane-wall border along the symbiotic interface at the early infection stage, and in the host cytoplasm/vacuoles in mature, infected cells. • It is likely that AGPs play a significant role in Alnus root nodules, especially in early nodulation stages.

Numerical responses of different trophic groups of invertebrates to manipulations of plant diversity in grasslands.

We studied the effects of plant diversity on abundance of invertebrate herbivores, parasitoids and predators in two grassland communities (one in Switzerland and one in Sweden) in which plant species richness and functional diversity have been experimentally manipulated. Among herbivores, the abundance of only the most sessile and specialised groups (leafhoppers and wingless aphids) was affected by plant diversity. At both sites, numbers of leafhoppers in sweep net samples showed a linear, negative relationship with plant species number whereas numbers of wingless aphids in suction samples increased with the number of plant functional groups (grasses, legumes, and non-legume forbs) present in the plot. Activity of carabid beetles and spiders (as revealed by pitfall catches) and the total number of predators in pitfalls at the Swiss site decreased linearly with increases in the number of plant species and plant functional groups. Abundance of more specialised enemies, hymenopteran parasitoids, was not affected by the manipulations of plant diversity. Path analysis and analysis of covariance indicated that plant diversity effects on invertebrate abundance were mostly indirect and mediated by changes in plant biomass and cover. At both sites, plant species composition (i.e. the identity of plant species in a mixture) affected numbers of most of the examined groups of invertebrates and was, therefore, a more important determinant of invertebrate abundance in grasslands than plant species richness per se or the number of plant functional groups. The presence of legumes in a mixture was especially important and led to higher numbers of most invertebrate groups. The similarity of invertebrate responses to plant diversity at the two study sites indicates that general patterns in abundance of different trophic groups can be detected across plant diversity gradients under different environmental conditions.

Actinorhizal symbioses and their N2 fixation.

More than 200 angiosperms, distributed in 25 genera, develop root nodule symbioses (actinorhizas) with soil bacteria of the actinomycetous genus Frankia. Although most soils studied contain infective Frankia, cultured strains are available only after isolation from root nodules. Frankia infects roots via root hairs in some hosts or via intercellular penetration in others. The nodule originates in the pericycle. The number of nodules in Alnus is determined by the plant in an autoregulated process that, in turn, is modulated by nutrients such as nitrogen and phosphate. Except in the genera Allocausarina and Casuarina, Frankia in nodules develops so-called vesicles where nitrogenase is localized. Sporulation of Frankia occurs in some symbioses. As a group, actinorhizal plants show a large range of anatomical and biochemical adaptations in order to balance the oxygen tension near nitrogenase. In symbioses with well aerated nodule tissue like Alnus, the vesicles have a multilayered envelope composed mainly of lipids, bacterio-hopanetetrol and their derivatives. This envelope is assumed to retard the diffusion of oxygen into the nitrogenase-containing vesicle. In symbioses like Casuarina, the infected plant cells themselves, rather than Frankia, appear to retard oxygen diffusion, and high concentrations of haemoglobin indicate an infected region with a low oxygen tension. At least in Alnus spp., ammonia resulting from N2 fixation is assimilated by glutamine synthetase in the plant. The carbon compound(s) used by Frankia in nodules is not yet known. Nitrogenase activity decreases in response to a number of environmental factors but recovers upon return to normal conditions. This dynamism in nitrogenase activity is often explained by loss and recovery of active nitrogenase and has been traced to loss and recovery of the nitrogenase proteins themselves. Recovery is partly due to growth of Frankia and to development of new vesicles in the Alnus nodules. In the field, varying conditions continuously affect the plants and the measured rate of N2 fixation is a result not only of the conditions prevailing at the moment but also of the conditions experienced over preceding days. N2 fixed by actinorhizal plants is substantial and actinorhizal plants have great potential in soil reclamation and in various types of forestry. Several species are also useful in horticulture. CONTENTS Summary 375 I. Introduction 376 II. The partners of actinorhizal symbioses 377 III. Root nodules 380 IV. Nitrogen fixation and related processes 385 V. Environmental effects on nitrogen fixation 389 VI. Ecological role 397 VII. Concluding remarks 398 Acknowledgements 398 References 398.

Fungal biomass in roots and extramatrical mycelium in relation to macronutrients and plant biomass of ectomycorrhizal Pinus sylvestris and Alnus incana.

We studied the effects of macronutrients on the production and distribution of fungal biomass and plant biomass in ectomycorrhizal (Paxillus involutus (Fr.) Fr.) or non-mycorrhiza] Pinus sylvestris L, and Alnus incana (L.) Moench. Fungal biomass was measured as ergosterol content in roots and extramatrical mycelium, Alnus infants was nodulated with Frankia. All six macronutrients were varied according to a two-level fractional factorial design, The plants were grown in pots during two growing periods in a growth chamber. Levels of N, P and sometimes K and interactions between them, had highly significant effects, whereas Ca. Mg and S had no significant effects. The production of extramatrical mycelial biomass peaked when P was low and other nutrients were high. This investment in extramatrical mycelium resulted in a 660%, higher biomass in mycorrhizal compared with non-mycorrhizal P. sylvestris at this nutrient regime. The proportion of fungal biomass in roots was stable in P. sylvestris hut more variable in A. incana. Alnus incana grew less when mycorrhizal then when non-mycorrhizal. The growth responses to mycorrhiza and to the different nutrient treatments were evident at the end of the first growing period. Non-mycorrhizal P. sylvestris did not respond to P limitation by a production of proportionally more roots. This might be a reflection of an obligate dependency on mycorrhiza for effective P uptake. By contrast, the root/shoot ratio in both mycorrhizal and non-mycorrhiza] P. sylvestris decreased strongly in response to increased N. The opposite root/shoot response was found in Alnus incana, and the ratio decreased strongly in response to increased P and increased in response to increased N.

Nitrogen fixation by Alnus incana and nitrogen transfer from A. incana to Pinus sylvestris influenced by macronutrients and ectomycorrhiza.

The aims of this study were to evaluate the effect of macronutrients on nitrogen fixation in mycorrhizal and non-mycorrhizal grey alder (Alnus incana (L.) Moench), and to evaluate the effect of ectomycorrhizal mycelium on the transfer of symbolically fixed nitrogen from grey alder to Scots pine (Pinus sylvestris L). One alder and one pine were grown together in pots with root systems separated by a 20µm mesh nylon filter which allowed hyphae but not roots to penetrate. Half the plants of both species were inoculated with Paxillus involutus (Ft.) Ft. and all alders were inoculated with Frankia. Nutrient solutions were added with macronutrient (N, K, P, Ca, Mg and S) concentrations varied according to a two-level fractional factorial design. The plants were harvested after two growing periods in a growth chamber. Nitrogen fixation by alder and transfer of symbiotically fixed N from alder to pine was measured by 15 N-dilution. Fixed N (mg) correlated with nodule biomass in both mycorrhizal and non-mycorrhizal alders. On average, specific nodule activity over the two periods was 510 mg N fixed R' nodule d. wt. This was not affected by mycorrhizal infection or by the different nutrient treatments. By contrast there was a strong nutrient effect on the proportion of N derived from fixation. These results indicate that the regulation of nitrogen fixation was via nodule growth rather than via nodule specific activity. Nitrogen had a strong negative effect and P a positive effect on the percentage of N derived from fixation (%Ndfa). However, the effect of N depended on the level of P. This N × P interaction resulted in a %Ndfa when N was high, of 5-10%, at low P and 45-48%, at high P. The highest value of 90% Ndfa was found at the combination of low N and high P. Potassium had a small but statistically significant effect on the %Ndfa but Ca, Mg and S had no significant effects. No mycorrhizal effect was found on the %Ndfa in alder. By contrast, the %Ndfa and biomass were lower in mycorrhizal than in non-mycorrhizal alders. The proportion of fixed N in pine, transferred from alder, was greatest (9%) when the pine was nitrogen starved and mycorrhizal and the alder was fixing maximally (low N and high P). However, the amount of fixed N transferred to pine was not statistically different from zero.

Influence of host (Alnus and Myrica) genotype on infectivity, N2 fixation, spore formation and hydrogenase activity in Frankia.

Nine species of Alnus, two of them represented by several provenances, and Myrica gale were inoculated with a 'local source' of Frankia. The inoculum is an effective (N2 -fixing), spore-Forming type of Frankia that lacks H, uptake (hydrogenase) activity in symbiosis with A. incana. Despite their wide geographical origin (Europe, China, North America) and their various taxonomie grouping within the genus, all Alnus species/provenances formed Infixing nodules. In all studied nodules Frankia had the phenotype Spore+ . None of the studied symbioses had hydrogenase activity. The Frankia inoculum was infective also on M. gale, and gave effective nodules without hydrogenase activity. Unlike some other spore-forming types of symbiotic Frankia, all studied symbiotic phenotypes of the 'local source' of Frankia were thus stable characters not influenced by the various host genotypes included in the study.

Nitrogenase in Frankia from root nodules of Alnus incana (L.) Moench: immunolocalization of the Fe- and MoFe-proteins during vesicle differentiation.

The localization of nitrogenase was studied in root nodules of Alnus incana (L.) Moench in symbiosis with a local source of Frankia. Nodules were fixed in glutaraldehyde and embedded in Epon or LR White. Ultrathin sections were examined by TEM after incubation with antisera against nitrogenase and gold-conjugated secondary antibodies. Antisera against dinitrogenase reductase (Fe-protein) from Rhodospirillum rubrum and dinitrogenase (MoFe-protein) from Azotobacter vinelandii were used. Immunogold label density in hyphae and in spores of Frankia was low and similar to that of plant tissue. Very young vesicles without septa showed a label density of Fe-protein similar to that of hyphae, while young vesicles with a few, usually non-transversing, septa had a significant but still low density of label. Mature vesicles, highly compartmentalised due to transversing septa, contained statistically significantly more Fe-protein label than all other cell types. Old degenerating vesicles always contained nitrogenase, about half as much as that of mature vesicles but often more than young vesicles. Similarly, MoFe label was not detected in hyphae, appeared in young but was greatest in mature vesicles. It is apparent that differentiation of vesicles reaches a certain stage of development prior to the onset of nitrogenase synthesis.