The evil within? Systemic fungicide application in trees enhances litter quality for an aquatic decomposer-detritivore system.

Waterborne exposure towards fungicides is known to trigger negative effects in aquatic leaf-associated microbial decomposers and leaf-shredding macroinvertebrates. We expected similar effects when these organisms use leaf material from terrestrial plants that were treated with systemic fungicides as a food source since the fungicides may remain within the leaves when entering aquatic systems. To test this hypothesis, we treated black alder (Alnus glutinosa) trees with a tap water control or a systemic fungicide mixture (azoxystrobin, cyprodinil, quinoxyfen, and tebuconazole) at two worst-case application rates. Leaves of these trees were used in an experiment targeting alterations in two functions provided by leaf-associated microorganisms, namely the decomposition and conditioning of leaf material. The latter was addressed via the food-choice response of the amphipod shredder Gammarus fossarum. During a second experiment, the potential impact of long-term consumption of leaves from trees treated with systemic fungicides on G. fossarum was assessed. Systemic fungicide treatment altered the resource quality of the leaf material resulting in trends of increased fungal spore production and an altered community composition of leaf-associated fungi. These changes in turn caused a significant preference of Gammarus for microbially conditioned leaves that had received the highest fungicide treatment over control leaves. This higher food quality ultimately resulted in a higher gammarid growth (up to 300% increase) during the long-term feeding assay. Although the underlying mechanisms still need to be addressed, the present study demonstrates a positive indirect response in aquatic organisms due to systemic pesticide application in a terrestrial system. As the effects from the introduction of plant material treated with systemic fungicides strongly differ from those mediated via other pathways (e.g., waterborne exposure), our study provides a novel perspective of fungicide-triggered effects in aquatic detritus-based food webs.

Exposure pathway-dependent effects of the fungicide epoxiconazole on a decomposer-detritivore system.

Shredders play a central role in the breakdown of leaf material in aquatic systems. These organisms and the ecological function they provide may, however, be affected by chemical stressors either as a consequence of direct waterborne exposure or through alterations in food-quality (indirect pathway). To unravel the biological relevance of these effect pathways, we applied a 2×2-factorial test design. Leaf material was microbially colonized for 10days in absence or presence of the fungicide epoxiconazole (15µg/L) and subsequently fed to the shredder Asellus aquaticus under exposure to epoxiconazole (15µg/L) or in fungicide-free medium over a 28-day period (n=40). Both effect pathways caused alterations in asselids' food processing, physiological fitness, and growth, although not always statistically significantly: assimilation either increased or remained at a similar level relative to the control suggesting compensatory behavior of A. aquaticus to cope with the enhanced energy demand for detoxification processes and decreased nutritional quality of the food. The latter was driven by lowered microbial biomasses and the altered composition of fatty acids associated with the leaf material. Even with increased assimilation, direct and indirect effects caused decreases in the growth and lipid (fatty acid) content of A. aquaticus with relative effect sizes between 10 and 40%. Moreover, the concentrations of two essential polyunsaturated fatty acids (i.e., arachidonic acid and eicosapentaenoic acid) were non-significantly reduced (up to ~15%) in asselids. This effect was, however, independent of the exposure pathway. Although waterborne effects were generally stronger than the diet-related effects, results suggest impaired functioning of A. aquaticus via both effect pathways.

Performance of ectomycorrhizal alders exposed to specific Canadian oil sands tailing stressors under in vivo bipartite symbiotic conditions.

Canadian oil sands tailings are predominately sodic residues contaminated by hydrocarbons such as naphthenic acids. These conditions are harsh for plant development. In this study, we evaluated the effect of inoculating roots of Alnus viridis ssp. crispa and Alnus incana ssp. rugosa with ectomycorrhizal fungi in the presence of tailings compounds. Seedlings were inoculated with 7 different strains of Paxillus involutus and Alpova diplophloeus and were grown under different treatments of NaCl, Na2SO4, and naphthenic acids in a growth chamber. Afterwards, seedling survival, height, dry biomass, leaf necrosis, and root mycorrhization rate were measured. Paxillus involutus Mai was the most successful strain in enhancing alder survival, health, and growth. Seedlings inoculated with this strain displayed a 25% increase in survival rate, 2-fold greater biomass, and 2-fold less leaf necrosis compared with controls. Contrary to our expectations, A. diplophloeus was not as effective as P. involutus in improving seedling fitness, likely because it did not form ectomycorrhizae on roots of either alder species. High intraspecific variation characterized strains of P. involutus in their ability to stimulate alder height and growth and to minimize leaf necrosis. We conclude that in vivo selection under bipartite symbiotic conditions is essential to select effective strains that will be of use for the revegetation and reclamation of derelict lands.

Change in ATP-binding cassette B1/19, glutamine synthetase and alcohol dehydrogenase gene expression during root elongation in Betula pendula Roth and Alnus glutinosa L. Gaertn in response to leachate and leonardite humic substances.

Humic substances (HS) are complex and heterogeneous compounds of humified organic matter resulting from the chemical and microbiological decomposition of organic residues. HS have a positive effect on plant growth and development by improving soil structure and fertility. They have long been recognized as plant growth-promoting substances, particularly with regard to influencing nutrient uptake, root growth and architecture. The biochemical and molecular mechanisms through which HS influence plant physiology are not well understood. This study evaluated the bioactivity of landfill leachate and leonardite HS on alder (Alnus glutinosa L. Gaertn) and birch (Betula pendula Roth) during root elongation in vitro. Changes in root development were studied in relation to auxin, carbon and nitrogen metabolisms, as well as to the stress adaptive response. The cDNA fragments of putative genes encoding two ATP-binding cassette (ABC) transporters (ABCB1 and ABCB19) belonging to the B subfamily of plant ABC auxin transporters were cloned and sequenced. Molecular data indicate that HS and their humic acid (HA) fractions induce root growth by influencing polar auxin transport (PAT), as illustrated by the modulation of the ABCB transporter transcript levels (ABCB1 and ABCB19). There were also changes in alcohol dehydrogenase (ADH) and glutamine synthetase (GS) gene transcript levels in response to HS exposure. These findings confirmed that humic matter affects plant growth and development through various metabolic pathways, including hormonal, carbon and nitrogen metabolisms and stress response or signalization.

Heavy metal stress in alders: Tolerance and vulnerability of the actinorhizal symbiosis.

Alders have already demonstrated their potential for the revegetation of both mining and industrial sites. These actinorhizal trees and shrubs and the actinobacteria Frankia associate in a nitrogen-fixing symbiosis which could however be negatively affected by the presence of heavy metals, and accumulate them. In our hydroponic assay with black alders, quantification of the roots and shoots metal concentrations showed that, in the absence of stress, symbiosis increases Mo and Ni root content and simultaneously decreases Mo shoot content. Interestingly, the Mo shoot content also decreases in the presence of Ni, Cu, Pb, Zn and Cd for symbiotic alders. In symbiotic alders, Pb shoot translocation was promoted in presence of Pb. On the other hand, Cd exclusion in symbiotic root tissues was observed with Pb and Cd. In the presence of symbiosis, only Cd and Pb showed translocation into aerial tissues when present in the nutrient solution. Moreover, the translocation of Ni to shoot was prevented by symbiosis in the presence of Cd, Ni and Pb. The hydroponic experiment demonstrated that alders benefit from the symbiosis, producing more biomass (total, root and shoot) than non nodulated alders in control condition, and in the presence of metals (Cu, Ni, Zn, Pb and Cd). Heavy metals did not reduce the nodule numbers (SNN), but the presence of Zn or Cd did reduce nodule allocation. Our study suggests that the Frankia-alder symbiosis is a promising (and a compatible) plant-microorganism association for the revegetation of contaminated sites, with minimal risk of metal dispersion.

Biomass of speckled alder on an air-polluted mountain site and its response to fertilization.

The article summarizes outcomes of a biomass study conducted in a young speckled alder plantation on a cold mountain site. At this location, the previously existing old forest was clear felled because of damage from air pollution, and present-day surface humus is in need of restoration. The intention of this study was to quantify the biomass and nutrients accumulated by alders and their components and assess whether the initial fertilization resulted in increased biomass production and nutrient accumulation in the biomass. Besides the control, two fertilized treatments were installed. In the surface treatment (SUT), the amendment was applied as a base dressing in small circles around trees. In the planting-hole treatment (PHT), the amendment was incorporated into soil inside the planting holes. Five growth seasons after planting and fertilization, six alders from each treatment were harvested including roots. Their biomass was quantified and analyzed for macroelements. The greatest pool of dry mass (DM) was branches in the control and stem wood in the fertilized treatments. The greatest pools of macroelements were leaves and branches. The most pronounced effects of fertilization were recorded in the DM and consequently in the absolute quantities of nutrients. The DM of an average tree in the control, SUT, and PHT was 85, 226, and 231 g, respectively. The absolute contents of nutrients per tree in the fertilized treatments showed the following increases, as compared with the control: (N) 2.5-2.6 times; (P) 1.6-2.4 times; (K) 1.8-2.1 times; and (Mg) 1.8-2.0 times, respectively. Speckled alder responded positively to fertilization.

From tolerance to acute metabolic deregulation: contribution of proteomics to dig into the molecular response of alder species under a polymetallic exposure.

Alnus spp. are actinorhizal trees commonly found in wet habitats and able to grow effectively in soil slightly contaminated with metal trace- elements. Two clones belonging to two Alnus species, namely, A. incana and A. glutinosa, were grown in hydroponics and exposed for 9 weeks to a Cd + Ni + Zn polymetallic constraint. Although responding by a similar decrease in total biomass production, the proteomic analysis associated with the study of various biochemical parameters including carbohydrate and mineral analyses revealed that the two clones have a distinct stress-responsive behavior. All parameters indicated that the roots, the organ in direct contact with the media, are more affected than the leaves. In fact, in A. glutinosa the response was almost completely confined to the roots, whereas many proteins change significantly in the roots and in the leaves of the treated A. incana. In both clones, the changes affected a broad range of metabolic processes such as redox regulation and energy metabolism and induced the production of pathogenesis-related proteins. In particular, changes in the accumulation of bacterial proteins that were not identified as coming from the known symbionts of Alnus were reported. Further investigation should be performed to identify their origin and exact role in the plant response to the polymetallic exposure tested here.

The effect of limited availability of N or water on C allocation to fine roots and annual fine root turnover in Alnus incana and Salix viminalis.

The effect of limited nitrogen (N) or water availability on fine root growth and turnover was examined in two deciduous species, Alnus incana L. and Salix viminalis L., grown under three different regimes: (i) supply of N and water in amounts which would not hamper growth, (ii) limited N supply and (iii) limited water supply. Plants were grown outdoors during three seasons in covered and buried lysimeters placed in a stand structure and filled with quartz sand. Computer-controlled irrigation and fertilization were supplied through drip tubes. Production and turnover of fine roots were estimated by combining minirhizotron observations and core sampling, or by sequential core sampling. Annual turnover rates of fine roots <1 mm (5-6 year(-1)) and 1-2 mm (0.9-2.8 year(-1)) were not affected by changes in N or water availability. Fine root production (<1 mm) differed between Alnus and Salix, and between treatments in Salix; i.e., absolute length and biomass production increased in the order: water limited < unlimited < N limited. Few treatment effects were detected for fine roots 1-2 mm. Proportionally more C was allocated to fine roots (≤2 mm) in N or water-limited Salix; 2.7 and 2.3 times the allocation to fine roots in the unlimited regime, respectively. Estimated input to soil organic carbon increased by ca. 20% at N limitation in Salix. However, future studies on fine root decomposition under various environmental conditions are required. Fine root growth responses to N or water limitation were less pronounced in Alnus, thus indicating species differences caused by N-fixing capacity and slower initial growth in Alnus, or higher fine root plasticity in Salix. A similar seasonal growth pattern across species and treatments suggested the influence of outer stimuli, such as temperature and light.

N2 fixation and cycling in Alnus glutinosa, Betula pendula and Fagus sylvatica woodland exposed to free air CO2 enrichment.

We measured the effect of elevated atmospheric CO(2) on atmospheric nitrogen (N(2)) fixation in the tree species Alnus glutinosa growing in monoculture or in mixture with the non-N(2)-fixing tree species Betula pendula and Fagus sylvatica. We addressed the hypotheses that (1) N(2) fixation in A. glutinosa will increase in response to increased atmospheric CO(2) concentrations, when growing in monoculture, (2) the impact of elevated CO(2) on N(2) fixation in A. glutinosa is the same in mixture and in monoculture and (3) the impacts of elevated CO(2) on N cycling will be evident by a decrease in leaf δ(15)N and by the soil-leaf enrichment factor (EF), and that these impacts will not differ between mixed and single species stands. Trees were grown in a forest plantation on former agricultural fields for four growing seasons, after which the trees were on average 3.8 m tall and canopy closure had occurred. Atmospheric CO(2) concentrations were maintained at either ambient or elevated (by 200 ppm) concentrations using a free-air CO(2) enrichment (FACE) system. Leaf δ(15)N was measured and used to estimate the amount (N(dfa)) and proportion (%N(dfa)) of N derived from atmospheric fixation. On average, 62% of the N in A. glutinosa leaves was from fixation. The %N(dfa) and N(dfa) for A. glutinosa trees in monoculture did not increase under elevated CO(2), despite higher growth rates. However, N(2) fixation did increase for trees growing in mixture, despite the absence of significant growth stimulation. There was evidence that fixed N(2) was transferred from A. glutinosa to F. sylvatica and B. pendula, but no evidence that this affected their CO(2) response. The results of this study show that N(2) fixation in A. glutinosa may be higher in a future elevated CO(2) world, but that this effect will only occur where the trees are growing in mixed species stands.

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.

Molecular diversity of Frankia in root nodules of Alnus incana grown with inoculum from polluted urban soils.

ABSTRACT The establishment and growth of trees can be compromised by soil contamination which can reduce populations of key microbial symbionts. We describe the colonisation of grey alder (Alnus incana) by Frankia from 10 urban soils with varying degrees of organic and inorganic pollution. Principal components analysis (PCA) of soil chemical profiles showed a separation of remediated and unremediated soils. A. incana seedlings were used as trap plants to capture the microsymbiont from soil. After 6 months growth, nodulation was lowest on trees grown with the most contaminated soils. Plant biomass was positively correlated with root nodule biomass and negatively correlated with PAH concentration. DNA was isolated from nodules for the analysis of Frankia genetic diversity. The polymerase chain reaction (PCR) was used to amplify the 16S-23S intergenic spacer (IGS) of Frankia ribosomal DNA. PCR products were subject to restriction digestion yielding 10 restriction fragment length polymorphism (RFLP) types from 72 nodules analysed. Our results demonstrate that each soil supports a distinct nodulating Frankia community. Partial 16S sequencing placed most strains in Frankia clusters 1a and 1b, which are typically Alnus-infecting, but sequences from several nodules obtained from a gasworks soil belonged to cluster 3, normally associated with Elaeagnus. These results show for the first time that polluted soils can be an effective source of Alnus-infective Frankia. Inoculation with site-adapted Frankia under greenhouse conditions could thus be an appropriate strategy to increase the symbiotic capacity of A. incana and to improve its chances of survival and growth when planted on polluted soils.

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.