Comparative transcriptome analyses reveal different mechanism of high- and low-tillering genotypes controlling tiller growth in orchardgrass (Dactylis glomerata L.).

BACKGROUND: Tillering is an important agronomic trait underlying the yields and reproduction of orchardgrass (Dactylis glomerata), an important perennial forage grass. Although some genes affecting tiller initiation have been identified, the tillering regulatory network is still largely unknown, especially in perennial forage grasses. Thus, unraveling the regulatory mechanisms of tillering in orchardgrass could be helpful in developing selective strategies for high-yield perennial grasses. In this study, we generated high-throughput RNA-sequencing data from multiple tissues of tillering stage plants to identify differentially expressed genes (DEGs) between high- and low-tillering orchardgrass genotypes. Gene Ontology and pathway enrichment analyses connecting the DEGs to tillering number diversity were conducted. RESULTS: In the present study, approximately 26,282 DEGs were identified between two orchardgrass genotypes, AKZ-NRGR667 (a high-tillering genotype) and D20170203 (a low-tillering genotype), which significantly differed in tiller number. Pathway enrichment analysis indicated that DEGs related to the biosynthesis of three classes of phytohormones, i.e., strigolactones (SLs), abscisic acid (ABA), and gibberellic acid (GA), as well as nitrogen metabolism dominated such differences between the high- and low-tillering genotypes. We also confirmed that under phosphorus deficiency, the expression level of the major SL biosynthesis genes encoding DWARF27 (D27), 9-cis-beta-carotene 9',10'-cleaving dioxygenase (CCD7), carlactone synthase (CCD8), and more axillary branching1 (MAX1) proteins in the high-tillering orchardgrass genotype increased more slowly relative to the low-tillering genotype. CONCLUSIONS: Here, we used transcriptomic data to study the tillering mechanism of perennial forage grasses. We demonstrated that differential expression patterns of genes involved in SL, ABA, and GA biosynthesis may differentiate high- and low-tillering orchardgrass genotypes at the tillering stage. Furthermore, the core SL biosynthesis-associated genes in high-tillering orchardgrass were more insensitive than the low-tillering genotype to phosphorus deficiency which can lead to increases in SL biosynthesis, raising the possibility that there may be distinct SL biosynthesis way in tillering regulation in orchardgrass. Our research has revealed some candidate genes involved in the regulation of tillering in perennial grasses that is available for establishment of new breeding resources for high-yield perennial grasses and will serve as a new resource for future studies into molecular mechanism of tillering regulation in orchardgrass.

Genome-wide identification, structural analysis and expression profiles of GRAS gene family in orchardgrass.

The GRAS gene family is a family of transcription factors that regulates plant growth and development. Despite being well-studied in many plant species, little is known about this gene family in orchardgrass (Dactylis glomerata L.), one of the top four economically important perennial forage grasses cultivated worldwide. We identified 46 GRAS genes in orchardgrass and analyzed their characteristics by phylogenetic, gene structural, motifs and expression patterns analysis. The phylogenetic analysis of eight species revealed that DgGRAS family had the evolutional conservation and closer homology relationship with the GRAS family of rice, barley and Brachypodium distachyon. Moreover, 46 DgGRAS proteins were divided into eight subfamilies based on the tree topology and rice or Arabidopsis classification, and LISCL subfamily was the largest one. Besides, we found that the motif 15 may be unique to the orchardgrass LISCL subfamily, and the motif 6 and motif 17 had indispensable functions in the orchardgrass LISCL subfamily. We further analyzed the expression profiles of DgGRAS genes at mature and seeding stage. And we found that DgGRAS17 played an important role in the growth and development no matter what stage it was at. DgGRAS5, DgGRAS28, DgGRAS31, DgGRAS42 and DgGRAS44 got involved in processes of the growth and development at seeding stage instead of mature stage. These results indicated that the major expression patterns and detailed functions of the DgGRAS genes varied with developmental stages. Taken together, this is the first systematic analysis of the GRAS gene family in the orchardgrass genome and the results provide insights into the potential functions of GRAS genes.

Integration of small RNAs and transcriptome sequencing uncovers a complex regulatory network during vernalization and heading stages of orchardgrass (Dactylis glomerata L.).

BACKGROUND: Flowering is a critical reproductive process in higher plants. Timing of optimal flowering depends upon the coordination among seasonal environmental cues. For cool season grasses, such as Dactylis glomerata, vernalization induced by low temperature provides competence to initiate flowering after prolonged cold. We combined analyses of the transcriptome and microRNAs (miRNAs) to generate a comprehensive resource for regulatory miRNAs and their target circuits during vernalization and heading stages. RESULTS: A total of 3,846 differentially expressed genes (DEGs) and 69 differentially expressed miRNAs were identified across five flowering stages. The expression of miR395, miR530, miR167, miR396, miR528, novel_42, novel_72, novel_107, and novel_123 demonstrated significant variations during vernalization. These miRNA targeted genes were involved in phytohormones, transmembrane transport, and plant morphogenesis in response to vernalization. The expression patterns of DEGs related to plant hormones, stress responses, energy metabolism, and signal transduction changed significantly in the transition from vegetative to reproductive phases. CONCLUSIONS: Five hub genes, c136110_g1 (BRI1), c131375_g1 (BZR1), c133350_g1 (VRN1), c139830_g1 (VIN3), and c125792_g2 (FT), might play central roles in vernalization response. Our comprehensive analyses have provided a useful platform for investigating consecutive transcriptional and post-transcriptional regulation of critical phases in D. glomerata and provided insights into the genetic engineering of flowering-control in cereal crops.

Local plant responses to global problems: Dactylis glomerata responses to different traffic pollutants on roadsides.

The growing number of road vehicles is a major source of regional and global atmospheric pollution increasing concentrations of CO2 in the air, and levels of metals in air and soil. Nevertheless, the effects of these pollutants on plants growing at roadsides are poorly documented. We carried out an observational study of unmanipulated plants growing by the road, to identify the morpho-physiological responses in a perennial grass Dactylis glomerata. Firstly, we wanted to know the general effect of traffic intensity and ambient CO2 and its interactions on different plant traits. Accordingly, we analyzed the photosynthetic response by field A/Ci Response Curves, SLA, pigment pools, foliar nitrogen, carbohydrates and morphological traits in plants at three distances to the road. Secondly, we wanted to know if Dactylis glomerata plants can accumulate metals present on the roadside (Pb, Zn, Cu, and Sr) in their tissues and rhizosphere, and the effect of these metals on morphological traits. The MANCOVA whole model results shown: 1) a significant effect of road ambient CO2 concentration on morphological traits (not affected by traffic intensity, P interaction CO2 x traffic intensity>0.05), that was mainly driven by a significant negative relationship between the inflorescence number and ambient CO2; 2) a positive and significant relationship between ambient CO2 and the starch content in leaves (unaffected by traffic intensity); 3) a reduction in Jmax (electron transport rate) at high traffic intensity. These lines of evidences suggest a decreased photosynthetic capacity due to high traffic intensity and high levels of ambient CO2. In addition, Pb, Cu, Zn and Sr were detected in Dactylis glomerata tissues, and Cu accumulated in roots. Finally, we observed that Dactylis glomerata individuals growing at the roadside under high levels of CO2 and in the presence of metal pollutants, reduced their production of inflorescences.

Association of candidate genes with heading date in a diverse Dactylis glomerata population.

Flowering occurs in response to cues from both temperature and photoperiod elicitors in cool-season, long-day forage grasses, and genes involved in sensing the elicitors and inducing downstream flowering responses have been associated with heading date and flowering time in perennial forage grasses as well as cereal grasses. In this study we test for association between orchardgrass (Dactylis glomerata L.) heading date and polymorphisms in the CONSTANS (DgCO1), FLOWERING TIME (DgFT1), a VRN1 like MADS-box (DgMADS), and PHOTOPERIOD (DgPPD1-like) containing genes. A diverse population of 150 genotypes was measured for heading date across three years, genotyped, and candidate genes sequenced. Although pairwise population kinship values were generally low, the genotypes fit into a two-group structure model. Linkage disequilibrium decayed rapidly, reaching r2 levels below 0.2 within the 500bp of each gene. SNPs significantly associated with heading date were detected in equal-dose and tetraploid dosage models. The DgCO1 gene had the most significant polymorphisms and those with the largest effects, while DgMADS had several significant polymorphisms in its first intron with smaller effects. These polymorphisms can be used for further validation, selection, and development of breeding lines of orchardgrass.

Comprehensive transcriptome analysis reveals distinct regulatory programs during vernalization and floral bud development of orchardgrass (Dactylis glomerata L.).

BACKGROUND: Vernalization and the transition from vegetative to reproductive growth involve multiple pathways, vital for controlling floral organ formation and flowering time. However, little transcription information is available about the mechanisms behind environmental adaption and growth regulation. Here, we used high-throughput sequencing to analyze the comprehensive transcriptome of Dactylis glomerata L. during six different growth periods. RESULTS: During vernalization, 4689 differentially expressed genes (DEGs) significantly increased in abundance, while 3841 decreased. Furthermore, 12,967 DEGs were identified during booting stage and flowering stage, including 7750 up-regulated and 5219 down-regulated DEGs. Pathway analysis indicated that transcripts related to circadian rhythm, photoperiod, photosynthesis, flavonoid biosynthesis, starch, and sucrose metabolism changed significantly at different stages. Coexpression and weighted correlation network analysis (WGCNA) analysis linked different stages to transcriptional changes and provided evidence of inner relation modules associated with signal transduction, stress responses, cell division, and hormonal transport. CONCLUSIONS: We found enrichment in transcription factors (TFs) related to WRKY, NAC, AP2/EREBP, AUX/IAA, MADS-BOX, ABI3/VP1, bHLH, and the CCAAT family during vernalization and floral bud development. TFs expression patterns revealed intricate temporal variations, suggesting relatively separate regulatory programs of TF modules. Further study will unlock insights into the ability of the circadian rhythm and photoperiod to regulate vernalization and flowering time in perennial grass.

Water use efficiency and shoot biomass production under water limitation is negatively correlated to the discrimination against 13C in the C3 grasses Dactylis glomerata, Festuca arundinacea and Phalaris arundinacea.

Climate change impacts rainfall patterns which may lead to drought stress in rain-fed agricultural systems. Crops with higher drought tolerance are required on marginal land with low precipitation or on soils with low water retention used for biomass production. It is essential to obtain plant breeding tools, which can identify genotypes with improved drought tolerance and water use efficiency (WUE). In C3 plant species, the variation in discrimination against 13C (Δ13C) during photosynthesis has been shown to be a potential indicator for WUE, where discrimination against 13C and WUE were negatively correlated. The aim of this study was to determine the variation in the discrimination against 13C between species and cultivars of three perennial C3 grasses (Dactylis glomerata (cocksfoot), Festuca arundinacea (tall fescue) and Phalaris arundinacea (reed canary grass)) and test the relationships between discrimination against 13C, season-long water use WUEB, shoot and root biomass production in plants grown under well-watered and water-limited conditions. The grasses were grown in the greenhouse and exposed to two irrigation regimes, which corresponded to 25% and 60% water holding capacity, respectively. We found negative relationships between discrimination against 13C and WUEB and between discrimination against 13C and shoot biomass production, under both the well-watered and water-limited growth conditions (p < 0.001). Discrimination against 13C decreased in response to water limitation (p < 0.001). We found interspecific differences in the discrimination against 13C, WUEB, and shoot biomass production, where the cocksfoot cultivars showed lowest and the reed canary grass cultivars highest values of discrimination against 13C. Cocksfoot cultivars also showed highest WUEB, shoot biomass production and potential tolerance to water limitation. We conclude that discrimination against 13C appears to be a useful indicator, when selecting C3 grass crops for biomass production under drought conditions.

Five species, many genotypes, broad phenotypic diversity: When agronomy meets functional ecology.

PREMISE OF THE STUDY: Current ecological theory can provide insight into the causes and impacts of plant domestication. However, just how domestication has impacted intraspecific genetic variability (ITV) is unknown. We used 50 ecotypes and 35 cultivars from five grassland species to explore how selection drives functional trait coordination and genetic differentiation. METHODS: We quantified the extent of genetic diversity among different sets of functional traits and determined how much genetic diversity has been generated within populations of natural ecotypes and selected cultivars. KEY RESULTS: In general, the cultivars were larger (e.g., greater height, faster growth rates) and had larger and thinner leaves (greater SLA). We found large (average 63%) and trait-dependent (ranging from 14% for LNC to 95.8% for growth rate) genetic variability. The relative extent of genetic variability was greater for whole-plant than for organ-level traits. This pattern was consistent within ecotypes and within cultivars. However, ecotypes presented greater ITV variability. CONCLUSIONS: The results indicated that genetic diversity is large in domesticated species with contrasting levels of heritability among functional traits and that selection for high yield has led to indirect selection of some associated leaf traits. These findings open the way to define which target traits should be the focus in selection programs, especially in the context of community-level selection.

Coupling Between and Among Ammonia Oxidizers and Nitrite Oxidizers in Grassland Mesocosms Submitted to Elevated CO2 and Nitrogen Supply.

Many studies have assessed the responses of soil microbial functional groups to increases in atmospheric CO2 or N deposition alone and more rarely in combination. However, the effects of elevated CO2 and N on the (de)coupling between different microbial functional groups (e.g., different groups of nitrifiers) have been barely studied, despite potential consequences for ecosystem functioning. Here, we investigated the short-term combined effects of elevated CO2 and N supply on the abundances of the four main microbial groups involved in soil nitrification: ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (belonging to the genera Nitrobacter and Nitrospira) in grassland mesocosms. AOB and AOA abundances responded differently to the treatments: N addition increased AOB abundance, but did not alter AOA abundance. Nitrobacter and Nitrospira abundances also showed contrasted responses to the treatments: N addition increased Nitrobacter abundance, but decreased Nitrospira abundance. Our results support the idea of a niche differentiation between AOB and AOA, and between Nitrobacter and Nitrospira. AOB and Nitrobacter were both promoted at high N and C conditions (and low soil water content for Nitrobacter), while AOA and Nitrospira were favored at low N and C conditions (and high soil water content for Nitrospira). In addition, Nitrobacter abundance was positively correlated to AOB abundance and Nitrospira abundance to AOA abundance. Our results suggest that the couplings between ammonia and nitrite oxidizers are influenced by soil N availability. Multiple environmental changes may thus elicit rapid and contrasted responses between and among the soil ammonia and nitrite oxidizers due to their different ecological requirements.

Efficacy of biosolids in assisted phytostabilization of metalliferous acidic sandy soils with five grass species.

The role of sewage sludge as an immobilising agent in the phytostabilization of metal-contaminated soil was evaluated using five grass species viz., Dactylis glomerata L., Festuca arundinacea Schreb., F. rubra L., Lolium perenne L., L. westerwoldicum L. The function of metal immobilization was investigated by monitoring pH, Eh and Cd, Pb, and Zn levels in column experiment over a period of 5-months. Grasses grown on sewage sludge-amendments produced high biomass in comparison to controls. A significant reduction in metal uptake by plants was also observed as a result of sewage sludge application, which was attributed to decreased bioavailability through soil stabilisation. We have observed that the sludge amendment decreased metal bioavailability and concentrations in soil at a depth of 25 cm, in contrast to untreated columns, where metal concentrations in the soil solution were very high.

Morphological and genetic characteristics of hybrid combinations of Dactylis glomerata.

Six F1 populations derived from crosses among 4 orchardgrass (Dactylis glomerata L.) cultivars were studied by morphological and simple sequence repeat molecular markers to test for hybrid vigor and a correlation between genetic distance and heterosis. Heterosis was observed for days to length of culm, leaf traits, tiller numbers, etc. Significant differences between obverse and inverse crosses were found for eleven traits. A cytoplasmic effect existed for the agronomic traits considered in this study. The correlations between genetic distance and heterosis were investigated by analyzing the performance of 3 crosses. The results showed that genetic distance was significantly correlated with tiller number (r = 0.834) and negatively correlated with length of culm (r = -0.889). However, there was no significant correlation with heterosis for the other traits, including yield; the correlation coefficient were too small to allow prediction of orchardgrass heterosis from the parental genetics.

Plants suppress their emission of volatiles when growing with conspecifics.

Plant volatiles mediate interactions with herbivores, herbivore enemies, and abiotic stresses, but these interactions mostly have been studied with individual isolated plants. It is not yet known how intra- and interspecific plant competition influence volatile emission. In a greenhouse experiment, we investigated the volatile emission by red clover (Trifolium pratense) growing alone, with a conspecific, or with an individual of the naturally co-occurring orchard grass, Dactylis glomerata. The individual and combined effects of above- and below ground plant contact were investigated. When T. pratense grew together with a conspecific, both total and herbivore-induced emission of volatiles was significantly reduced as compared to T. pratense growing with D. glomerata or growing alone. This reduction in emission occurred despite the fact that there was a significant reduction in T. pratense biomass due to competition with D. glomerata. The suppression of T. pratense volatile emission growing next to a conspecific was a general pattern observed for all major herbivore-induced volatiles and independent of whether plants were in contact above ground, below ground, or both above- and below ground. The reduction in volatile emission from plants growing with conspecifics may serve to reduce attack by specialist herbivores and minimize exploitation of herbivore attack information by neighbors.

Relationship between water soluble carbohydrate content, aphid endosymbionts and clonal performance of Sitobion avenae on cocksfoot cultivars.

Aphids feed on plant phloem sap, rich in sugars but poor in essential amino acids. However, sugars cause osmotic regulation problems for aphids, which they overcome by hydrolysing the sugars in their gut and polymerising the hydrolysis products into oligosaccharides, excreted with honeydew. Aphids harbour primary bacterial endosymbionts, which supply them with essential amino acids necessary for survival. They also harbour secondary (facultative) endosymbionts (sfS), some of which have a positive impact on life history traits, although it is not yet known whether they also play a role in providing effective tolerance to differing levels of water soluble carbohydrates (WSCs). We investigated the relationship between WSC content of cocksfoot cultivars and performance of clones of the English grain aphid Sitobion avenae F. We evaluated how clone genotype and their sfS modulate performance on these different cultivars. We therefore examined the performance of genetically defined clones of S. avenae, collected from different host plants, harbouring different sfS. The performance was tested on 10 Dactylis glomerata L. cultivars with varying WSC content. D. glomerata is known as a wild host plant for S. avenae and is also commercially planted. We found that high WSCs levels are responsible for the resistance of D. glomerata cultivars to specific S. avenae clones. The minimum level of WSCs conferring resistance to D. glomerata cultivars was 1.7% dw. Cultivars with a WSC content of 2.2% or higher were resistant to S. avenae and did not allow reproduction. Our results further indicate that sfS modulate to some extend host plant cultivar adaptation in S. avenae. This is the first study revealing the importance of WSCs for aphid performance. Cocksfoot cultivars with a high content of WSCs might be therefore considered for aphid control or used for resistance breeding in this and other grass species, including cereals.

[Effects of drought stress on the root growth and photosynthetic characters of Dactylis glomerata seedlings].

Taking the drought-sensitive Dactylis glomerata line "01998" and drought-tolerant cultivar "Baoxing" as test materials, a pot experiment was conducted to study the effects of drought stress on the seedlings root growth, physiological characteristics, and leaf photosynthesis, aimed to approach the differences in the drought-tolerance mechanism of different D. glomerata lines (cultivars). Under drought stress, the root vitality and root number of "01998" and "Baoxing" presented a trend of increased first and decreased then. When the soil relative moisture content decreased to 30%, the root vitality and root number of "01998" and "Baoxing" increased significantly and reached the maximum. Drought stress decreased the relative water content, leaf chlorophyll content, net photosynthesis, transpiration, and stomatal conductance, but increased the electric conductivity and intercellular CO2 concentration of "01998" and "Baoxing". Under drought stress, the leaf area per plant of both "01998" and "Baoxing" decreased, and the underground and aboveground plant biomass of "01998" decreased while that of "Baoxing" had less change.

Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L.

The tripeptide antioxidant γ-L-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), serves a central role in ROS scavenging and oxidative signalling. Here, GSH, glutathione disulphide (GSSG), and other low-molecular-weight (LMW) thiols and their corresponding disulphides were studied in embryogenic suspension cultures of Dactylis glomerata L. subjected to moderate (0.085 M NaCl) or severe (0.17 M NaCl) salt stress. Total glutathione (GSH + GSSG) concentrations and redox state were associated with growth and development in control cultures and in moderately salt-stressed cultures and were affected by severe salt stress. The redox state of the cystine (CySS)/2 cysteine (Cys) redox couple was also affected by developmental stage and salt stress. The glutathione half-cell reduction potential (E(GSSG/2 GSH)) increased with the duration of culturing and peaked when somatic embryos were formed, as did the half-cell reduction potential of the CySS/2 Cys redox couple (E(CySS/2 Cys)). The most noticeable relationship between cellular redox state and developmental state was found when all LMW thiols and disulphides present were mathematically combined into a 'thiol-disulphide redox environment' (E(thiol-disulphide)), whereby reducing conditions accompanied proliferation, resulting in the formation of pro-embryogenic masses (PEMs), and oxidizing conditions accompanied differentiation, resulting in the formation of somatic embryos. The comparatively high contribution of E(CySS/2 Cys) to E(thiol-disulphide) in cultures exposed to severe salt stress suggests that Cys and CySS may be important intracellular redox regulators with a potential role in stress signalling.

Dynamic trajectories of growth and nitrogen capture by competing plants.

Although dynamic, plant competition is usually estimated as biomass differences at a single, arbitrary time; resource capture is rarely measured. This restricted approach perpetuates uncertainty. To address this problem, we characterized the competitive dynamics of Dactylis glomerata and Plantago lanceolata as continuous trajectories of biomass production and nitrogen (N) capture. Plants were grown together or in isolation. Biomass and N content were measured at 17 harvests up to 76 d after sowing. Data were fitted to logistic models to derive instantaneous growth and N capture rates. Plantago lanceolata was initially more competitive in terms of cumulative growth and N capture, but D. glomerata was eventually superior. Neighbours reduced maximum biomass, but influenced both maximum N capture and its rate constant. Timings of maximal instantaneous growth and N capture rates were similar between species when they were isolated, but separated by 16 d when they were competing, corresponding to a temporal convergence in maximum growth and N capture rates in each species. Plants processed N and produced biomass differently when they competed. Biomass and N capture trajectories demonstrated that competitive outcomes depend crucially on when and how 'competition' is measured. This potentially compromises the interpretation of conventional competition experiments.

Root-shoot growth responses during interspecific competition quantified using allometric modelling.

BACKGROUND AND AIMS: Plant competition studies are restricted by the difficulty of quantifying root systems of competitors. Analyses are usually limited to above-ground traits. Here, a new approach to address this issue is reported. METHODS: Root system weights of competing plants can be estimated from: shoot weights of competitors; combined root weights of competitors; and slopes (scaling exponents, α) and intercepts (allometric coefficients, ß) of ln-regressions of root weight on shoot weight of isolated plants. If competition induces no change in root : shoot growth, α and ß values of competing and isolated plants will be equal. Measured combined root weight of competitors will equal that estimated allometrically from measured shoot weights of each competing plant. Combined root weights can be partitioned directly among competitors. If, as will be more usual, competition changes relative root and shoot growth, the competitors' combined root weight will not equal that estimated allometrically and cannot be partitioned directly. However, if the isolated-plant α and ß values are adjusted until the estimated combined root weight of competitors matches the measured combined root weight, the latter can be partitioned among competitors using their new α and ß values. The approach is illustrated using two herbaceous species, Dactylis glomerata and Plantago lanceolata. KEY RESULTS: Allometric modelling revealed a large and continuous increase in the root : shoot ratio by Dactylis, but not Plantago, during competition. This was associated with a superior whole-plant dry weight increase in Dactylis, which was ultimately 2·5-fold greater than that of Plantago. Whole-plant growth dominance of Dactylis over Plantago, as deduced from allometric modelling, occurred 14-24 d earlier than suggested by shoot data alone. CONCLUSION: Given reasonable assumptions, allometric modelling can analyse competitive interactions in any species mixture, and overcomes a long-standing problem in studies of competition.

Enzymatic activity of a mine soil varies according to vegetation cover and level of compost applied.

We applied three doses of compost from mixed municipal solid waste (0, 15, and 30 g kg(-1) of soil) to a soil developed on pyrite mine wastes. Part of the soil was planted with young Erica australis L. collected at the mine; part was fertilized with N-P-K-Mg and sown with Dactylis glomerata L .Bare soil without mineral fertilization was included in the experiment, as well. Compost application to bare soil increased pH, provided plant nutrients, and enhanced the activity of the six soil enzymes tested. Growth of D. glomerata, and E. australis was stimulated in compost-amended soil compared with unamended controls. The presence of D. glomerata led to the greatest activities of soil acid phosphatase, beta-glucosidase, and cellulase compared with bare soil or with soil with E. australis. The presence of E. australis increased the activities of protease and cellulase in amended soil, compared with control, but it impaired dehydrogenase, fl-glucosidase, and acid phosphatase activities. These negative impacts probably derived from phenolic compounds known to be released from roots of this species. The survival strategy of this species seems to include a small need for P in the shoots, and the release of exudates that impair microbial activity and P cycling.

Optimization of culture conditions of Arnica montana L.: effects of mycorrhizal fungi and competing plants.

Arnica montana is a rare plant that needs special protection because of its intensive harvesting for medicinal purposes. The present work was aimed at finding optimal culture conditions for Arnica plants in order to enable their successful reintroduction into their natural stands. Plants were cultivated under controlled greenhouse conditions on substrata with different nitrogen (N) concentration. As Arnica is always colonized by arbuscular mycorrhizal fungi (AMF) in nature, a fact that has been overlooked in other similar projects, we, here, applied and tested different inocula. We found that they differed in their effectiveness, both in establishing symbiosis, assessed by the colonization parameters, and in improving the performance of Arnica, evaluated by the photosynthetic parameters derived from the fluorescence transients (JIP-test), with the inocula containing G. intraradices or composed of several Glomus strains being the most effective. The comparison was possible only on substrata with medium N, since high N did not permit the formation of mycorrhiza, while at low N, few nonmycorrhizal plants survived until the measurements and mycorrhizal plants, which were well growing, exhibited a high heterogeneity. Analysis of secondary metabolites showed clearly that mycorrhization was associated with increased concentrations of phenolic acids in roots. For some of the inocula used, a tendency for increase of the level of phenolic acids in shoots and of sesquiterpene lactones, both in roots and in shoots, was also observed. We also studied the interactions between A. montana and Dactylis glomerata, known to compete with Arnica under field conditions. When specimens from both species were cultured together, there was no effect on D. glomerata, but Arnica could retain a photosynthetic performance that permitted survivability only in the presence of AMF; without AMF, the photosynthetic performance was lower, and the plants were eventually totally outcompeted.

Effects of management regime and plant species on the enzyme activity and genetic structure of N-fixing, denitrifying and nitrifying bacterial communities in grassland soils.

Management by combined grazing and mowing events is commonly used in grasslands, which influences the activity and composition of soil bacterial communities. Whether observed effects are mediated by management-induced disturbances, or indirectly by changes in the identity of major plant species, is still unknown. To address this issue, we quantified substrate-induced respiration (SIR), and the nitrification, denitrification and free-living N(2)-fixation enzyme activities below grass tufts of three major plant species (Holcus lanatus, Arrhenatherum elatius and Dactylis glomerata) in extensively or intensively managed grasslands. The genetic structures of eubacterial, ammonia oxidizing, nitrate reducing, and free-living N(2)-fixing communities were also characterized by ribosomal intergenic spacer analysis, and denaturing gradient gel electrophoresis (DGGE) or restriction fragment length polymorphism (RFLP) targeting group-specific genes. SIR was not influenced by management and plant species, whereas denitrification enzyme activity was influenced only by plant species, and management-plant species interactions were observed for fixation and nitrification enzyme activities. Changes in nitrification enzyme activity were likely largely explained by the observed changes in ammonium concentration, whereas N availability was not a major factor explaining changes in denitrification and fixation enzyme activities. The structures of eubacterial and free-living N(2)-fixing communities were essentially controlled by management, whereas the diversity of nitrate reducers and ammonia oxidizers depended on both management and plant species. For each functional group, changes in enzyme activity were not correlated or were weakly correlated to overall changes in genetic structure, but around 60% of activity variance was correlated to changes in five RFLP or DGGE bands. Although our conclusions should be tested for other ecosystems and seasons, these results show that predicting microbial changes induced by management in grasslands requires consideration of management-plant species interactions.