Effects of the Clonostachys epichloë fungal hyperparasite on the symbiotic interaction between Botanophila flies and Epichloë fungus.

Botanophila flies are associated with Epichloë fungi in a symbiotic relationship in which the flies benefit from stromata as a food source for both adults and larvae, and the fungus benefits from the transfer of conidia by the flies, resulting in fertilization. Derogations from this pattern indicate that the Epichloë-Botanophila interaction cannot be clearly defined. The situation may be complicated by reports of new elements of the interaction, e.g., Wolbachia bacteria present in Botanophila larvae. The present study investigates the impact of Clonostachys epichloë (Speg.) Schroers, the fungal hyperparasite of Epichloë stromata, on the Botanophila-Epichloë interaction. The interaction between C. epichloë and Botanophila flies associated with Epichloë typhina subsp. clarkii (J.F. White) Leuchtm. & Schardl stromata was studied in the Holcus lanatus L. grass population. C. epichloë was present on 76.5% of stromata, covering on average 44.8 ± 32.1% of its surface and influencing the final perithecial coverage to the same extent as larval feeding. C. epichloë began to appear on stromata much later than the fly eggs and did not affect the preference for Botanophila egg laying. On the other hand, C. epichloë reduced larval hatching success and increased the mortality of the larvae. Clonostachys was responsible for 76.0% of all deaths, overgrowing brood chambers, and its mycelium was present both on and within larvae in all cases. Overall, as a result of the presence of C. epichloë, the number of Botanophila fly offspring decreased by 52.7%. Of the 26 surviving larvae, 10 (38.5%) were affected by C. epichloë, and their weight was significantly lower than that of unaffected larvae. Results show that C. epichloë, a new element of the interaction between E. typhina fungus and Botanophila flies, negatively affects both fungal reproduction and the offspring success of flies. This is the first report on the entomopathogenic activity of C. epichloë against Epichloë-associated Botanophila flies.

Microbiome and ecotypic adaption of Holcus lanatus (L.) to extremes of its soil pH range, investigated through transcriptome sequencing.

BACKGROUND: Plants can adapt to edaphic stress, such as nutrient deficiency, toxicity and biotic challenges, by controlled transcriptomic responses, including microbiome interactions. Traditionally studied in model plant species with controlled microbiota inoculation treatments, molecular plant-microbiome interactions can be functionally investigated via RNA-Seq. Complex, natural plant-microbiome studies are limited, typically focusing on microbial rRNA and omitting functional microbiome investigations, presenting a fundamental knowledge gap. Here, root and shoot meta-transcriptome analyses, in tandem with shoot elemental content and root staining, were employed to investigate transcriptome responses in the wild grass Holcus lanatus and its associated natural multi-species eukaryotic microbiome. A full factorial reciprocal soil transplant experiment was employed, using plant ecotypes from two widely contrasting natural habitats, acid bog and limestone quarry soil, to investigate naturally occurring, and ecologically meaningful, edaphically driven molecular plant-microbiome interactions. RESULTS: Arbuscular mycorrhizal (AM) and non-AM fungal colonization was detected in roots in both soils. Staining showed greater levels of non-AM fungi, and transcriptomics indicated a predominance of Ascomycota-annotated genes. Roots in acid bog soil were dominated by Phialocephala-annotated transcripts, a putative growth-promoting endophyte, potentially involved in N nutrition and ion homeostasis. Limestone roots in acid bog soil had greater expression of other Ascomycete genera and Oomycetes and lower expression of Phialocephala-annotated transcripts compared to acid ecotype roots, which corresponded with reduced induction of pathogen defense processes, particularly lignin biosynthesis in limestone ecotypes. Ascomycota dominated in shoots and limestone soil roots, but Phialocephala-annotated transcripts were insignificant, and no single Ascomycete genus dominated. Fusarium-annotated transcripts were the most common genus in shoots, with Colletotrichum and Rhizophagus (AM fungi) most numerous in limestone soil roots. The latter coincided with upregulation of plant genes involved in AM symbiosis initiation and AM-based P acquisition in an environment where P availability is low. CONCLUSIONS: Meta-transcriptome analyses provided novel insights into H. lanatus transcriptome responses, associated eukaryotic microbiota functions and taxonomic community composition. Significant edaphic and plant ecotype effects were identified, demonstrating that meta-transcriptome-based functional analysis is a powerful tool for the study of natural plant-microbiome interactions.

Arsenic extractability and uptake by velvetgrass Holcus lanatus and ryegrass Lolium perenne in variously treated soils polluted by tailing spills.

Phytostabilization should be considered as an appropriate phytoremediation technique to restore the area affected by tailing spills in Zloty Stok, where arsenic ores were mined and processed for several centuries. The study aimed to compare the suitability of velvetgrass (Holcus lanatus L.) and ryegrass (Lolium perenne L.) for development of plant cover in that area. Various treatments commonly applied to support phytostabilization were examined. A pot experiment was carried out to assess the effects of soil amendment with phosphate (P), sewage sludge (SS) and iron salts (Fe) on arsenic extractability and its uptake by grass. Four kinds of soil material, containing 356-5350 mg kg(-1) As, were examined. Velvetgrass proved to be more resistant than ryegrass to the toxicity of soil arsenic. Ammonium sulphate extractability of As in soils correlated well with As concentrations in the biomass of both grass species. In three of four tested soils, application of Fe failed to decrease As extractability and to reduce its concentrations in the aboveground parts of grasses. Application of P and SS resulted in increased As solubility in soils, but their effects on plant biomass and As uptake were ambiguous. SS had a strong beneficial influence on the growth of velvetgrass, while such an effect was not observed for ryegrass.

Multiple mechanisms enable invasive species to suppress native species.

PREMISE OF THE STUDY: Invasive plants represent a significant threat to ecosystem biodiversity. To decrease the impacts of invasive species, a major scientific undertaking of the last few decades has been aimed at understanding the mechanisms that drive invasive plant success. Most studies and theories have focused on a single mechanism for predicting the success of invasive plants and therefore cannot provide insight as to the relative importance of multiple interactions in predicting invasive species' success. METHODS: We examine four mechanisms that potentially contribute to the success of invasive velvetgrass Holcus lanatus: direct competition, indirect competition mediated by mammalian herbivores, interference competition via allelopathy, and indirect competition mediated by changes in the soil community. Using a combination of field and greenhouse approaches, we focus on the effects of H. lanatus on a common species in California coastal prairies, Erigeron glaucus, where the invasion is most intense. KEY RESULTS: We found that H. lanatus had the strongest effects on E. glaucus via direct competition, but it also influenced the soil community in ways that feed back to negatively influence E. glaucus and other native species after H. lanatus removal. CONCLUSIONS: This approach provided evidence for multiple mechanisms contributing to negative effects of invasive species, and it identified when particular strategies were most likely to be important. These mechanisms can be applied to eradication of H. lanatus and conservation of California coastal prairie systems, and they illustrate the utility of an integrated set of experiments for determining the potential mechanisms of invasive species' success.

Species-specific effects of live roots and shoot litter on soil decomposer abundances do not forecast plant litter-nitrogen uptake.

Plant species produce litter of varying quality and differ in the quality and quantity of compounds they release from live roots, which both can induce different decomposer growth in the soil. To test whether differences in decomposer growth can forecast the amount of N species acquire from plant litter, as suggested by theory, we grew individuals of three grassland plants-Holcus lanatus, Plantago lanceolata and Lotus corniculatus-in soils into which (15)N-labelled litter of either Holcus, Plantago or Lotus was added. We measured the effects of live roots and litter of each species on soil microbes and their protozoan and nematode feeders, and to link decomposer growth and plant nutrient uptake, we measured the amount of N taken up by plants from the added litter. We hypothesised that those species that induce the highest growth of microbes, and especially that of microbial feeders, will also take up the highest amount of N from the litter. We found, however, that although numbers of bacterial-feeding Protozoa and nematodes were on average lower after addition of Holcus than Plantago or Lotus litter, N uptake was higher from Holcus litter. Further, although the effects on Protozoa and bacterial- and fungal-feeding nematodes did not differ between the live plants, litter-N uptake differed, with Holcus being the most efficient compared to Plantago and Lotus. Hence, although microbes and their feeders unquestionably control N mineralization in the soil, and their growth differs among plant species, these differences cannot predict differences in litter-N uptake among plant species. A likely reason is that for nutrient uptake, other species-specific plant traits, such as litter chemistry, root proliferation ability and competitiveness for soil N, override in significance the species-specific ability of plants to induce decomposer growth.

Potential contribution of natural enemies to patterns of local adaptation in plants.

Genetic differentiation among plant populations and adaptation to local environmental conditions are well documented. However, few studies have examined the potential contribution of plant antagonists, such as insect herbivores and pathogens, to the pattern of local adaptation. Here, a reciprocal transplant experiment was set up at three sites across Europe using two common plant species, Holcus lanatus and Plantago lanceolata. The amount of damage by the main above-ground plant antagonists was measured: a rust fungus infecting Holcus and a specialist beetle feeding on Plantago, both in low-density monoculture plots and in competition with interspecific neighbours. Strong genetic differentiation among provenances in the amount of damage by antagonists in both species was found. Local provenances of Holcus had significantly higher amounts of rust infection than foreign provenances, whereas local provenances of Plantago were significantly less damaged by the specialist beetle than the foreign provenances. The presence of surrounding vegetation affected the amount of damage but had little influence on the ranking of plant provenances. The opposite pattern of population differentiation in resistance to local antagonists in the two species suggests that it will be difficult to predict the consequences of plant translocations for interactions with organisms of higher trophic levels.

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.

Nutrient availability and atmospheric CO2 partial pressure modulate the effects of nutrient heterogeneity on the size structure of populations in grassland species.

BACKGROUND AND AIMS: Size-asymmetric competition occurs when larger plants have a disproportionate advantage in competition with smaller plants. It has been hypothesized that nutrient heterogeneity may promote it. Experiments testing this hypothesis are inconclusive, and in most cases have evaluated the effects of nutrient heterogeneity separately from other environmental factors. The aim of this study was to test, using populations of Lolium perenne, Plantago lanceolata and Holcus lanatus, two hypotheses: (a) nutrient heterogeneity promotes size-asymmetric competition; and (b) nutrient heterogeneity interacts with both atmospheric CO2 partial pressure (P(CO2)) and nutrient availability to determine the magnitude of this response. METHODS: Microcosms consisting of monocultures of the three species were grown for 90 d in a factorial experiment with the following treatments: P(CO2) (37.5 and 70 Pa) and nutrient availability (NA; 40 and 120 mg of N added as organic material) combined with different spatial distribution of the organic material (NH; homogeneous and heterogeneous). Differences in the size of individual plants within populations (size inequality) were quantified using the coefficient of variation of individual above-ground biomass and the combined biomass of the two largest individuals in each microcosm. Increases in size inequality were associated with size-asymmetric competition. KEY RESULTS: Size inequality increased when the nutrients were heterogeneously supplied in the three species. The effects of NH on this response were more pronounced under high nutrient supply in both Plantago and Holcus (significant NA x NH interactions) and under elevated P(CO2) in Plantago (significant P(CO2) x NA x NH interaction). No significant two- and three-way interactions were found for Lolium. CONCLUSIONS: Our first hypothesis was supported by our results, as nutrient heterogeneity promoted size-asymmetric competition in the three species evaluated. Nutrient supply and P(CO2) modified the magnitude of this effect in Plantago and Holcus, but not in Lolium. Thus, our second hypothesis was partially supported.

Effects of gamma irradiation on Holcus lanatus (Yorkshire fog grass) and associated soil microorganisms.

An investigation was conducted to determine the impact of acute doses of gamma radiation on the microbial community structure of a Holcus lanatus dominated grassland soil. Mesocosms containing soil and established grass were irradiated using a sealed (137)Cs source (7.0 Gy min(-1)). Doses ranged from 5 to 160 Gy, analyses were conducted on the day of irradiation, then 7 and 30 days later. Plant growth and arbuscular mycorrhizal fungal colonisation of roots were reduced by irradiation. Gram-negative bacteria, and microbial metabolic capacity were also negatively affected by treatment. Microbial biomass measured by phospholipid fatty acid (PLFA) analysis, showed an increase at doses above 20 Gy, 7 and 30 days after treatment. Proportions of Gram-positive bacterial and fungal PLFAs fluctuated inversely to each other, in response to both sampling time and radiation dose. We hypothesise that many of the observed soil microbial responses are indirect effects mediated by the influence of ionising radiation on the plants in this system.

Impact of temperature on the arbuscular mycorrhizal (AM) symbiosis: growth responses of the host plant and its AM fungal partner.

The growth response of the hyphae of mycorrhizal fungi has been determined, both when plant and fungus together and when only the fungus was exposed to a temperature change. Two host plant species, Plantago lanceolata and Holcus lanatus, were grown separately in pots inoculated with the mycorrhizal fungus Glomus mosseae at 20/18 degrees C (day/night); half of the pots were then transferred to 12/10 degrees C. Plant and fungal growth were determined at six sequential destructive harvests. A second experiment investigated the direct effect of temperature on the length of the extra-radical mycelium (ERM) of three mycorrhizal fungal species. Growth boxes were divided in two equal compartments by a 20 micro m mesh, allowing only the ERM and not roots to grow into a fungal compartment, which was either heated (+8 degrees C) or kept at ambient temperature. ERM length (LERM) was determined on five sampling dates. Growth of H. lanatus was little affected by temperature, whereas growth of P. lanceolata increased with temperature, and both specific leaf area (SLA) and specific root length (SRL) increased independently of plant size. Percentage of colonized root (LRC) and LERM were positively correlated with temperature when in symbiosis with P. lanceolata, but differences in LRC were a function of plant biomass. Colonization was very low in H. lanatus roots and there was no significant temperature effect. In the fungal compartment LERM increased over time and was greatest for Glomus mosseae. Heating the fungal compartment significantly increased LERM in two of the three species but did not affect LRC. However, it significantly increased SRL of roots in the plant compartment, suggesting that the fungus plays a regulatory role in the growth dynamics of the symbiosis. These temperature responses have implications for modelling carbon dynamics under global climate change.

The distribution of arsenate and arsenite in shoots and roots of Holcus lanatus is influenced by arsenic tolerance and arsenate and phosphate supply.

The recent discovery that phytochelatins are important for arsenic (As) detoxification in terrestrial plants results in the necessity to understand As speciation and metabolism in plant material. A hydroponic study was therefore conducted to examine the effects of different levels of phosphate and arsenate [As(V)] on As speciation and distribution in tolerant and non-tolerant clones of Holcus lanatus. Speciation of As in tissue (using high-performance liquid chromatography-inductively coupled plasma mass spectrometry) revealed that the predominant species present were the inorganic As species (As(V) and arsenite [As(III)]), although small levels (<1%) of organic As species (dimethylarsinic acid and monomethylarsonic acid) were detected in shoot material. In roots, the proportion of total As present as As(III) generally increased with increasing levels of As(V) in the nutrient solution, whereas in shoots, the proportion of total As present as As(III) generally decreased with increasing levels of As(V). H. lanatus plants growing in the high-phosphorus (P) (100 micro M) solution contained a higher proportion of As(V) (with regard to total As) in both roots and shoots than plants supplied with low P (10 micro M); in addition, tolerant clones generally contained a higher proportion of As(V) with regard to total As than non-tolerant clones. The study further revealed that As(V) can be reduced to As(III) in both roots and shoots. Although the reduction capacity was limited, the reduction was closely regulated by As influx for all treatments. The results therefore provide a new understanding about As metabolism in H. lanatus.

Ameliorating effects of industrial sugar residue on the Jales gold mine spoil (NE Portugal) using Holcus lanatus and Phaseolus vulgaris as indicators.

Phytostabilisation of bare heavily contaminated substrate, such as abandoned mine sites, is considered a very appropriate technology in order to diminish erosion and dispersion of contaminants into the surroundings. In this short-term pot study, application of industrial sugar residue (ISR), a waste product of the sugar industry, proved to ameliorate spoils conditions for plant performance by elevating pH and immobilising several metals. Although arsenate concentrations were positively correlated to spoil pH and spoil treatment with ISR mobilised As, growth of both Phaseolus vulgaris and Holcus lanatus improved significantly after applications of 3.75 g ISR kg(-1) dry spoil. Nutrient uptake from the substrate, with the exception of potassium, was elevated by ISR. As a remediation technique ISR application could be effective although in As-contaminated sites application might be restricted to areas where leaching to (ground) water does not form a risk.