Plant growth promotion of Miscanthus × giganteus by endophytic bacteria and fungi on non-polluted and polluted soils.

Putative endophytes of Miscanthus × giganteus were isolated, and screened in the laboratory, greenhouse and field for their plant growth promoting properties in this host. Pantoea ananatis and Pseudomonas savastanoi were the predominant bacteria in leaves whereas other pseudomonads prevailed in roots. Almost all fungal endophytes belonged to the Pezizomycotina and most were isolated from roots; Fusarium oxysporum was most abundant, followed by the genera Periconia, Exophiala, Microdochium and Leptodontidium. All endophytic groups produced phytohormones and some bacteria also produced siderophores, solubilised P and exhibited ACC-deaminase activity in vitro. In subsequent pot experiments with pre-selected endophytes, several isolates including pseudomonads, Variovorax paradoxus, Verticillium leptobactrum, Halenospora sp. and Exophiala sp. enhanced Miscanthus growth in gamma-sterilised soil. These promising Miscanthus-derived isolates were tested either as single or mixed inocula along with a mixed bacterial inoculum originating from poplar. No significant effects of inocula were detected in a pot experiment in non-sterilised soil. On two marginal field sites the mixture of bacterial endophytes from poplar had a consistently negative effect on survival and growth of Miscanthus. Contrarily, mixtures consisting of bacteria or fungi originating from Miscanthus promoted growth of their host, especially on the heavy metals-polluted site. The combination of bacteria and fungi was inferior to the mixtures consisting of bacteria or fungi alone. Our observations indicate extensive potential of mixed bacterial and fungal endophytic inocula to promote establishment and yield of Miscanthus grown on marginal and polluted land and emphasise the necessity to test particular microbial-plant host combinations. Morphotypes of fungi isolates from Miscanthus × giganteus.

Combined effects of fungal inoculants and the cytokinin-like growth regulator thidiazuron on growth, phytohormone contents and endophytic root fungi in Miscanthus × giganteus.

Aim of this study was to investigate main effects and interactions between symbiotic fungi and the cytokinin-like growth regulator thidiazuron (TDZ) in Miscanthus × giganteus. The arbuscular mycorrhiza fungus Rhizophagus intraradices (AMF) and the endophyte Piriformospora indica (PI) were chosen as model symbionts. The fungal inoculants and TDZ had no significant effect on plant growth but modulated phytohormone levels in the leaves. TDZ induced accumulation of salicylic acid in controls, but not in plants inoculated with fungi. Leaf concentrations of abscisic acid (ABA) derivatives, auxin (indole-3-acetic acid) precursors and catabolites and numerous cytokinins were increased by R. intraradices but lowered by P. indica. TDZ raised concentrations of ABA compounds, the non-indole auxin phenylacetic acid, jasmonate and some cytokinins, but decreased cis-zeatin and N6-(Δ2-isopentenyl)adenine levels. Inoculation with AMF reduced abundance of endogenous clampless endophytes. TDZ application strongly reduced formation of arbuscular mycorrhiza and increased occurrence of clamped mycelia (i.e. basidiomycetous endophytes). Our study provides a thorough outline of the phytohormone homeostasis under the combined influence of beneficial inoculants and a growth regulator, highlighting the necessity to study their interaction in the whole plant-microbial context.

Selenium hyperaccumulators harbor a diverse endophytic bacterial community characterized by high selenium resistance and plant growth promoting properties.

Selenium (Se)-rich plants may be used to provide dietary Se to humans and livestock, and also to clean up Se-polluted soils or waters. This study focused on endophytic bacteria of plants that hyperaccumulate selenium (Se) to 0.5-1% of dry weight. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to compare the diversity of endophytic bacteria of hyperaccumulators Stanleya pinnata (Brassicaceae) and Astragalus bisulcatus (Fabaceae) with those from related non-accumulators Physaria bellii (Brassicaceae) and Medicago sativa (Fabaceae) collected on the same, seleniferous site. Hyperaccumulators and non-accumulators showed equal T-RF diversity. Parsimony analysis showed that T-RFs from individuals of the same species were more similar to each other than to those from other species, regardless of plant Se content or spatial proximity. Cultivable endophytes from hyperaccumulators S. pinnata and A. bisulcatus were further identified and characterized. The 66 bacterial morphotypes were shown by MS MALDI-TOF Biotyper analysis and 16S rRNA gene sequencing to include strains of Bacillus, Pseudomonas, Pantoea, Staphylococcus, Paenibacillus, Advenella, Arthrobacter, and Variovorax. Most isolates were highly resistant to selenate and selenite (up to 200 mM) and all could reduce selenite to red elemental Se, reduce nitrite and produce siderophores. Seven isolates were selected for plant inoculation and found to have plant growth promoting properties, both in pure culture and when co-cultivated with crop species Brassica juncea (Brassicaceae) or M. sativa. There were no effects on plant Se accumulation. We conclude that Se hyperaccumulators harbor an endophytic bacterial community in their natural seleniferous habitat that is equally diverse to that of comparable non-accumulators. The hyperaccumulator endophytes are characterized by high Se resistance, capacity to produce elemental Se and plant growth promoting properties.

The impact of different grazing periods in dry grasslands on the expansive grass Arrhenatherum elatius L. and on woody species.

Dry grasslands are one of the most species rich and endangered types of vegetation in Europe. In the Czech Republic, dry grasslands are mainly of anthropogenic origin and were formed as a result of grazing after the clear-cutting of thermophilous oak woods. Gradual changes in the farming landscape throughout the 20th century, particularly in the 1960s, resulted in the abandonment of the relatively infertile habitats of dry grasslands. After abandonment, dry grasslands decline and degrade due to the gradual overgrowth of woody species and expansion of perennial tall grasses. In the year 2000, a grazing management program was introduced in the protected areas within the territory of Prague City to maintain the species diversity of dry grasslands. The responses of the expansive grass species, Arrhenatherum elatius L. and multiple woody species (especially, Prunus spinosa L.) to differences in grazing periods were monitored for over a decade. Grazing in spring through the end of June had the greatest impact on the reduction of A. elatius and woody species. Grazing in the height of summer through autumn did not reduce the cover of these plants, and may support the prosperity of both A. elatius and the woody species due to higher levels of nutrients.

Effect of clone selection, nitrogen supply, leaf damage and mycorrhizal fungi on stilbene and emodin production in knotweed.

BACKGROUND: Fallopia japonica and its hybrid, F. xbohemica, due to their fast spread, are famous as nature threats rather than blessings. Their fast growth rate, height, coverage, efficient nutrient translocation between tillers and organs and high phenolic production, may be perceived either as dangerous or beneficial features that bring about the elimination of native species or a life-supporting source. To the best of our knowledge, there have not been any studies aimed at increasing the targeted production of medically desired compounds by these remarkable plants. We designed a two-year pot experiment to determine the extent to which stilbene (resveratrol, piceatannol, resveratrolosid, piceid and astringins) and emodin contents of F. japonica, F. sachalinensis and two selected F. xbohemica clones are affected by soil nitrogen (N) supply, leaf damage and mycorrhizal inoculation. RESULTS: 1) Knotweeds are able to grow on substrates with extremely low nitrogen content and have a high efficiency of N translocation. The fast-spreading hybrid clones store less N in their rhizomes than the parental species. 2) The highest concentrations of stilbenes were found in the belowground biomass of F. japonica. However, because of the high belowground biomass of one clone of F. xbohemica, this hybrid produced more stilbenes per plant than F. japonica. 3) Leaf damage increased the resveratrol and emodin contents in the belowground biomass of the non-inoculated knotweed plants. 4) Although knotweed is supposed to be a non-mycorrhizal species, its roots are able to host the fungi. Inoculation with mycorrhizal fungi resulted in up to 2% root colonisation. 5) Both leaf damage and inoculation with mycorrhizal fungi elicited an increase of the piceid (resveratrol-glucoside) content in the belowground biomass of F. japonica. However, the mycorrhizal fungi only elicited this response in the absence of leaf damage. Because the leaf damage suppressed the effect of the root fungi, the effect of leaf damage prevailed over the effect of the mycorrhizal fungi on the piceid content in the belowground biomass. CONCLUSIONS: Two widely spread knotweed species, F. japonica and F. xbohemica, are promising sources of compounds that may have a positive impact on human health. The content of some of the target compounds in the plant tissues can be significantly altered by the cultivation conditions including stress imposed on the plants, inoculation with mycorrhizal fungi and selection of the appropriate plant clone.

Factors influencing the production of stilbenes by the knotweed, Reynoutria x bohemica.

BACKGROUND: Japanese knotweed, Reynoutria japonica, is known for its high growth rate, even on adverse substrates, and for containing organic substances that are beneficial to human health. Its hybrid, Reynoutria x bohemica, was described in the Czech Republic in 1983 and has been widespread ever since. We examined whether Reynoutria x bohemica as a medicinal plant providing stilbenes and emodin, can be cultivated in spoil bank substrates and hence in the coalmine spoil banks changed into arable fields. We designed a pot experiment and a field experiment to assess the effects of various factors on the growth efficiency of Reynoutria x bohemica on clayish substrates and on the production of stilbenes and emodin in this plant. RESULTS: In the pot experiment, plants were grown on different substrates that varied in organic matter and nutrient content, namely the content of nitrogen and phosphorus. Nitrogen was also introduced into the substrates by melilot, a leguminous plant with nitrogen-fixing rhizobia. Melilot served as a donor of mycorrhizal fungi to knotweed, which did not form any mycorrhiza when grown alone. As expected, the production of knotweed biomass was highest on high-nutrient substrates, namely compost. However, the concentration of the organic constituents studied was higher in plants grown on clayish low-nutrient substrates in the presence of melilot. The content of resveratrol including that of its derivatives, resveratrolosid, piceatannol, piceid and astringin, was significantly higher in the presence of melilot on clay, loess and clayCS. Nitrogen supplied to knotweed by melilot was correlated with the ratio of resveratrol to resveratrol glucosides, indicating that knotweed bestowed some of its glucose production upon covering part of the energy demanded for nitrogen fixation by melilot's rhizobia, and that there is an exchange of organic substances between these two plant species. The three-year field experiment confirmed the ability of Reynoutria x bohemica to grow on vast coalmine spoil banks. The production of this species reached 2.6 t of dry mass per hectare. CONCLUSIONS: Relationships between nitrogen, phosphorus, emodin, and belowground knotweed biomass belong to the most interesting results of this study. Compared with melilot absence, its presence increased the number of significant relationships by introducing those of resveratrol and its derivatives, and phosphorus and nitrogen. Knotweed phosphorus was predominantly taken up from the substrate and was negatively correlated with the content of resveratrol and resveratrol derivatives, while knotweed nitrogen was mainly supplied by melilot rhizobia and was positively correlated with the content of resveratrol and resveratrol derivatives.