Heavy-ion beam mutagenesis of the ectomycorrhizal agaricomycete Tricholoma matsutake that produces the prized mushroom "matsutake" in conifer forests.

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces the prized mushroom "matsutake" in Pinaceae forests. Currently, there are no available cultivars or cultivation methods that produce fruiting bodies. Heavy-ion beams, which induce mutations through double-stranded DNA breaks, have been used widely for plant breeding. In the present study, we examined whether heavy-ion beams could be useful in isolating T. matsutake mutants. An argon-ion beam gave a suitable lethality curve in relation to irradiation doses, accelerating killing at 100-150 Gy. Argon-ion beam irradiation of the agar plate cultures yielded several transient mutants whose colony morphologies differed from that of the wild-type strain at the first screening, but which did not persist following culture transfer. It also generated a mutant whose phenotype remained stable after repeated culture transfers. The stable pleiotropic mutant not only exhibited a different colony morphology to the wild type, but also showed increased degradation of dye-linked water-insoluble amylose and cellulose substrates. Thus, heavy-ion beams may be useful for isolating mutants of T. matsutake, although precautions may be required to maintain the mutants, without phenotypic reversion, during repetitive culture of their mycelia.

Rhizophagus irregularis MUCL 41833 can colonize and improve P uptake of Plantago lanceolata after exposure to ionizing gamma radiation in root organ culture.

Long-lived radionuclides such as (90)Sr and (137)Cs can be naturally or accidentally deposited in the upper soil layers where they emit ß/γ radiation. Previous studies have shown that arbuscular mycorrhizal fungi (AMF) can accumulate and transfer radionuclides from soil to plant, but there have been no studies on the direct impact of ionizing radiation on AMF. In this study, root organ cultures of the AMF Rhizophagus irregularis MUCL 41833 were exposed to 15.37, 30.35, and 113.03 Gy gamma radiation from a (137)Cs source. Exposed spores were subsequently inoculated to Plantago lanceolata seedlings in pots, and root colonization and P uptake evaluated. P. lanceolata seedlings inoculated with non-irradiated AMF spores or with spores irradiated with up to 30.35 Gy gamma radiation had similar levels of root colonization. Spores irradiated with 113.03 Gy gamma radiation failed to colonize P. lanceolata roots. P content of plants inoculated with non-irradiated spores or of plants inoculated with spores irradiated with up to 30.35 Gy gamma radiation was higher than in non-mycorrhizal plants or plants inoculated with spores irradiated with 113.03 Gy gamma radiation. These results demonstrate that spores of R. irregularis MUCL 41833 are tolerant to chronic ionizing radiation at high doses.

Interactive effects of juvenile defoliation, light conditions, and interspecific competition on growth and ectomycorrhizal colonization of Fagus sylvatica and Pinus sylvestris seedlings.

Seedlings of forest tree species are exposed to a number of abiotic (organ loss or damage, light shortage) and biotic (interspecific competition) stress factors, which may lead to an inhibition of growth and reproduction and, eventually, to plant death. Growth of the host and its mycorrhizal symbiont is often closely linked, and hence, host damage may negatively affect the symbiont. We designed a pot experiment to study the response of light-demanding Pinus sylvestris and shade-tolerant Fagus sylvatica seedlings to a set of abiotic and biotic stresses and subsequent effects on ectomycorrhizal (ECM) root tip colonization, seedling biomass, and leaf nitrogen content. The light regime had a more pronounced effect on ECM colonization than did juvenile damage. The interspecific competition resulted in higher ECM root tip abundance for Pinus, but this effect was insignificant in Fagus. Low light and interspecific competition resulted in lower seedling biomass compared to high light, and the effect of the latter was partially masked by high light. Leaf nitrogen responded differently in Fagus and Pinus when they grew in interspecific competition. Our results indicated that for both light-demanding (Pinus) and shade-tolerant (Fagus) species, the light environment was a major factor affecting seedling growth and ECM root tip abundance. The light conditions favorable for the growth of seedlings may to some extent compensate for the harmful effects of juvenile organ loss or damage and interspecific competition.

Exploring the transfer of recent plant photosynthates to soil microbes: mycorrhizal pathway vs direct root exudation.

Plants rapidly release photoassimilated carbon (C) to the soil via direct root exudation and associated mycorrhizal fungi, with both pathways promoting plant nutrient availability. This study aimed to explore these pathways from the root's vascular bundle to soil microbial communities. Using nanoscale secondary ion mass spectrometry (NanoSIMS) imaging and (13) C-phospho- and neutral lipid fatty acids, we traced in-situ flows of recently photoassimilated C of (13) CO2 -exposed wheat (Triticum aestivum) through arbuscular mycorrhiza (AM) into root- and hyphae-associated soil microbial communities. Intraradical hyphae of AM fungi were significantly (13) C-enriched compared to other root-cortex areas after 8 h of labelling. Immature fine root areas close to the root tip, where AM features were absent, showed signs of passive C loss and co-location of photoassimilates with nitrogen taken up from the soil solution. A significant and exclusively fresh proportion of (13) C-photosynthates was delivered through the AM pathway and was utilised by different microbial groups compared to C directly released by roots. Our results indicate that a major release of recent photosynthates into soil leave plant roots via AM intraradical hyphae already upstream of passive root exudations. AM fungi may act as a rapid hub for translocating fresh plant C to soil microbes.

Are carbon and nitrogen exchange between fungi and the orchid Goodyera repens affected by irradiance?

BACKGROUND AND AIMS: The green orchid Goodyera repens has been shown to transfer carbon to its mycorrhizal partner, and this flux may therefore be affected by light availability. This study aimed to test whether the C and N exchange between plant and fungus is dependent on light availability, and in addition addressed the question of whether flowering and/or fruiting individuals of G. repens compensate for changes in leaf chlorophyll concentration with changes in C and N flows from fungus to plant. METHODS: The natural abundances of stable isotopes of plant C and N were used to infer changes in fluxes between orchid and fungus across natural gradients of irradiance at five sites. Mycorrhizal fungi in the roots of G. repens were identified by molecular analyses. Chlorophyll concentrations in the leaves of the orchid and of reference plants were measured directly in the field. KEY RESULTS: Leaf δ(13)C values of G. repens responded to changes in light availability in a similar manner to autotrophic reference plants, and different mycorrhizal fungal associations also did not affect the isotope abundance patterns of the orchid. Flowering/fruiting individuals had lower leaf total N and chlorophyll concentrations, which is most probably explained by N investments to form flowers, seeds and shoot. CONCLUSIONS: The results indicate that mycorrhizal physiology is relatively fixed in G. repens, and changes in the amount and direction of C flow between plant and fungus were not observed to depend on light availability. The orchid may instead react to low-light sites through increased clonal growth. The orchid does not compensate for low leaf total N and chlorophyll concentrations by using a (13)C- and (15)N-enriched fungal source.

Positive diversity-invasibility relationship in species-rich semi-natural grassland at the neighbourhood scale.

BACKGROUND AND AIMS: Attempts to answer the old question of whether high diversity causes high invasion resistance have resulted in an invasion paradox: while large-scale studies often find a positive relationship between diversity and invasibility, small-scale experimental studies often find a negative relationship. Many of the small-scale studies are conducted in artificial communities of even-aged plants. Species in natural communities, however, do not represent one simultaneous cohort and occur at various levels of spatial aggregation at different scales. This study used natural patterns of diversity to assess the relationship between diversity and invasibility within a uniformly managed, semi-natural community. METHODS: In species-rich grassland, one seed of each of ten species was added to each of 50 contiguous 16 cm(2) quadrats within seven plots (8 × 100 cm). The emergence of these species was recorded in seven control plots, and establishment success was measured in relation to the species diversity of the resident vegetation at two spatial scales, quadrat (64 cm(2)) within plots (800 cm(2)) and between plots within the site (approx. 400 m(2)) over 46 months. KEY RESULTS: Invader success was positively related to resident species diversity and richness over a range of 28-37 species per plot. This relationship emerged 7 months after seed addition and remained over time despite continuous mortality of invaders. CONCLUSIONS: Biotic resistance to plant invasion may play only a sub-ordinate role in species-rich, semi-natural grassland. As possible alternative explanations for the positive diversity-invasibility relationship are not clear, it is recommended that future studies elaborate fine-scale environmental heterogeneity in resource supplies or potential resource flows from resident species to seedlings by means of soil biological networks established by arbuscular mycorrhizal fungi.

Exposure to ionizing radiation affects the growth of ectomycorrhizal fungi and induces increased melanin production and increased capacities of reactive oxygen species scavenging enzymes.

Ectomycorrhizal (EM) fungi form symbioses with dominant tree families in boreal, temperate and tropical ecosystems and are important drivers of ecosystem function. EM fungal hyphae extend over a large area making them susceptible to enhanced radiation levels from naturally occurring or anthropogenically originating radioisotopes in the rhizosphere. In this study, the in-vitro effects of ionizing radiation on the growth and biomass of EM fungi Suillus luteus, S. bovinus and Rhizopogon luteolus were investigated. EM fungal cultures were exposed to gamma radiation from a 137Cs source for 137 h in darkness at 21 °C at dose rates of 404, 108.5 and 54.9 mGy h-1 resulting in total absorbed doses of 55.21, 14.82 and 7.50 Gy respectively. Cultures grown in the dark at 21 °C but not exposed to the 137Cs source served as the control. Our results show that EM fungi vary in their sensitivity to ionizing radiation. EM fungi used in this study produced melanin and reactive oxygen species scavenging enzymes such as catalase and superoxide dismutase as a response to ionizing radiation.

CASIROZ: Root parameters and types of ectomycorrhiza of young beech plants exposed to different ozone and light regimes.

Tropospheric ozone (O(3)) triggers physiological changes in leaves that affect carbon source strength leading to decreased carbon allocation below-ground, thus affecting roots and root symbionts. The effects of O(3) depend on the maturity-related physiological state of the plant, therefore adult and young forest trees might react differently. To test the applicability of young beech plants for studying the effects of O(3) on forest trees and forest stands, beech seedlings were planted in containers and exposed for two years in the Kranzberg forest FACOS experiment (Free-Air Canopy O(3) Exposure System, http://www.casiroz.de ) to enhanced ozone concentration regime (ambient [control] and double ambient concentration, not exceeding 150 ppb) under different light conditions (sun and shade). After two growing seasons the biomass of the above- and below-ground parts, beech roots (using WinRhizo programme), anatomical and molecular (ITS-RFLP and sequencing) identification of ectomycorrhizal types and nutrient concentrations were assessed. The mycorrhization of beech seedlings was very low ( CA. 5 % in shade, 10 % in sun-grown plants), no trends were observed in mycorrhization (%) due to ozone treatment. The number of Cenococcum geophilum type of ectomycorrhiza, as an indicator of stress in the forest stands, was not significantly different under different ozone treatments. It was predominantly occurring in sun-exposed plants, while its majority share was replaced by Genea hispidula in shade-grown plants. Different light regimes significantly influenced all parameters except shoot/root ratio and number of ectomycorrhizal types. In the ozone fumigated plants the number of types, number of root tips per length of 1 to 2 mm root diameter, root length density per volume of soil and concentration of Mg were significantly lower than in control plants. Trends to a decrease were found in root, shoot, leaf, and total dry weights, total number of root tips, number of vital mycorrhizal root tips, fine root (mass) density, root tip density per surface, root area index, concentration of Zn, and Ca/Al ratio. Due to the general reduction in root growth indices and nutrient cycling in ozone-fumigated plants, alterations in soil carbon pools could be predicted.

Caesium inhibits the colonization of Medicago truncatula by arbuscular mycorrhizal fungi.

Contamination of soils with radioisotopes of caesium (Cs) is of concern because of their emissions of harmful ß and γ radiation. Radiocaesium enters the food chain through vegetation and the intake of Cs can affect the health of organisms. Arbuscular mycorrhizal (AM) fungi form mutualistic symbioses with plants through colonization of the roots and previous studies on the influence of AM on Cs concentrations in plants have given inconsistent results. These studies did not investigate the influence of Cs on AM fungi and it is therefore not known if Cs has a direct effect on AM colonization. Here, we investigated whether Cs influences AM colonization and if this effect impacts on the influence of Rhizophagus intraradices on Cs accumulation by Medicago truncatula. M. truncatula was grown with or without R. intraradices in pots containing different concentrations of Cs. Here, we present the first evidence that colonization of plants by AM fungi can be negatively affected by increasing Cs concentrations in the soil. Mycorrhizal colonization had little effect on root or shoot Cs concentrations. In conclusion, the colonization by AM fungi is impaired by high Cs concentrations and this direct effect of soil Cs on AM colonization might explain the inconsistent results reported in literature that have shown increased, decreased or unaffected Cs concentrations in AM plants.

Effects of flower dimorphism and light environment on arbuscular mycorrhizal colonisation in a cleistogamous herb.

Although it is known that floral dimorphism contributes to the maintenance of mixed breeding systems, the consequences of producing progeny of a contrasting genetic background and seeds with differential resource allocation has been practically ignored regarding establishment of belowground organisms-plant interactions. This article evaluates the combined effect of floral dimorphism with cross type and light environment on interactions between Ruellia nudiflora and arbuscular mycorrhizal fungi (AMF). R. nudiflora produces cleistogamous (CL) flowers that exhibit obligate self-pollination and chasmogamous (CH) flowers with facultative self- (CHs) or cross- (CHc) pollination. We evaluated the establishment of the plant-AMF interaction in progeny derived from each floral type, under two light conditions (shaded versus open). We established different scenarios depending on the existence of inbreeding depression (ID) and whether the differential resource allocation (DRA) to CH and CL flowers affected the R. nudiflora-AMF interaction. We predicted that under shaded light conditions there might be an intensification of ID, having a negative effect on AMF colonisation. The percentages of hyphae and vesicles in the harvested roots was significantly higher in the shaded plants (F ≥ 4.11, P < 0.05), while progeny of CHc and CHs presented a higher percentage of hyphae and vesicle colonisation compared to CL progeny (F = 15.26, P < 0.01). The results show that DRA to CH flowers and light availability both determines the establishment of R. nudiflora-AMF interaction. The results also suggest that even under stressful light conditions, endogamy does not affect this interaction, which may explain the success of R. nudiflora as an invasive species.

Reducing the infectivity and richness of ectomycorrhizal fungi in a calcareous Quercus ilex forest through soil preparations for truffle plantation establishment: A bioassay study.

In the early years of a black truffle plantation, the field proliferation of the nursery-inoculated fungi can be hampered by native ectomycorrhizal fungi colonising the seedling roots. Reducing the soil ectomycorrhizal infectivity in the planting hole before introducing the inoculated seedling could be an effective strategy to reduce this problem. Three bioassays were conducted to evaluate the impact of several soil preparations on the ectomycorrhizal infectivity and richness of a Quercus ilex soil in a truffle-producing region. Microwaves, quicklime, and acetic acid significantly decreased the percent root colonisation and morphotype richness of the native ectomycorrhizal fungi. However, they also decreased seedling survival or growth. Peracetic acid, hydrogen peroxide, and sodium hypochlorite did not show a significant negative effect on the soil ectomycorrhizal community. The results support the potential of soil preparation for reducing the ectomycorrhizal infectivity of forest soils, thus being a promising strategy to reduce the early colonisation by native fungi in truffle plantations. However, the indications of damage to the seedling development must be addressed.

Response of terrestrial microorganisms to ultraviolet-B radiation in ecosystems.

Recent studies suggest that ultraviolet-B radiation, a major component of global climate change, can affect the community structure, biomass and functioning of terrestrial microorganisms. In this report, these studies are reviewed and gaps in our understanding highlighted so that future research can provide more conclusive evidence on the mechanisms and wider ecological implications of the impacts of elevated ultraviolet-B radiation on terrestrial microorganisms.

High temperature effect on microflora of radish root-inhabited zone and nutrient solutions for radish growth.

The effect of high temperatures (35 and 45 degrees C) on microflora of the root zone of radish plants grown in phytotron was evaluated by the response of microorganisms from 9 indicator groups. Phytotron air temperature elevated to 35 degrees C for 20 hours caused no significant changes in qualitative and quantitative composition of the root microflora in experimental plants. By the end of the experiment, the species diversity of microflora had changed. The amount of phytopathogenic microorganisms decreased which can be interpreted as more stable co-existence of microflora with plants. The numbers of microbes from other indicator groups was in dynamic equilibrium. The plants' condition did not deteriorate either. Exposure to the temperature of 45 degrees C for 7 hours have been found to change the numbers and species diversity in the radish root zone microflora. The microorganisms were observed to increase their total numbers at the expense of certain indicator groups. Bacteria increased spore forms at the stage of spores. Colon bacillus bacteria of increased their numbers by the end of experiment by an order. By the end of experiment the roots of experiment plants had microscopic fungi from Mucor, Aspergillus, Trichoderma, Cladosporium genera. The observed changes in the microbial complex seem to be associated with the changes of root emissions and general deterioration of the plants' condition. It is suggested that the response of the microorganisms can be indicative of the condition of plants under investigation.

Mycorrhizal symbiosis effects on growth of chalk false-brome (Brachypodium pinnatum) are dependent on the environmental light regime.

AMF (arbuscular mycorrhizal fungi) colonization of the grass chalk false-brome (Brachypodium pinnatum (L.) P. B.) was studied in selected habitats under spatially different light regimes: (a) shade condition under oak trees, (b) half shade in a shrubby area and (c) full-sun conditions on unshaded grassland. This study assessed the variations in AMF colonization of the grass dependent on the light supply in field habitats. Soil, root and shoot samples were collected four times during the vegetation period (in June, July, September and October). Root colonization, root and shoot biomass as well as soil water content were determined. The highest rate of AMF colonization was detected in June under half-sun and full-sun conditions, where about 50% of the roots were colonized. The average amount of arbuscules was less than 20% in the roots at the three sites, with the highest number of arbuscules in June, under half-sun and full-sun conditions, however, not under the trees. Overall, best mycorrhizal colonization occurred during summer, and its rate decreased in autumn. This tendency inversely correlated with the amount of precipitation, and thus with the water content of soils. The high colonization rate of the examined root samples, and also its seasonal fluctuation, might reflect the importance of the symbiosis where inorganic nutrients and water are the growth-limiting factors. The marginal AMF colonization of chalk false-brome under shade conditions indicates that plants do not use AMF under all stress conditions. When low light limits photosynthesis and thus growth of the plants, they dispense with the colonization of AMF in order to save the expenditure of organic carbon.

Preservation at ultra-low temperature of in vitro cultured arbuscular mycorrhizal fungi via encapsulation-drying.

At present, over 300 species of arbuscular mycorrhizal fungi (AMF) have been identified, most of which being stored in international collections. Their maintenance is mostly achieved in greenhouse via continuous culture on trap plants or in vitro in association with excised root organs. Both methods are work-intensive and for the former present the risk of unwanted contaminations. The in vitro root organ culture of AMF has become an alternative preventing contamination. Nevertheless, the risk for somaclonal variation during the sub-cultivation process cannot be excluded. A method for the long-term conservation that guarantees the stability of the biological material is thus highly demanded to preserve the microorganisms and their genetic stability. Here, 12 AMF isolates cultured in vitro in association with excised carrot roots were encapsulated in alginate beads and subsequently cryopreserved. Several protocols were tested taking into consideration culture age, alginate bead pre-drying, and rate of decrease in temperature. The viability of the AMF isolates was estimated by the percentage of potentially infective beads (%PIB) that measure the % of beads that contain at least one germinated propagule. Thermal behaviour of alginate beads was analysed by a differential thermal calorimeter before and after drying to estimate the frozen and unfrozen water during the cryopreservation process. It was shown that the spore damage was directly related to ice formation during cryopreservation. The encapsulation and culture age were also determinant parameters for the successful cryopreservation. Irrespective of the AMF isolate, the optimal procedure for cryopreservation comprised five steps: (1) the encapsulation of propagules (i.e. spores and mycorrhizal root pieces) isolated from 5m old cultures, (2) the incubation overnight in trehalose (0.5M), (3) the drying during 48h at 27°C, (4) the cryopreservation in the freezer at -130°C following a two-step decrease in temperature: a fast decrease (∼12°Cmin(-1)) from room temperature (+20°C) to -110°C followed by a slow decrease in temperature (∼1°Cmin(-1)) from -110°C to -130°C, and (5) the direct thawing in a water bath (+35°C). The % PIB was above 70 % for all the isolates and even above 95% for 11 out of the 12 isolates after several months of storage at ultra-low temperature. All the isolates kept their capacity to associate to an excised carrot root in vitro and to reproduce the fungal life cycle with the production of several hundreds to thousands of spores after 2m. This method opens the door for the long-term maintenance at ultra-low temperature of AMF isolates within international repositories.

Métodos físicos y químicos para la eliminación de propágulos de hongos formadores de micorrizas nativos de muestras de suelo / Physical and chemical methods for eliminating propagules of indigenous mycorrhizal fungi from soil samples

El objetivo de este trabajo fue evaluar métodos para eliminar hongos nativos formadores de micorrizas arbusculares (HMA) o reducir su número en muestras de suelo, sin afectar sus propiedades edáficas y microbiológicas. Se estudió la aplicación de calor húmedo (autoclave), de calor seco (estufa), de hipoclorito de sodio (NaClO) y de formaldehído, en concentraciones entre 100,0 y 3,3 µl/g y 16,7 y 3,3 µl/g, respectivamente. Las semillas de raigrás (Lolium multiflorum Lam.) sembradas en sustratos que recibieron NaClO (100,0-33,3 µl/g) no germinaron y el autoclave incrementó el contenido de fósforo en el sustrato. Estos tratamientos no eliminaron la micorrización por HMA y ambos fueron descartados. En un segundo ensayo se analizaron los tratamientos estufa y formaldehído (10,0 µl/g), asociados o no a la descontaminación de las semillas y a la reinoculación con HMA. Ambos procedimientos redujeron o eliminaron la micorrización por HMA nativos en suelos con 12 a 29 mg/kg de fósforo y permitieron la multiplicación de inóculos de HMA. El tiempo de ventilación de las muestras y los requisitos de seguridad fueron mayores con la aplicación de formaldehído

The objective of this work was to evaluate methods to eliminate or reduce the number of indigenous arbuscular mycorrhizal fungi (AMF) from soil samples without affecting their edaphic or microbiological properties. At an early trial we evaluated moist heat (autoclaving), dry heat (oven), sodium hypochlorite (NaClO) and formaldehyde at a range of 100.0-3.3 µl/g and 16.7-3.3 µl/g respectively. There was no germination in plants of ryegrass (Lolium multiflorum Lam.) sown on substrates receiving NaClO (100.0-33.3 ul/g), whereas autoclaving significantly increased the available soil phosphorous content. Both treatments failed to eradicate AMF colonization at 9 weeks; therefore, they were discarded. In a second trial, oven and formaldehyde (10.0 µl/g) treatments were analyzed to assess the effects of seed decontamination and AMF reinoculation. Both procedures were effective in reducing or eliminating indigenous AMF at a range of soil P availability of 12-29 mg/kg. However, the time between soil treatment and AMF multiplication and safety requirements were greater in the case of formaldehyde application

Action spectrum for the induction of hyphal branches of an arbuscular mycorrhizal fungus: exposure sites versus branching sites.

The first action spectrum for a photo-induced response of an arbuscular mycorrhizal fungus is reported. At low light intensity, the responsive wavelengths for light-induced hyphal branching of the primary germ tube of Gigaspora gigantea were determined to be in the blue to uv-A range. The action spectrum showed the greatest stimulation of branching occurred around 390 nm although a shoulder was observed between 360-370 nm. A second major peak of light-induced branching occurred at 430 nm. The exposure of specific areas of the germ tube to high intensity blue light for a short period led to several interesting observations. By exposing 2 mm segments (0-2, 2-4, 4-6, etc.) or 3 mm segments away from the tip, it was determined that photoinduction of hyphal branches could occur anywhere along the axis of a growing germ tube except in the apical 2 mm. When 3 mm segments were exposed at greater distances from the tip (6-9, 9-12, and up to 33-36 mm), branches frequently formed in areas not directly exposed to light. The branches were usually in clusters which were spaced approximately 3 or 6 mm apart. Since light scattering could be ruled out, these results indicated that the exposure sites and sites of hyphal branching did not necessarily coincide and suggested the probable involvement of a second messenger during this blue light-induced event.