Fine root dynamics in a Norway spruce forest (Picea abies (L.) Karst) in eastern Sweden

The annual dynamics of live and dead fine roots for trees and the field layer species and live/dead ratios were investigated at a coniferous fern forest (Picea abies L. Karts) in Sweden. Our methods of estimating the average amount of fine roots involved the periodic sampling of fine roots in sequential cores on four sampling occasions. The highest live/dead ratio was found in the upper part of the humus layer for both tree and field-layer species and decreased with depth. Most tree fine roots on the four sampling occasions were found in the mineral soil horizon, where 86, 81, 85 and 89% of <1 mm and 89, 88, 89 and 92% of <2 mm diameter of the total amounts of live fine roots in the soil profile were found. The mean amounts of live fine roots of tree species for the total soil profile on the four sampling occasions was 317, 150, 139 and 248 g m^?2 for <1 mm and 410, 225, 224 and 351 g m^?2 for <2 mm diameter fine roots. The related amount of dead fine roots was 226, 321, 176 and 299 g m^?2 and 294, 424, 282 and 381 g m^?2, respectively. Average amounts of live and dead fine-roots and live/dead ratios from other Picea abies forest ecosystems were within the range of our estimates. The production of fine roots, <1 and <2 mm in diameter, estimated from the annual increments in live fine roots, was 207 and 303 g m^?2. The related accumulation of dead fine roots was 257 and 345 g m^?2, The turnover rate of tree fine roots <1 mm in diameter in the total soil profile amounted to 0.7 yr^?1 for live and 0.8 yr^?1 for dead fine roots. The related turnover rates for tree fine roots <2 mm were 0.4 yr^?1 and 0.7 yr^?1. Our data, although based on minimum estimates of the annual fluxes of live and dead fine roots, suggests a carbon flow to the forest soil from dead fine-roots even more substantial than from the needle litter fall. Fine-root data from several Picea abies forest ecosystems, suggest high turnover rates of both live and dead tree fine-roots.     

Plant neighbor effects mediated by rhizosphere factors along a simulated aridity gradient

Aims

We investigated how rhizosphere factors (total rhizosphere, roots, arbuscular mycorrhizal fungal hyphae [AMF], and soil solution) and water availability affect interactions between neighboring Medicago sativa plants.

Methods

A three-compartment mesocosm was used to test the effects of rhizosphere factors on plant–plant interactions. A relative interaction index (RII) was calculated to indicate whether effects of neighbor plant on target plant were positive or negative (facilitative or competitive). Isotope tracers were used to test whether AMF hyphae mediated competition for nitrogen (N) between target and neighbor plants.

Results

The effects of rhizosphere factors on the interactions between neighboring M. sativa plants depended on water availability. The effects of total rhizosphere shifted RII from negative to positive as water availability increased. Interaction with the roots and rhizosphere soil solution of neighbor plants shifted RII from negative to positive as water availability increased but the opposite was true for AMF hyphae. AMF hyphae helped neighbor plants compete for ¹⁵N when water was available but not when water was limiting.

Conclusions

The effect of total rhizosphere on plant–plant interaction of M. sativa shifted from competitive to facilitative as water availability increased. Competition was reduced by neighboring soil solution and roots but was increased by AMF hyphae.     

Infection Structure–Specific Expression of β-1,3-Glucan Synthase Is Essential for Pathogenicity of Colletotrichum graminicola and Evasion of β-Glucan–Triggered Immunity in Maize

β-1,3-Glucan and chitin are the most prominent polysaccharides of the fungal cell wall. Covalently linked, these polymers form a scaffold that determines the form and properties of vegetative and pathogenic hyphae. While the role of chitin in plant infection is well understood, the role of β-1,3-glucan is unknown. We functionally characterized the β-1,3-glucan synthase gene GLS1 of the maize (Zea mays) pathogen Colletotrichum graminicola, employing RNA interference (RNAi), GLS1 overexpression, live-cell imaging, and aniline blue fluorochrome staining. This hemibiotroph sequentially differentiates a melanized appressorium on the cuticle and biotrophic and necrotrophic hyphae in its host. Massive β-1,3-glucan contents were detected in cell walls of appressoria and necrotrophic hyphae. Unexpectedly, GLS1 expression and β-1,3-glucan contents were drastically reduced during biotrophic development. In appressoria of RNAi strains, downregulation of β-1,3-glucan synthesis increased cell wall elasticity, and the appressoria exploded. While the shape of biotrophic hyphae was unaffected in RNAi strains, necrotrophic hyphae showed severe distortions. Constitutive expression of GLS1 led to exposure of β-1,3-glucan on biotrophic hyphae, massive induction of broad-spectrum defense responses, and significantly reduced disease symptom severity. Thus, while β-1,3-glucan synthesis is required for cell wall rigidity in appressoria and fast-growing necrotrophic hyphae, its rigorous downregulation during biotrophic development represents a strategy for evading β-glucan–triggered immunity.     

Lipid metabolism of the endomycorrhizal fungus: Glomus intraradices

The use of monoxenic cultures of the obligately biotrophic vesicular arbuscular fungus Glomus intraradices now permits investigation of the lipid metabolism of this organism. In bicompartmental culture plates, sporulating extraradical hyphae can be obtained, totally free of roots, and then provided with 14C-acetate as lipid precursor. Three experimental stages were studied: i) stage A, symbiotic stage corresponding to the fungus still attached to the host plant roots, ii) stage B, consisting of the fungus detached from the host roots, iii) stage C, germinating spores. In each case, the fungus proved to be able to synthesise its own lipids: 1,2- and 1,3-diacylglycerols, triacylglycerols, phospholipids, sterols and free fatty acids, de novo. Lipid metabolism varied with the experimental conditions. Phospholipid synthesis was intensive in germinating spores. Thus the obligately biotrophic status of this fungus cannot be explained by a deficiency in synthesis of these various lipid classes.     

A fluorescent antibody assay for hyphae and glomalin from arbuscular mycorrhizal fungi

Studies on the role of arbuscular mycorrhizal (AM) fungi in soil have been aided by the use of a monoclonal antibody that detects a molecule common to all isolates of these fungi studied to date. The molecule, glomalin, is a glycoprotein that forms on hyphae, but apparently sloughs off and adheres to soil particles or imbedded plastic mesh. An indirect immunofluorescence (IF) assay is described for detection of glomalin on hyphae attached to roots, in roots, on hyphae traps and on the surface of soil aggregates. Small sieves are used to process hyphae attached to roots and soil aggregates. Glomalin on hyphae and glomalin attached to plastic or nylon are assayed on a 1 cm² section of meshes. Examples of IF assay results are shown and discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Conservation of the Ustilago maydis effector See1 in related smuts

Ustilago maydis is a biotrophic fungus that induces formation of tumors in maize (Zea mays L). In a recent study we identified See1 (Seedling efficient effector 1) as an U. maydis organ-specific effector required for tumor formation in leaves. See1 is required for U. maydis induced reactivation of plant DNA synthesis during leaf tumor progression. The protein is secreted from biotrophic hyphae and localizes to the cytoplasm and nucleus of plant cell. See1 interacts with maize SGT1, a cell cycle and immune regulator, interfering with its MAPK-triggered phosphorylation. Here, we present new data on the conservation of See1 in other closely related smuts and experimental data on the functionality of See1 ortholog in Ustilago hordei, the causal agent of barley covered smut disease.     

Carbon and nitrogen fluxes between beech and their ectomycorrhizal assemblage

To determine the exchange of nitrogen and carbon between ectomycorrhiza and host plant, young beech (Fagus sylvatica) trees from natural regeneration in intact soil cores were labelled for one growing season in a greenhouse with ¹³CO₂ and ¹⁵NO₃ ¹⁵NH₄. The specific enrichments of ¹⁵N and ¹³C were higher in ectomycorrhizas (EMs) than in any other tissue. The enrichments of ¹³C and ¹⁵N were also higher in the fine-root segments directly connected with the EM (mainly second-order roots) than that in bulk fine or coarse roots. A strict, positive correlation was found between the specific ¹⁵N enrichment in EM and the attached second-order roots. This finding indicates that strong N accumulators provide more N to their host than low N accumulators. A significant correlation was also found for the specific ¹³C enrichment in EM and the attached second-order roots. However, the specific enrichments for ¹⁵N and ¹³C in EM were unrelated showing that under long-term conditions, C and N exchange between host and EMs are uncoupled. These findings suggest that EM-mediated N flux to the plant is not the main control on carbon flux to the fungus, probably because EMs provide many different services to their hosts in addition to N provision in their natural assemblages.     

The infection process ofFusarium oxysporum in cotton root tips

Summary Conidia ofFusarium oxysporum f. sp.vasinfectum started to germinate on the roots of cotton (Gossypium barbadense L.) 6 h after inoculation and formed a compact mycelium covering the root surface. 18 h later, penetration hyphae branched off and infected the root. The number of penetration hyphae increased with the number of conidia used for inoculation. The optimal temperature for penetration was between 28 and 30 °C. The highest numbers of penetration hyphae were found in the meristematic zone, 40 percent less in the elongation and root hair zones, and none in the lateral root zone. The fine structure of the infection process was studied in protodermal cells of the meristematic zone and in rhizodermal cells of the elongation zone. The penetration hyphae were well preserved after freeze substitution and showed a Golgi equivalent consisting of three populations of smooth cisternae. Plant reactions were found already during fungal growth on the root surface. In the meristematic zone, a thickening of the plant cell wall due to an apposition of dark and lightly staining material below the hyphae occurred. This wall apposition increased in size around the hypha invading the plant cell and led to the formation of a prominent wall apposition with finger-like projections into the host cytoplasm. In the elongation zone, the deposits around the penetration hypha appeared less thick and the dark inclusions were less pronounced. High pressure freezing of infected cells revealed, thatF. oxysporum penetrates and grows within the host cells without inducing damages such as plasmolysis, cell degeneration or even host necrosis. We suggest thatF. oxysporum has an endophytic or biotrophic phase during colonization of the root tips.Abbreviation Ph penetration hyphae     

Effects of the mycorrhizal fungus Glomus intraradices on uranium uptake and accumulation by Medicago truncatula L. from uranium-contaminated soil

Phytostabilization strategies may be suitable to reduce the dispersion of uranium (U) and the overall environmental risks of U-contaminated soils. The role of Glomus intraradices, an arbuscular mycorrhizal (AM) fungus, in such phytostabilization of U was investigated with a compartmented plant cultivation system facilitating the specific measurement of U uptake by roots, AM roots and extraradical hyphae of AM fungi and the measurement of U partitioning between root and shoot. A soil-filled plastic pot constituted the main root compartment (C_A) which contained a plastic vial filled with U-contaminated soil amended with 0, 50 or 200 mg KH₂PO₄−P kg^–1soil (C_B). The vial was sealed by coarse or fine nylon mesh, permitting the penetration of both roots and hyphae or of just hyphae. Medicago truncatula plants grown in C_A were inoculated with G. intraradices or remained uninoculated. Dry weight of shoots and roots in C_A was significantly increased by G. intraradices, but was unaffected by mesh size or by P application in C_B. The P amendments decreased root colonization in C_B, and increased P content and dry weight of those roots. Glomus intraradices increased root U concentration and content in C_A, but decreased shoot U concentrations. Root U concentrations and contents were significantly higher when only hyphae could access U inside C_B than when roots could also directly access this U pool. The proportion of plant U content partitioned to shoots was decreased by root exclusion from C_B and by mycorrhizas (M) in the order: no M, roots in C_B > no M, no roots in C_B > M, roots in C_B > M, no roots in C_B. Such mycorrhiza-induced retention of U in plant roots may contribute to the phytostabilization of U contaminated environments.     

The symbiotic recapture of nitrogen from dead mycorrhizal and non-mycorrhizal roots of tomato plants

Aims

The aim was to quantify the nitrogen (N) transferred via the extra-radical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices from both a dead host and a dead non-host donor root to a receiver tomato plant. The effect of a physical disruption of the soil containing donor plant roots and fungal mycelium on the effectiveness of N transfer was also examined.

Methods

The root systems of the donor (wild type tomato plants or the mycorrhiza-defective rmc mutant tomato) and the receiver plants were separated by a 30 μm mesh, penetrable by hyphae but not by the roots. Both donor genotypes produced a similar quantity of biomass and had a similar nutrient status. Two weeks after the supply of ¹⁵?N to a split-root part of donor plants, the shoots were removed to kill the plants. The quantity of N transferred from the dead roots into the receiver plants was measured after a further 2 weeks.

Results

Up to 10.6 % of donor-root ¹⁵N was recovered in the receiver plants when inoculated with the arbuscular mycorrhizal fungus (AMF). The quantity of ¹⁵N derived from the mycorrhizal wild type roots clearly exceeded that from the only weakly surface-colonised rmc roots. Hyphal length in the donor rmc root compartments was only about half that in the wild type compartments. The disruption of the soil led to a significantly increased AMF-mediated transfer of N to the receiver plants.

Conclusions

The transfer of N from dead roots can be enhanced by AMF, especially when the donor roots have been formerly colonised by AMF. The transfer can be further increased with higher hyphae length densities, and the present data also suggest that a direct link between receiver mycelium and internal fungal structures in dead roots may in addition facilitate N transfer. The mechanical disruption of soil containing dead roots may increase the subsequent availability of nutrients, thus promoting mycorrhizal N uptake. When associated with a living plant, the external mycelium of G. intraradices is readily able to re-establish itself in the soil following disruption and functions as a transfer vessel.     

Phosphorus and carbon availability regulate structural composition and complexity of AM fungal mycelium

The regulation of the structural composition and complexity of the mycelium of arbuscular mycorrhizal (AM) fungi is not well understood due to their obligate biotrophic nature. The aim of this study was to investigate the structure of extraradical mycelium at high and low availability of carbon (C) to the roots and phosphorus (P) to the fungus. We used monoxenic cultures of the AM fungus Rhizophagus irregularis (formerly Glomus intraradices) with transformed carrot roots as the host in a cultivation system including a root-free compartment into which the extraradical mycelium could grow. We found that high C availability increased hyphal length and spore production and anastomosis formation within individual mycelia. High P availability increased the formation of branched absorbing structures and reduced spore production and the overall length of runner hyphae. The complexity of the mycelium, as indicated by its fractal dimensions, increased with both high C and P availability. The results indicate that low P availability induces a growth pattern that reflects foraging for both P and C. Low C availability to AM roots could still support the explorative development of the mycelium when P availability was low. These findings help us to better understand the development of AM fungi in ecosystems with high P input and/or when plants are subjected to shading, grazing or any management practice that reduces the photosynthetic ability of the plant.     

Nucleoside diphosphate kinase-1 regulates hyphal development via the transcriptional regulation of catalase in Neurospora crassa

A nucleoside diphosphate kinase-1-disrupted (ndk-1^RIP-1) mutant was observed to be defective in aerial hyphal and conidial development. In this study, two types of hyphae, fine and thick, were observed in wild-type (Wt) strains. However, only fine-type hyphae were observed in the ndk-1^RIP-1 mutants. The ndk-1^RIP-1 mutants were stimulated by oxidative stress and constitutively expressed an antioxidant enzyme catalase (CAT)-3. Furthermore the ndk-1^RIP-1 mutants could form thick hyphae by oxidative stress and a disruption of cat-3. These results suggest that the loss of thick hyphae in the ndk-1^RIP-1 mutants may be caused by the over-expression of cat-3.     

Dynamics of fine root carbon in Amazonian tropical ecosystems and the contribution of roots to soil respiration

Radiocarbon (¹⁴C) provides a measure of the mean age of carbon (C) in roots, or the time elapsed since the C making up root tissues was fixed from the atmosphere. Radiocarbon signatures of live and dead fine (<2 mm diameter) roots in two mature Amazon tropical forests are consistent with average ages of 4–11 years (ranging from <1 to >40 years). Measurements of ¹⁴C in the structural tissues of roots known to have grown during 2002 demonstrate that new roots are constructed from recent (<2‐year‐old) photosynthetic products. High Δ¹⁴C values in live roots most likely indicate the mean lifetime of the root rather than the isotopic signature of inherited C or C taken up from the soil. Estimates of the mean residence time of C in forest fine roots (inventory divided by loss rate) are substantially shorter (1–3 years) than the age of standing fine root C stocks obtained from radiocarbon (4–11 years). By assuming positively skewed distributions for root ages, we can effectively decouple the mean age of C in live fine roots (measured using ¹⁴C) from the rate of C flow through the live root pool, and resolve these apparently disparate estimates of root C dynamics. Explaining the ¹⁴C values in soil pore space CO₂, in addition, requires that a portion of the decomposing roots be cycled through soil organic matter pools with decadal turnover time.     

Formvar membrane laid on artificial medium induces haustorium-like structure formation in powdery mildew fungi

We aimed to develop an artificial membrane system to observe the infection process of the obligate biotrophic powdery mildew fungi without the use of living plant cells. The conidia of Blumeria graminis and Erysiphe pisi conidia were inoculated on a formvar membrane laid on an artificial medium. Germinated conidia frequently formed appressoria and then penetrated the membrane to form haustorium-like structures in the artificial medium. Secondary hyphae elongation was also observed after the formation of haustorium-like structures. These results suggested that the formvar membrane laid on artificial medium induced the formation of haustorium-like structures that have roles in the formation of secondary hyphae.     

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.     

Time-course study and partial characterization of a protein on hyphae of arbuscular mycorrhizal fungi during active colonization of roots

Material on the surface of hyphal walls of arbuscular mycorrhizal fungi (AMF) during active colonization of plant roots was detected by a monoclonal antibody. Pot-cultured isolates of Glomus, Acaulospora, Gigaspora, Scutellospora, and Entrophospora had immunofluorescent material (IM) on younger, thinner, intact hyphae, but IM was scant to absent on thicker, melanized or lysing hyphae. Colonization of corn (Zea mays L.), Sudangrass (Sorghum sudanense (Piper) Staph.) or red clover (Trifolium pratense L.) was examined during 5 months of plant growth by removing cores and performing an indirect immunoassay on roots with attached hyphae. Fresh spores of some Glomus spp. had IM on the outer layer of the spore wall. Abundant IM was seen on root hairs of plants colonized by some isolates, and some IM was detected on root surfaces of all plants examined even during early colonization. After cultures were dried, hyphae, roots and spores had little to no IM. Uninoculated control roots had very rare, small patches of IM. An immunoreactive protein was extracted from hyphae of Gigaspora and Glomus isolates by using 20mM citrate (pH 7.0) at 121°C for 90 min. Gel electrophoresis profiles indicated that all isolates tested had the same banding patterns. Lectin-binding of extracted protein is suggestive of a glycoprotein. The immunofluorescence assay can be used to examine root sections for active colonization by AMF, and the potential use of the protein to quantify AMF activity in soil is discussed.