Laccaria Bicolor S238N Research Articles

Role of secondary metabolites in the interaction between Pseudomonas fluorescens and soil microorganisms under iron-limited conditions.

Microorganisms can be versatile in their interactions with each other, being variously beneficial, neutral or antagonistic in their effect. Although this versatility has been observed among many microorganisms and in many environments, little is known regarding the mechanisms leading to these changes in behavior. In the present work, we analyzed the mechanism by which the soil bacterium Pseudomonas fluorescens BBc6R8 shifts from stimulating the growth of the ectomycorrhizal fungus Laccaria bicolor S238N to killing the fungus. We show that among the three secondary metabolites produced by the bacterial strain-the siderophores enantio-pyochelin and pyoverdine, and the biosurfactant viscosin-the siderophores are mainly responsible for the antagonistic activity of the bacterium under iron-limited conditions. While the bacterial strain continues to produce beneficial factors, their effects are overridden by the action of their siderophores. This antagonistic activity of the strain P. fluorescens BBC6R8 in iron-depleted environments is not restricted to its influence on L. bicolor, since it was also seen to inhibit the growth of the actinomycete Streptomyces ambofaciens ATCC23877. We show that the strain P. fluorescens BBc6R8 uses different strategies to acquire iron, depending on certain biotic and abiotic factors.

Pairwise transcriptomic analysis of the interactions between the ectomycorrhizal fungus Laccaria bicolor S238N and three beneficial, neutral and antagonistic soil bacteria.

Ectomycorrhizal fungi are surrounded by bacterial communities with which they interact physically and metabolically during their life cycle. These bacteria can have positive or negative effects on the formation and the functioning of ectomycorrhizae. However, relatively little is known about the mechanisms by which ectomycorrhizal fungi and associated bacteria interact. To understand how ectomycorrhizal fungi perceive their biotic environment and the mechanisms supporting interactions between ectomycorrhizal fungi and soil bacteria, we analysed the pairwise transcriptomic responses of the ectomycorrhizal fungus Laccaria bicolor (Basidiomycota: Agaricales) when confronted with beneficial, neutral or detrimental soil bacteria. Comparative analyses of the three transcriptomes indicated that the fungus reacted differently to each bacterial strain. Similarly, each bacterial strain produced a specific and distinct response to the presence of the fungus. Despite these differences in responses observed at the gene level, we found common classes of genes linked to cell-cell interaction, stress response and metabolic processes to be involved in the interaction of the four microorganisms.

Ethylene and jasmonic acid act as negative modulators during mutualistic symbiosis between Laccaria bicolor and Populus roots

The plant hormones ethylene, jasmonic acid and salicylic acid have interconnecting roles during the response of plant tissues to mutualistic and pathogenic symbionts. We used morphological studies of transgenic- or hormone-treated Populus roots as well as whole-genome oligoarrays to examine how these hormones affect root colonization by the mutualistic ectomycorrhizal fungus Laccaria bicolor S238N. We found that genes regulated by ethylene, jasmonic acid and salicylic acid were regulated in the late stages of the interaction between L.bicolor and poplar. Both ethylene and jasmonic acid treatments were found to impede fungal colonization of roots, and this effect was correlated to an increase in the expression of certain transcription factors (e.g. ETHYLENE RESPONSE FACTOR1) and a decrease in the expression of genes associated with microbial perception and cell wall modification. Further, we found that ethylene and jasmonic acid showed extensive transcriptional cross-talk, cross-talk that was opposed by salicylic acid signaling. We conclude that ethylene and jasmonic acid pathways are induced late in the colonization of root tissues in order to limit fungal growth within roots. This induction is probably an adaptive response by the plant such that its growth and vigor are not compromised by the fungus.

Erratum to: Laccaria bicolor S238N improves Scots pine mineral nutrition by increasing root nutrient uptake from soil minerals but does not increase mineral weathering

Erratum to: Laccaria bicolor S238N improves Scots pine mineral nutrition by increasing root nutrient uptake from soil minerals but does not increase mineral weathering

Role of fungal trehalose and bacterial thiamine in the improved survival and growth of the ectomycorrhizal fungus Laccaria bicolor S238N and the helper bacterium Pseudomonas fluorescens BBc6R8

The mycorrhiza helper bacterial strain Pseudomonas fluorescens BBc6R8 enhances the establishment of Laccaria bicolor S238N ectomycorrhizae by improving the pre-symbiotic growth and survival of the fungus. Nothing is known about the effect of the ectomycorrhizal fungus on the helper bacteria or the molecules that are involved in the interaction. In this study, we have monitored the population density of the helper strain P. fluorescens BBc6R8 in soils inoculated with L. bicolor and in control soils and found that the ectomycorhizal fungus improves the survival of the helper bacteria. We investigated the identity of the fungal and bacterial metabolites involved in this reciprocal growth-promoting effect using a combination of growth measurements, chemoattractant assays, HPLC and in silico genome analyses. We showed that trehalose, a disaccharide that accumulates to high levels in the fungal hyphae, chemoattracted and promoted the growth of the helper bacteria. Meanwhile, P. fluorescens BBc6R8 produced thiamine at concentrations that enhanced the fungal growth in vitro. Altogether our data indicate that the interaction between the two microorganisms is beneficial for both species and relies, at least in part, on trophic mutualism.

Laccaria bicolor S238N improves Scots pine mineral nutrition by increasing root nutrient uptake from soil minerals but does not increase mineral weathering

The role of ectomycorrhizal fungi on mineral nutrient mobilization and uptake is crucial for tree nutrition and growth in temperate forest ecosystems. By using a “mineral weathering budget” approach, this study aims to quantify the effect of the symbiosis with the ectomycorrhizal model strain Laccaria bicolor S238N on mineral weathering and tree nutrition, carrying out a column experiment with a quartz/biotite substrate. Each column was planted with one Scots pine (Pinus sylvestris L.) non-mycorrhizal or mycorrhizal with L. bicolor, with exception of the abiotic control treatment. The columns were continuously supplied with a nutrient-poor solution. A mineral weathering budget was calculated for K and Mg. The pine shoot growth was significantly increased (73%) when plants were mycorrhizal with L. bicolor. Whatever their mycorrhizal status, pines increased mineral weathering by factors 1.5 to 2.1. No difference between non-mycorrhizal and mycorrhizal pine treatments was revealed, however, mycorrhizal pines assimilated significantly more K and Mg. This suggests that in our experimental conditions, L. bicolor S238N improved shoot growth and K and Mg assimilation in Scots pine mainly by increasing the uptake of dissolved nutrients, linked to a better exploration and exploitation of the soil by the mycorrhizal roots.

The ecological significance of the coarse soil fraction for Picea abies (L.) Karst. seedling nutrition

Recent studies showed that the coarse fraction of soils can have a considerable stock of short-term available nutrients. However, a direct proof that coarse soil fragments have significant nutritional functions in forest ecosystems is missing. In a 23 week microcosm experiment with controlled in- and output, mycorrhizal (Laccaria bicolor S238N) and non-mycorrhizal Norway spruce seedlings (Picea abies (L.) Karst.) were grown on either coarse soil fragments (dark paragneiss, Black Forest, S-W Germany) or quartz only. The microcosms were irrigated with a solution adjusted to the mean ionic composition of the soil solution in the reference profile, but devoid of Ca and Mg. The uptake rates of Ca and Mg by spruce seedlings were sufficient to allow normal root and shoot growth. Mycorrhization was low and did not have a significant influence on seedling nutrition. The oxalic anion was found in concentrations below 1.5 mg L−1 in the leachate, approximately corresponding to a C2O42−-saturation in equilibrium with precipitated calcium oxalate. Cation budgets and relationships among the cation species suggest that exchange processes were the main trigger of Ca and Mg mobilisation and uptake rather than protolytic weathering by exudation of carboxylic acids. The exchange processes may be attributed to weathering cracks filled with fine material of high base saturation. Therefore it is concluded that the short-term nutritional functions of coarse soils in forest sites are significant, and exchange mechanisms in the “stone protected fine earth” are in principal the same as they are known for the “free fine earth”.

The mycorrhiza helper Pseudomonas fluorescens BBc6R8 has a specific priming effect on the growth, morphology and gene expression of the ectomycorrhizal fungus Laccaria bicolor S238N

The mycorrhiza helper Pseudomonas fluorescens BBc6R8 promotes the presymbiotic survival and growth of the ectomycorrhizal fungus Laccaria bicolor S238N in the soil. An in vitro fungal-bacterial confrontation bioassay mimicking the promoting effects of the bacteria on fungal growth was set up to analyse the fungal morphological and transcriptional changes induced by the helper bacteria at three successive stages of the interaction. The specificity of the P. fluorescens BBc6R8 effect was assessed in comparison with six other rhizobacterial strains possessing mycorrhiza helper or pathogen antagonistic abilities. The helper BBc6R8 strain was the only strain to induce increases in the radial growth of the colony, hyphal apex density and branching angle. These morphological modifications were coupled with pleiotropic alterations of the fungal transcriptome, which varied throughout the interaction. Early stage-responsive genes were presumably involved in recognition processes and transcription regulation, while late stage-responsive genes encoded proteins of primary metabolism. Some of the responsive genes were partly specific to the interaction with P. fluorescens BBc6R8, whereas others were mutually regulated by different rhizobacteria. The results highlight the fact that the helper BBc6R8 strain has a specific priming effect on growth, morphology and gene expression of its fungal associate L. bicolor S238N.

Agrobacterium-mediated transformation of the ectomycorrhizal symbiont Laccaria bicolor S238N

The development of an efficient transformation system is required to alter the expression of symbiosis-regulated genes and to develop insertional mutagenesis in the ectomycorrhizal basidiomycete Laccaria bicolor S238N. Vegetative mycelium of this fungus was transformed by Agrobacterium tumefaciens-mediated gene transfer. The selection marker was the hygromycin resistance gene of Escherichia coli (hph) under the control of the gpd promoter from Agaricus bisporus and the CaMV 35S terminator as part of the T-DNA. PCR amplification of hph and Southern blot analyses showed that the genome of the hygromycin-resistant transformants contained the cassette. The latter proved mostly single copy and random integration of part of the transgene into the fungal genome. A. tumefaciens-mediated gene transfer should facilitate future development of insertional mutagenesis, targeted gene disruption and RNA interference technology in L. bicolor.

Occurrence and distribution of endobacteria in the plant‐associated mycelium of the ectomycorrhizal fungus Laccaria bicolor S238N

Fluorescence in situ hybridization, associated with confocal laser scanning microscopy or epifluorescence microscopy with deconvolution system, has allowed the detection of a community of intracellular bacteria in non-axenic samples of the ectomycorrhizal fungus Laccaria bicolor S238N. The endobacteria, mainly alpha-proteobacteria, were present in more than half of the samples, which consisted of ectomycorrhizae, fungal mats and fruit bodies, collected in the glasshouse or in the forest. Acridine orange staining suggests that the endobacteria inhabit both live and dead fungal cells. The role of these endobacteria remains to be clarified.

Fate, tree growth effect and potential impact on soil microbial communities of mycorrhizal and bacterial inoculation in a forest plantation

The knowledge of the survival of inoculated beneficial fungal and bacterial strains in the field and the effects of their release on the indigenous microbial communities has been of great interest since the practical use of selected natural or genetically modified microorganisms has been developing. The aim of this study was to monitor, 4 years after plantation into the field site, the effects of Douglas fir ( Pseudotsuga menziesii) co-inoculation with the mycorrhiza helper bacterial strain Pseudomonas fluorescens BBc6R8 and/or the fungal strain Laccaria bicolor S238N on seedling growth and on the indigenous bacterial and ectomycorrhizal communities using quantitative and qualitative approaches. The field persistence of the inoculated strains was also monitored. The seedling shoot volume estimate was statistically significantly higher in the fungal inoculated plots in comparison to the non-inoculated plots but no treatment-related changes in the quantitave or qualitative microbial measurements were observed and the inoculated strains could not be detected after 4 years.

Comparative effects of auxin transport inhibitors on rhizogenesis and mycorrhizal establishment of spruce seedlings inoculated with Laccaria bicolor.

We compared the effects of two auxin transport inhibitors (2,3,5-triiodobenzoic acid (TIBA) and 1-N-naphthylphthalamic acid (NPA)) on rhizogenesis and mycorrhizal establishment of Picea abies L. (Karst.) seedlings inoculated with Laccaria bicolor S238N (Maire) Orton. Inoculation of seedlings with L. bicolor under in vitro conditions strongly increased host root and shoot growth. Although TIBA had no effect on taproot growth, NPA decreased taproot growth and deformed the root apex into a globular shape in both non-inoculated seedlings and seedlings inoculated with L. bicolor. Inoculation with L. bicolor strongly increased lateral rhizogenesis of the seedlings, and application of 100 microM indole-3-acetic acid (IAA) partially reproduced this effect. Although TIBA completely inhibited the stimulatory effect of L. bicolor on lateral root formation, NPA inhibited it only partially. Both TIBA and NPA counteracted the effect of exogenous IAA on lateral rhizogenesis. Inoculation with L. bicolor significantly increased shoot growth and seedling dry biomass, whereas application of exogenous IAA had no effect on either parameter. There was no effect of NPA on shoot growth and biomass production. The presence of TIBA completely prevented the development of ectomycorrhizal structures (mantle and Hartig net). In the presence of NPA, the number of seedlings colonized by the fungus was reduced and the degree of development of ectomycorrhizal structures was variable, but not completely prevented. In medium lacking tryptophan, neither TIBA nor NPA inhibited the release of IAA produced by L. bicolor in pure culture. When 100 microM tryptophan was added to the medium, TIBA significantly increased the amount of IAA released by the fungus, whereas NPA had no significant effect. We conclude that fungal IAA plays an important role in plant rhizogenesis and in the establishment of ectomycorrhizal symbiosis.

Survival after outplanting of the ectomycorrhizal fungus Laccaria bicolor S238N inoculated on Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) cuttings

Selected strains of ectomycorrhizal fungi can be inoculated in forest nurseries to improve survival and growth of seedlings or cuttings after field transplantation. The survival of the American strain Laccaria bicolorS238N on Douglas fir cuttings was evaluated in nursery and field conditions three years after outplanting using morphological and PCR/RFLP of nuclear rDNA spacers. The compari- son of the mycorrhizal status of Douglas fir cuttings at the end of the nursery phase and two years after outplanting shown several beha- viours among the ectomycorrhizal fungi naturally occurring in the nursery or artificially introduced. The naturally occurring Rhizopogon type disappeared after outplanting, while the inoculated strain Laccaria bicolorS238N and an unknown type (1/2 ITS ribotype) survived and competed with the naturally occurring fungi of the outplanting site. Only one indigenous type (1/3 ITS ribotype) seemed occurring in the outplanting site where Cenococcum geophilumwas almost completely absent.

Survival in the soil of the ectomycorrhizal fungus Laccaria bicolor and the effects of a mycorrhiza helper Pseudomonas fluorescens

In disinfected forest nursery soils, inoculating Douglas fir ( Pseudotsuga menziesii) seedlings with the ectomycorrhizal fungal strain Laccaria bicolor S238N significantly increases tree growth after outplantating. However, the success of the inoculation depends on survival of the fungal inoculum in the soil during the pre-symbiotic life of the fungus. We followed the survival of L. bicolor S238N in autoclaved nursery soil in the glasshouse, and under gnotobiotic conditions in autoclaved or γ-irradiated nursery soil. We also studied the effect of the mycorrhiza helper bacterium Pseudomonas fluorescens BBc6R8, which promotes the Douglas fir- L. bicolor S238N symbiosis, on fungal viability. In the glasshouse, fungal viability was assessed by trapping with Douglas fir seedlings. We showed that the fungus retained its viability in a pre-symbiotic state in the soil at least for 23 weeks, which is much longer than that reported in the literature for other ectomycorrhizal fungi. The bacterium did not significantly modify the survival of the fungus. In the gnotobiotic experiments, ergosterol, a specific fungal membrane component, was used to quantify fungal biomass. Fungal behaviour differed with the disinfection technique used, which modified the chemical characteristics of the initial soil. There was no fungal growth in the autoclaved soil but there was a rapid increase of fungal biomass in the irradiated soil. The effect of the bacterium on fungal biomass also varied with a significant stimulation in the autoclaved soil vs. a significant inhibition in the irradiated soil. Our results show that the beneficial effect of the bacterium on the fungus depends on the condition of the fungus, i.e. the greatest benefit occurs when the fungus is growing under unfavourable conditions.

Structure and dynamics of experimentally introduced and naturally occurring laccaria sp. Discrete genotypes in a douglas fir plantation

Ectomycorrhizal fungi have been introduced in forest nurseries to improve seedling growth. Outplanting of inoculated seedlings to forest plantations raises the questions about inoculant persistence and its effects on indigenous fungal populations. We previously showed (M.-A. Selosse et al. Mol. Ecol. 7:561-573, 1998) that the American strain Laccaria bicolor S238N persisted 10 years after outplanting in a French Douglas fir plantation, without introgression or selfing and without fruiting on uninoculated adjacent plots. In the present study, the relevance of those results to sympatric strains was assessed for another part of the plantation, planted in 1985 with seedlings inoculated with the French strain L. bicolor 81306 or left uninoculated. About 720 Laccaria sp. sporophores, collected from 1994 to 1997, were typed by using randomly amplified polymorphic DNA markers and PCR amplification of the mitochondrial and nuclear ribosomal DNAs. All plots were colonized by small spontaneous discrete genotypes (genets). The inoculant strain 81306 abundantly fruited beneath inoculated trees, with possible introgression in indigenous Laccaria populations but without selfing. In contrast to our previous survey of L. bicolor S238N, L. bicolor 81306 colonized a plot of uninoculated trees. Meiotic segregation analysis verified that the invading genet was strain 81306 (P < 0.00058), implying a vegetative growth of 1.1 m. year-1. This plot was also invaded in 1998 by strain S238N used to inoculate other trees of the plantation. Five other uninoculated plots were free of these inoculant strains. The fate of inoculant strains thus depends less on their geographic origin than on unknown local factors.

Meiotic segregation and recombination of the intergenic spacer of the ribosomal DNA in the ectomycorrhizal basidiomycete Laccaria bicolor.

The aim of this study was to clarify the inheritance of the nuclear ribosomal DNA (rDNA) in the ectomycorrhizal basidiomycete Laccaria bicolor S238N in order to resolve inter- and within-strain relationships in forest ecosystems. PCR amplification of the intergenic spacer (IGS) was carried out in the dikaryotic mycelium and its haploid progeny. In the dikaryotic mycelium, multiple amplification products were produced for the 25s/5s (IGS1) and 5s/17s (IGS2) intergenic spacers. The 4.5- and 4.0-kb fragments of IGS2 (haplotypes alpha and beta, respectively) were observed to occur in a 1:1 ratio within the haploid progeny as a result of divergent IGS haplotypes in the two separate nuclei. Recombinant monokaryons having both types of IGS2 occurred at a low frequency (6.5%; 60 kb per centimorgan) during meiosis. Haplotypes alpha and beta of IGS1 cross-hybridized forming heteroduplexes during the PCR temperature cycle. The two IGS1 haplotypes differed only by the repeat number of a TA2C3 motif and co-segregated with the IGS2 haplotypes. Heteroduplex formation and IGS polymorphism provide information that is helpful in distinguishing between introduced exotic L. bicolor S238N and indigenous populations of Laccaria spp. in forest ecosystems.

Nitrogen source regulates the biosynthesis of NADP‐glutamate dehydrogenase in the ectomycorrhizal basidiomycete Laccaria bicolor

summaryNADP‐glutamate dehydrogenase (NADP‐GDH) is a key enzyme of N assimilation in ectomycorrhizal fungi, but little is known about its regulation. In the present study. the effects of N source and concentration cm NADP‐GDH specific activity, polypeptide content and biosynthesis have been investigated in the ectomycorrhizal basidiomycete Laccaria bicolor S238N. Mycelium was transferred from NH+4‐rich medium to either NO−3‐rich or N‐free medium. Under both these conditions, intracellular NH.−3 and glutamine content decreased dramatically. The specific activity of NADP‐GDH was simultaneously increased 3 4.5‐fold. Immunological analysis using anti‐GDH antibodies showed that depression of enzyme activity resulted from increased concentration of the GDH polypeptide. In addition, assessment of the steady‐state biosynthesis of NADP‐GDH by in vivo labelling of proteins demonstrated that the observed changes in enzyme concentration are explained by differences in the relative rate of enzyme biosynthesis. These results suggested that the source of N regulates synthesis of NADP‐GDH and that this regulation is under transitional and/or transcriptional control.

Monitoring the persistence of Laccaria bicolor as an ectomycorrhizal symbiont of nursery‐grown Douglas fir by PCR of the rDNA intergenic spacer

AbstractThe large‐scale inoculation of selected beneficial ectomycorrhizal fungi in forest nurseries has generated renewed interest in the ecology of these symbiotic fungi. However, information on the dissemination and persistence of introduced symbionts is scarce due to the limitation of the current identification methods. To identify ectomycorrhizal fungi on single root tips, we investigated the polymorphism of the PCR‐amplified ribosomal DNA intergenic spacer (IGS) from a wide range of ectomycorrhizal fungi. To investigate the reliability of this molecular approach in large‐scale surveys, the dissemination and persistence on Douglas fir seedlings of the introduced Laccaria bicolor S238N were assessed in a forest nursery in the Massif Central (France). Several hundred ectomycorrhizas and fruiting bodies were sampled from plots where control and L. bicolor inoculated‐Douglas fir seedlings were grown for 1.5 years. PCR typing of mycorrhizas indicated that trees inoculated with L. bicolor S238N remained exclusively colonized by that isolate (or sexually derived isolates) for the entire test period. In contrast, control seedlings were infected by indigenous isolates of Laccaria laccata and Thelephora terrestris. The molecular evidence for the persistence of the introduced mycobiont despite the competition from indigenous isolates of the same species provides further illustration of the potential of exotic species for large‐scale microbial application.