Temporal persistence and spatial distribution of an American inoculant strain of the ectomycorrhizal basidiomycete Laccaria bicolor in a French forest plantation

Selected strains of ectomycorrhizal fungi, such as the basidiomycete Laccaria bicolor , are currently being used as inoculants in nurseries to improve growth of forest trees after outplanting. Information is needed on the survival of these introduced strains in forests and their impact on indigenous biodiversity. Dissemination and persistence of an American strain, L. bicolor S238N, were studied 10 years after outplanting in a Douglas fir plantation located at Saint-Brisson (Morvan, France). About 430 Laccaria spp. sporophores were collected over 3 years. Inheritance of nuclear ribosomal DNA, as well as RAPD markers, was characterized in L. bicolor S238N, using a haploid progeny set of 91 monokaryons. More than 50 markers were identified (19 heterozygous and 33 homozygous or cytoplasmic markers), which unambiguously confirmed that the introduced strain was still present in the inoculated plots. Neither selfing ( P < 0.0008) nor introgression with indigenous strains was detected although in vitro interfertility between the American strain and indigenous L. bicolor was identified. No ingress of the introduced genet into adjacent uninoculated plots colonized by various local Laccaria genets was detected. It is proposed that the spatial distributions identified have developed through mycelial propagation of the introduced strain and intraspecific competition with native genets. Although longer-term data is still lacking, the stability of the inoculant strain and the limited disturbance to indigenous populations described support large-scale nursery production of this host-fungal combination.     

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.     

SCAR markers to detect mycorrhizas of an American Laccaria bicolor strain inoculated in European Douglas-fir plantations

The American strain S238N of the ectomycorrhizal fungus Laccaria bicolor (Maire) Orton has been used to inoculate Douglas-fir [Pseudotsuga menziesii (Mir.) Franco] plantations in France over the last two decades. Laccaria fruit bodies are scarce in mature plantations, which precludes further assessment of its persistence by fruit body surveys. Our objective was to develop new markers to identify this strain and its eventual non-fruiting progeny on root tips. We converted nine random amplified polymorphic DNA markers into sequence characterized amplified region (SCAR) markers. Two of these SCAR markers enabled us to detect S238N on roots of seedlings and mature trees. No amplification of non-fungal (host plant, bacterial, etc.) DNA was observed. Moreover, both SCARs were amplified from Laccaria-like mycorrhizas in a Douglas-fir plantation inoculated 14 years ago, demonstrating the long-term persistence of the inoculant strain. We also obtained a SCAR marker to detect one strain of European origin (L. bicolor 81306), indicating that SCARs are potential markers to type the naturally occurring genets. Thus, SCAR markers are of great value in studying the persistence of inoculant strains and the effects on local populations of introducing foreign strains.     

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.     

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

The 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.     

The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) inhibits the stimulation of in vitro lateral root formation and the colonization of the tap-root cortex of Norway spruce (Picea abies) seedlings by the ectomycorrhizal fungus Laccaria bicolor

Norway spruce ( Picea abies (L.) Karst.) seedlings were inoculated with the ectomycorrhizal fungus Laccaria bicolor ((Marie) Orton), strain S238 N, in axenic conditions. The presence of the fungus slowed tap–root elongation by 26% during the first 15 d after inoculation and then stimulated it by 136%. In addition, it multiplied in vitro lateral root formation by 4.3, the epicotyl growth of the seedlings by 8.4 and the number of needles by 2. These effects were maintained when the fungus was separated from the roots by a cellophane membrane preventing symbiosis establishment, thus suggesting that the fungus acted by non-nutritional effects. We tested the hypothesis that IAA produced by L. bicolor S238 N would be responsible for the stimulation of fungal induced rhizogenesis. We showed in previous work that L. bicolor S238 N can synthesize IAA in pure culture. Exogenous IAA supplies (100 and 500 μ m ) reproduced the stimulating effect of the fungus on root branching but inhibited root elongation. The presence of 2,3,5-triiodobenzoic acid (TIBA) in the culture medium significantly depressed lateral root formation of inoculated seedlings. As TIBA had no significant effect on IAA released in the medium by L. bicolor S238 N, but counteracted the stimulation of lateral rhizogenesis induced by an exogenous supply of IAA, we suggest that TIBA inhibited the transport of fungal IAA in the root. Furthermore TIBA blocked the colonization of the main root cortex by L. bicolor S238 N and the formation of the Hartig net. These results specified the role of fungal IAA in the stimulation of lateral rhizogenesis and in ectomycorrhizal symbiosis establishment.     

Effects of bacteria on mycorrhizal development and growth of container grown Eucalyptus diversicolor F. Muell. seedlings

The development of ectomycorrhizas on inoculated eucalypt seedlings in commercial nurseries is often slow so that only a small percentage of roots are mycorrhizal at the time of outplanting. If mycorrhizal formation could be enhanced by co-inoculation with bacteria which promote rapid root colonisation by specific ectomycorrhizal fungi, as demonstrated by certain bacteria in the Douglas fir-Laccaria bicolor association, this would be of advantage to the eucalypt forest industry. Two bacterial isolates with a demonstrated Mycorrhization Helper Bacteria (MHB) effect on ectomycorrhiza formation between Pseudotsuga menziesii and Laccaria bicolor (S238), and seven Western Australian bacterial isolates from Laccaria fraterna sporocarps or ectomycorrhizas were tested in isolation for their effect on ectomycorrhizal development by three Laccaria spp. with Eucalyptus diversicolor seedlings. Mycorrhizal formation by L. fraterna (E710) as measured by percentage infected root tips, increased significantly (p < 0.05) by up to 296% in treatments coinoculated with MHB isolates from France (Pseudomonas fluorescens Bbc6 or Bacillus subtilis MB3), or indigenous isolates (Bacillus sp. Elf28 or a pseudomonad Elf29). In treatments coinoculated with L. laccata (E766) and the MHB isolate P. fluorescens (Bbc6) mycorrhizal development was significantly inhibited (p < 0.05). A significant Plant Growth Promoting Rhizobacteria (PGPR) effect was observed where the mean shoot d.w. of seedlings inoculated only with an unidentified bacterium (Elf21), was 49% greater than the mean of uninoculated controls (-fungus, -bacterium). Mean shoot d.w. of seedlings coinoculated with L. bicolor (S-238), which did not form ectomycorrhizas with E. diversicolor, and an unidentified bacterium (Slf14) or Bacillus sp. (Elf28) were significantly higher than uninoculated seedlings or seedlings inoculated with L. bicolor (S-238) alone. This is the first time that an MHB effect has been demonstrated in a eucalypt-ectomycorrhizal fungus association. These organisms have the potential to improve ectomycorrhizal development on eucalypts under nursery conditions and this is particularly important for fast growing eucalypt species where the retention time of seedlings in the nursery is of short duration (2–3 months).     

Mechanisms for the development of genetically variable mycorrhizal mycelia in the ectomycorrhizal fungus Laccaria bicolor.

An in vitro study investigated mechanisms for the development of genetically variable mycorrhizal mycelia for Laccaria bicolor. Seedlings of jack pine (Pinus banksiana) grown nonaseptically in an autoclaved soil substrate were given different L. bicolor inoculum treatments. These included (i) a dikaryotic mycelium genotype (D); (ii) D and basidiospores collected from one group of five sporophores (T1); (iii) D and basidiospores collected from 10 sporophores, two from each of five different groups (T5); (iv) T1 alone; (v) T5 alone; and (vi) a noninoculated control. Dikaryotic mycelial inoculum was provided at the time of sowing, while basidiospore inoculum was added at 10 weeks after seed germination. Sporophore formation was induced after 20 weeks of growth, and dikaryotic cultures were isolated from their tissue. Seedlings were harvested, and growth and mycorrhization were assessed. Levels of both were generally lower for T1-treated seedlings, compared with seedlings receiving D, while levels for T5-treated seedlings were intermediate. Sporophore genotype variability was assessed for inoculum treatments by using the isoenzymatic marker leucine aminopeptidase. The greatest genetic variability was seen with the basidiospore treatments T1 and T5, with up to four leucine aminopeptidase patterns per seedling. The mixed treatments D plus T1 and D plus T5 produced most frequently, but not exclusively, the inoculated dikaryon genotype. After isoenzyme results were assessed, variable sporophore isolates of mixed treatments were analyzed with randomly amplified polymorphic DNA and PCR mitochondrial DNA markers to determine if they were formed by dikaryon-monokaryon crosses between the inoculated dikaryon and monosporous mycelia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cation exchange capacity and lead sorption in ectomycorrhizal fungi

Two ectomycorrhizal fungi, Paxillus involutus 533 and Laccaria bicolor S238, differing greatly in their mycelial characteristics, were investigated with regard to their cation exchange capacity and Pb-binding capacity in vitro after growth with either NO₃ ^- or NH₄ ⁺ as N source. The CECs of 800–1200 mol g-1 dry weight for Paxillus involutus 533 and 2000–3000 mol g-1 dry weight for Laccaria bicolor S238, were high compared to plant roots. The fungal mycelium also had a high Pb sorption capacity. It was higher in Laccaria bicolor S238 than in Paxillus involutus 533 and higher after pregrowth in NO₃ ^- compared to NH₄ ⁺. Both the higher CEC and the higher Pb sorption capacity of Laccaria bicolor S238 compared to Paxillus involutus 533 might have been the result of the hydrophilic nature of the of Laccaria bicolor S238 mycelium. It would have absorbed the solutions better than the hydrophobic mycelium of Paxillus involutus 533. X-ray microanalysis of the cell walls revealed that the Pb content of the cell walls was higher in Paxillus involutus 533 than in Laccaria bicolor S238. Nevertheless, electron dense deposits in the cell walls of Laccaria bicolor S238 contained large amounts of Pb, P and S. Thus, while Pb was evenly distributed in the cell walls of Paxillus involutus 533, Pb was accumulated in electron dense deposits in Laccaria bicolor S238. The results are discussed in view of their significance for the mycorrhizal symbiosis.     

Phylogenetic, genomic organization and expression analysis of hydrophobin genes in the ectomycorrhizal basidiomycete Laccaria bicolor

Hydrophobins are morphogenetic, small secreted hydrophobic fungal proteins produced in response to changing development and environmental conditions. These proteins are important in the interaction between certain fungi and their hosts. In mutualistic ectomycorrhizal fungi several hydrophobins form a subclass of mycorrhizal-induced small secreted proteins that are likely to be critical in the formation of the symbiotic interface with host root cells. In this study, two genomes of the ectomycorrhizal basidiomycete Laccaria bicolor strains S238N-H82 (from North America) and 81306 (from Europe) were surveyed to construct a comprehensive genome-wide inventory of hydrophobins and to explore their characteristics and roles during host colonization. The S238N-H82 L. bicolor hydrophobin gene family is composed of 12 genes while the 81306 strain encodes nine hydrophobins, all corresponding to class I hydrophobins. The three extra hydrophobin genes encoded by the S238N-H82 genome likely arose via gene duplication and are bordered by transposon rich regions. Expression profiles of the hydrophobin genes of L. bicolor varied greatly depending on life stage (e.g. free living mycelium vs. root colonization) and on the host root environment. We conclude from this study that the complex diversity and range of expression profiles of the Laccaria hydrophobin multi-gene family have likely been a selective advantage for this mutualist in colonizing a wide range of host plants.     

In situ identification of intracellular bacteria related to Paenibacillus spp. in the mycelium of the ectomycorrhizal fungus Laccaria bicolor S238N

Bacterial proliferations have recurrently been observed for the past 15 years in fermentor cultures of the ectomycorrhizal fungus Laccaria bicolor S238N, suggesting the presence of cryptic bacteria in the collection culture of this fungus. In this study, intracellular bacteria were detected by fluorescence in situ hybridization in combination with confocal laser scanning microscopy in several collection subcultures of L. bicolor S238N. They were small (0.5 micro m in diameter), rare, and heterogeneously distributed in the mycelium and were identified as Paenibacillus spp. by using a 16S rRNA-directed oligonucleotide probe initially designed for bacteria isolated from a fermentor culture of L. bicolor S238N.     

A genetic linkage map for the ectomycorrhizal fungus Laccaria bicolor and its alignment to the whole-genome sequence assemblies

A genetic linkage map for the ectomycorrhizal basidiomycete Laccaria bicolor was constructed from 45 sib-homokaryotic haploid mycelial lines derived from the parental S238N strain progeny. For map construction, 294 simple sequence repeats (SSRs), single-nucleotide polymorphisms (SNPs), amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNA (RAPD) markers were employed to identify and assay loci that segregated in backcross configuration. Using SNP, RAPD and SSR sequences, the L. bicolor whole-genome sequence (WGS) assemblies were aligned onto the linkage groups. A total of 37.36 Mbp of the assembled sequences was aligned to 13 linkage groups. Most mapped genetic markers used in alignment were colinear with the sequence assemblies, indicating that both the genetic map and sequence assemblies achieved high fidelity. The resulting matrix of recombination rates between all pairs of loci was used to construct an integrated linkage map using JoinMap. The final map consisted of 13 linkage groups spanning 812 centiMorgans (cM) at an average distance of 2.76 cM between markers (range 1.9-17 cM). The WGS and the present linkage map represent an initial step towards the identification and cloning of quantitative trait loci associated with development and functioning of the ectomycorrhizal symbiosis.     

铝对外生菌根真菌草酸分泌及磷、钾、铝吸收的影响

试验研究了在铝胁迫条件下,6种(株)外生菌根真菌(ECMF)的生长、草酸分泌,以及磷、钾、铝的吸收状况。结果表明,铝对抗(耐)型菌种Pt715、HrSp、CgSIV的生长无抑制作用,但显著抑制敏感型菌株LbS238N、LbS238A和Lb270的生长,说明ECMF对铝胁迫的生长反应可能是筛选抗(耐)铝的指标之一。在铝胁迫条件下,无论是抗(耐)型还是敏感型菌种(株),都会发生一系列有益于抗(耐)铝的生化反应,包括草酸分泌量、菌丝磷和钾含量增加,H+分泌改变等。在培养液中,草酸电离产生的H+仅占H+总浓度的少数,说明溶液中H+的主要来源不是ECMF所分泌的草酸,而是菌丝细胞为保持吸收阳离子的电荷平衡排出的H+或分泌的其它有机酸。     

High genetic diversity in a population of the ectomycorrhizal basidiomycete laccaria amethystina in a 150-year-old beech forest

The genetic structure of a population of the ectomycorrhizal basidiomycete Laccaria amethystina (Bolt. ex Hooker) Murr. was assessed in a closed 150-year-old beech (Fagus sylvatica L.) forest in the Vosges Mountains in northeastern France. During the autumn of 1994 and 1997, sporophores were collected from three 100-m2 sampling plots located along a 120-m transect crossing the beech stand. The genetic variation of 676 sporophores was initially estimated using heteroduplex analysis of the ribosomal DNA intergenic spacer (IGS1). Ten unique IGS1 heteroduplex/homoduplex patterns were identified, although three types represented most of the sporophores analysed. Each group of IGS1 type was then analysed using random amplified microsatellite analysis (RAMS). RAMS resolved 388 different genotypes amongst the 634 sporophores analysed from the three plots during the autumn of 1994 and 1997. Density as high as 130 genets per 100 m2 was observed during the autumn of 1994. The largest clone covered approximately 1 m2, but most genets covered a few cm2 and produced only one to three sporophores. Only eight genotypes identified in 1994 were found in 1997. Although L. amethystina has the capacity for vegetative persistence, the present study indicates that its populations maintain a genetic structure more consistent with a high frequency of sexual reproduction. This suggests that beech trees could be recolonized by new genotypes each year. Alternatively, this spatial distribution may also arise from erratic fruiting of underground persistent genets. These features (i.e. numerous genets of small size), typical of ruderal species, contrast with studies carried out on other ectomycorrhizal basidiomycetes occurring in mature closed forests.     

Ectomycorrhizal mycelia reduce bacterial activity in a sandy soil

Abstract: Bacterial activity was studied in a growth system containing Pinus contorta seedlings inoculated with different mycorrhizal fungi. Nylon nets enabled separation of soil compartments with extramatrical mycorrhizal hyphae from soil compartments with roots and mycelium. In three separate experiments bacterial activity, estimated as thymidine incorporation, was reduced in soils with Paxillus involutus hyphae compared to controls without mycorrhizal hyphae. This effect was found irrespective of compartments with and without roots were compared. Laccaria bicolor only reduced the activity in one of these three experiments. Thelephora terrestris (tested in two experiments), Laccaria proxima, Suillus variegatus and Hebeloma crustuliniforme (one experiment), also reduced the thymidine and leucine incorporation rates of bacteria. The reduction for these fungi varied between 20% and 50% in all experiments. Numbers of viable bacteria appeared to be reduced by T. terrestris, L. proxima, S. variegatus and H. crustuliniforme in one experiment, while no effect was seen in the other experiments.     

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.     

离体条件下日本冷杉/二色蜡蘑的外生菌根合成

本实验报道了以离体培养方式诱导日本冷杉(Abies firma Sieb.et Zucc.)与二色蜡蘑(Laccaria bicolor(Maire)Orton)的外生菌根合成的方法.日本冷杉能否与广谱性的外生菌根菌二色蜡蘑形成人工菌根?菌根结构的诱导是否只发生在植物体整体水平上?这些是本实验的关注点.研究结果显示:无菌幼苗接种10周后,在光学显微镜下可观察到典型的外生菌根的特征,即由高度分叉的外延菌丝体形成的厚实的菌套以及由内延菌丝体形成的哈蒂氏网.将来源于下胚轴的愈伤组织与二色蜡蘑的菌丝体进行共同培养,3周后,菌丝体开始接触愈伤组织的表面,并且侵入到愈伤组织的细胞间,形成拟哈蒂氏网结构.愈伤组织可以作为一种菌根学研究的有效的培养体系.这一方法可望为冷杉类植物菌根学的研究提供有效的实验工具.     

离体条件下日本冷杉／二色蜡蘑的外生菌根合成

本实验报道了以离体培养方式诱导日本冷杉(AbiesfirmaSieb.etZucc.)与二色蜡蘑(Laccariabicolor(Maire)Orton)的外生菌根合成的方法。日本冷杉能否与广谱性的外生菌根菌二色蜡蘑形成人工菌根?菌根结构的诱导是否只发生在植物体整体水平上?这些是本实验的关注点。研究结果显示:无菌幼苗接种10周后,在光学显微镜下可观察到典型的外生菌根的特征,即由高度分叉的外延菌丝体形成的厚实的菌套以及由内延菌丝体形成的哈蒂氏网。将来源于下胚轴的愈伤组织与二色蜡蘑的菌丝体进行共同培养,3周后,菌丝体开始接触愈伤组织的表面,并且侵入到愈伤组织的细胞间,形成拟哈蒂氏网结构。愈伤组织可以作为一种菌根学研究的有效的培养体系。这一方法可望为冷杉类植物菌根学的研究提供有效的实验工具。