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 μm 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.     

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.     

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。     

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.     

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.     

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).     

Survival of an introduced ectomycorrhizal Laccaria bicolor strain in a European forest plantation monitored by mitochondrial ribosomal DNA analysis

Mitochondrial and nuclear genes have different inheritance, thus studies of fungal populations should use both mitochondrial and nuclear markers. Using nuclear markers, the S238N strain of the ectomycorrhizal basidiomycete Laccaria bicolor ((Maire) Orton) has been previously shown to persist for at least 10 yr after outplanting in a plantation of Douglas fir ( Pseudotsuga menziesii (Mir.) Franco) inoculated with this strain. In the present study, we have sampled 539 sporophores of Laccaria spp. from this plantation, some of which had the S238N nuclear genotype, to study mitochondrial DNA polymorphism and persistence of the inoculated S238N mitochondrial genome. Length polymorphism in fragments of the large subunit of mitochondrial ribosomal DNA (LrDNA) allowed distinction of the haplotypes present in the plantation at the species level. In addition, heteroduplex analysis and sequencing revealed intraspecific polymorphism of LrDNA among the L. bicolor sporophores and enabled specific identification of S238N LrDNA. This haplotype was only retained in sporophores carrying the S238N nuclear genome, confirming the survival of this introduced strain in a natural population.     

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.     

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.     

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.     

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.     

Polyamines in humic acid and their effect on radical growth of lettuce seedlings

Linum usitatissimum, Sorghum bicolor and Triticum aestivum plants were further colonised by the arbuscular mycorrhizal fungus, Glomus mosseae, during a four week period of hydroponic culture after a pre-culture period of three weeks with the fungus in perlite substrate. The viability of mycorrhizal colonisation of T. aestivum was indicated by an initial experiment where G. mosseae from mycorrhizal plants colonised non-mycorrhizal plants when the plants were grown together in the same hydroponic container using modified Long Ashton nutrient solution. Intermittant aeration of the plant roots (2 h periods, four times per day) provided a compromise between adequate aeration and minimal disturbance of the fungus. In a second experiment, two nutrient media, modified Long Ashton and modified Knop plus Hoagland medium were compared for culturing G. mosseae on T. aestivum. A significantly higher root dry weight was found for the mycorrhizal versus the non-mycorrhizal wheat plants in modified Long Ashton nutrient medium, which contained 10 µM P and an organic buffer. Modified Knop plus Hoagland nutrient medium contained a high P concentration (0.9 mM) and did not produce viable cultures of mycorrhizal colonisation. In a third experiment, modified Long Ashton medium was used for hydroponic culture of mycorrhizal L. usitatissimum, S. bicolor and T. aestivum. The root colonisation percentages for T. aestivum (73%), S. bicolor (36%) and L. usitatissimum (65%) were within the range of colonisation rates obtained with solid substrate culture in perlite. Viability of the mycorrhizal structures in hydroponic culture was assessed by monitoring activity of fungal succinate dehydrogenase and found to be similar to cultures in perlite. No difference in the P concentration of mycorrhizal and non-mycorrhizal plants was observed, possibly owing to the lack of diffusion limits for P in hydroponic solution. This report describes a system for the viable culture of G. mosseae with different plant species where a high mycorrhizal colonisation rate was produced under conditions of a short culture period using intermittent aeration, a low concentration of P supply and an organic buffer.     

New evidence for bacterial diversity in the ascoma of the ectomycorrhizal fungus Tuber borchii Vittad

The microbial community associated with ascocarps of the ectomycorrhizal fungus Tuber borchii Vittad. was studied by both cultivation and direct extraction of bacterial 16S rRNA gene (rDNA) sequence approaches. The inner part of six T. borchii ascoma collected in North-Central Italy was used to establish a bacterial culture collection and to extract the total genomic DNA to obtain a library of 16S rDNAs representative of the truffle bacterial community. Most of the isolates were affiliated to the gamma-Proteobacteria, mainly Fluorescent pseudomonads; some isolates were members of the Bacteroidetes group and Gram-positive bacteria, mostly Bacillaceae. The majority of the clones from the library were alpha-Proteobacteria showing significant similarity values, of greater than 97%, with members of the Sinorhizobium/Ensifer Group, Rhizobium and Bradyrhizobium spp. not previously identified as Tuber-associated bacteria. Only a few bacterial strains belonging to this bacterial subclass were found in the culture collection and isolated on a medium specific for Rhizobium-like organisms. A few clones were members of the beta- and gamma-Proteobacteria; as well as low and high G+C Gram-positive bacteria. Our findings clearly indicate that a dual approach increases the information obtained on the structural composition of a truffle bacterial community as compared to that derived via cultivation or direct recovery of 16S rDNA sequences alone.     

Impact of nitrate supply in C and N assimilation in the parasitic plant Striga hermonthica (Del.) Benth (Scrophulariaceae) and its host Sorghum bicolor L

The threshold of tolerance for nitrate of the parasitic weed Striga hermonthica (Del.) Benth and the host plant Sorghum bicolor L. was determined by estimating the impact of increasing nitrate loads on plant growth and various parameters of C and N assimilation. Nitrate supply improved chlorophyll (Chl) content and photosystem II (PSII) photochemistry of infected S. bicolor that, in comparison to S. hermonthica, displayed a low imbalance between C and N assimilation when nitrate was supplied up to 1500 mg N per plant. Indeed, nitrate supplies increased strongly the leaf N:C ratio of the parasite. The higher nitrate load induced strong accumulation of nitrate, nitrite and ammonium, and consequently the death of S. hermonthica. Nevertheless, lower nitrate loads (up to 500 mg N per S. bicolor in this study) promoted leaf expansion, PSII photochemistry and N metabolism of S. hermonthica mature (M) plants, as attested by the significant rise in soluble protein and free amino-acid contents. Following these N supplies, the nitrate tolerance of S. hermonthica was correlated with an increase in PSII activity and a high incorporation of N excess into asparagine. This confirmed the central role of asparagine in the N metabolism of S. hermonthica, although this detoxification pathway was insufficient to limit ammonium accumulation under higher nitrate loads.     

脱色希瓦氏菌(Shewanella decolorationis) S12还原不同电子受体的厌氧发酵罐培养方法

脱色希瓦氏菌Shewanella decolorationisS12在厌氧环境下能够使用多种电子受体进行厌氧呼吸。为了取得足够的细胞量用于膜蛋白质组学等科学研究的需要,本研究选取无机小分子(硝酸钠)、金属离子(柠檬酸铁)和有机大分子(偶氮染料苋菜红)作为电子受体,在使用确定成分的无机盐培养基条件下,使用不同浓度的电子供体和碳源对S12进行厌氧条件下静置和发酵罐的优化培养,采用连续补充电子受体的培养方式,确认了电子供体和碳源的合适浓度,建立了S12厌氧发酵罐培养方法。相比传统的静置厌氧培养,厌氧发酵罐培养方法在保证了严格厌氧条件下高效率还原电子受体的同时,还极大的提高了细胞生长密度。连续补充电子受体的厌氧发酵罐培养的S12最大细胞密度最大分别可达到静置厌氧培养细胞密度的325,304,369倍,而生长时间也比静置厌氧培养分别缩短了26.5%,17.6%,7.5%。这为需要大量细胞和蛋白的细菌厌氧呼吸生长实验建立了可行方法,对于进行兼性厌氧呼吸的微生物的大规模厌氧培养具有借鉴意义。     

The potential role of ectomycorrhizal fungi in determining Douglas-fir resistance to defoliation by the western spruce budworm (Lepidoptera: Tortricidae)

There is phenotypic variation among individual trees of interior Douglas-fir (Pseudotsuga menziesii var. glauca [Beissn.] Franco) in their resistance to defoliation by the western spruce budworm (Choristoneura occidentalis Freeman). We evaluated the potential role of ectomycorrhizal fungi in determining this resistance using half-sib seedlings derived from parent trees that are resistant versus susceptible to budworm defoliation in the field. The seedlings were inoculated with Laccaria bicolor ectomycorrhizal fungi, fertilized, or untreated. Approximately 48 d after treatment, late-instar larvae from a nondiapausing laboratory colony of C. occidentalis were allowed to feed on pairs of resistant versus susceptible seedlings for 1 wk. Chemical analyses of current-year shoots for nitrogen (N), phosphorus (P), magnesium (Mg), and zinc (Zn) indicated that the fungus increased foliar concentrations of P and Mg in resistant seedlings, but it did not increase their growth rate. However, L. bicolor had no effect on foliar concentrations of P or Mg in susceptible seedlings, even though seedling growth rates increased slightly in response to the inoculation. L. bicolor had no effect on foliar levels of N or Zn in any of the seedlings. As expected, fertilization increased levels of N and P in the foliage of both resistant and susceptible seedlings, but it did not affect levels of Mg and Zn. Surprisingly, the fertilizer treatment had no effect on seedling growth rates. Despite these differences, late-instar budworms showed no feeding preference among untreated, mycorrhizal, or fertilized seedlings. The fact that seedlings from resistant versus susceptible Douglas-firs responded differently to the L. bicolor treatment lends preliminary support to the hypothesis that ecotmycorrhizae might play a role in Douglas-fir resistance to damage from the western spruce budworm. Finally, differences in foliar concentrations of N and P among untreated seedlings from different maternal trees suggested that foliar nutritional chemistry is influenced by the tree's genotype.