Comparative genomics reveals a dynamic genome evolution in the ectomycorrhizal milk-cap (Lactarius) mushrooms.

Ectomycorrhizal fungi play a key role in forests by establishing mutualistic symbioses with woody plants. Genome analyses have identified conserved symbiosis-related traits among ectomycorrhizal fungal species, but the molecular mechanisms underlying host specificity remain poorly known. We sequenced and compared the genomes of seven species of milk-cap fungi (Lactarius, Russulales) with contrasting host specificity. We also compared these genomes with those of symbiotic and saprotrophic Russulales species, aiming to identify genes involved in their ecology and host specificity. The size of Lactarius genomes is significantly larger than other Russulales species, owing to a massive accumulation of transposable elements and duplication of dispensable genes. As expected, their repertoire of genes coding for plant cell wall-degrading enzymes is restricted, but they retained a substantial set of genes involved in microbial cell wall degradation. Notably, Lactarius species showed a striking expansion of genes encoding proteases, such as secreted ectomycorrhiza-induced sedolisins. A high copy number of genes coding for small secreted LysM proteins and Lactarius-specific lectins were detected, which may be linked to host specificity. This study revealed a large diversity in the genome landscapes and gene repertoires within Russulaceae. The known host specificity of Lactarius symbionts may be related to mycorrhiza-induced species-specific genes, including secreted sedolisins.

Bacteria of the genus Rhodopseudomonas (Bradyrhizobiaceae): obligate symbionts in mycelial cultures of the black truffles Tuber melanosporum and Tuber brumale.

BACKGROUND: This work aimed at characterizing 12 isolates of the genus Tuber including Tuber melanosporum (11 isolates) and Tuber brumale (one isolate). This was done using internal transcribed spacer (ITS) sequences, confirming their origin. RESULTS: Analysis of their mating type revealed that both MAT1-1 and MAT1-2 exist within these isolates (with 3 and 8 of each, respectively). We observed that each of these cultures was consistently associated with one bacterium that was intimately linked to fungal growth. These bacterial associates failed to grow in the absence of fungus. We extracted DNA from bacterial colonies in the margin of mycelium and sequenced a nearly complete 16S rDNA gene and a partial ITS fragment. We found they all belonged to the genus Rhodopseudomonas, fitting within different phylogenetic clusters. No relationships were evidenced between bacterial and fungal strains or mating types. Rhodopseudomonas being a sister genus to Bradyrhizobium, we tested the nodulation ability of these bacteria on a promiscuously nodulating legume (Acacia mangium), without success. We failed to identify any nifH genes among these isolates, using two different sets of primers. CONCLUSIONS: While the mechanisms of interaction between Tuber and Rhodopseudomonas remain to be elucidated, their interdependency for in vitro growth seems a novel feature of this fungus.

Local NO3- or NH4+ supply modifies the root system architecture of Cedrus atlantica seedlings grown in a split-root device.

To study the effects of local nitrate or ammonium supply on the architecture of the Cedrus atlantica root system, cedar seedlings were grown in split-root boxes in a growth chamber. In each box-compartment, roots were fertilized with a solution containing nitrogen, either as nitrate [Ca(NO(3))(2)] or ammonium (NH(4)Cl), supplied at 0.1 or 5.0mM. For each seedling, the shoot growth was measured twice a week for 3 months. The root system architecture was also recorded twice a week by tracing the root elongation through the transparent face of the root observation boxes. The apical diameter of the tap-root relay and that of a representative sample of lateral roots were recorded once a month using a monocular magnifier. The increase of ammonium or nitrate concentration in the nutrient solution has significantly enhanced the production of lateral roots on the tap-root relay. After 90 days of culture, percentages of short lateral roots obtained with nitrate were higher than those obtained using ammonium. A preferential carbon allocation to the shoots was also obtained with an increasing nitrogen supply. Until the 40th day of culture, the elongation of lateral roots was similar for all treatments and ranged from 0.25 to 0.5 cm day(-1). From the 40th day to the 95th day, significant differences were observed between the compared modes and maximum elongation rates were obtained with 5mM NH(4)(+) (2.18 cm day(-1)) and 5mM NO(3)(-) (1.18 cm day(-1)). Local applications of nitrate and ammonium at a low or a high concentration had local effects on elongation and branching of the root system in the fertilized compartment. Contrasting effects of ammonium and nitrate were observed on the apical diameter of tap-roots and lateral roots. The root-split culture device confirmed that nitrate had local effects on the architecture of the C. atlantica root system.

Molecular markers detecting an ectomycorrhizal Suillus collinitus strain on Pinus halepensis roots suggest successful inoculation and persistence in Mediterranean nursery and plantation.

Survival of the ectomycorrhizal fungal strain Suillus collinitus Sc-32 on Pinus halepensis after inoculation and outplanting was monitored in a Mediterranean plantation. Three molecular fingerprints were developed: RFLP of the internal transcribed spacer ribosomal DNA, intersimple sequence repeat, and a specific sequence-characterized amplified region marker. The inoculant was demonstrated to survive on inoculated seedlings 4 years after outplanting (56 months after inoculation), although S. collinitus was not fruiting. The designed markers set allows reliable and inexpensive monitoring of inoculated seedlings and suggests that S. collinitus is suitable for inoculation of Mediterranean Pinus. These data are discussed in the framework of suilloid population ecology.

Diversity of ectomycorrhizal fungi naturally established on containerised Pinus seedlings in nursery conditions.

The study examined the diversity of ectomycorrhizal fungi, naturally established on roots of containerised Pinus seedlings in a nursery, using PCR-RFLP and sequencing of the nuclear ribosomal internal transcribed spacer. Seventy-two samples, including ectomycorrhizae and fruit bodies, were examined. Molecular typing assigned the fungal symbionts to four ectomycorrhizal Boletales: Rhizopogon rubescens, Suillus bovinus, S. variegatus, and R. luteolus. R. rubescens was abundant (37.5%), while Suillus and R. luteolus species were moderately established (25-26%) and rare (2.8%), respectively. In addition, Rhizopogon species colonised P. nigra ssp. salzmannii seedlings, whereas Suillus species were identified on Pinus nigra ssp. nigra seedlings. The diversity and the ability of these naturally established symbionts under artificial nursery conditions were discussed. The molecular survey investigated here should contribute to successful monitoring of mycorrhizal application under both nursery and plantation conditions.

The ectomycorrhizal symbiosis between Lactarius deliciosus and Pinus sylvestris in forest soil samples: symbiotic efficiency and development on roots of a rDNA internal transcribed spacer-selected isolate of L. deliciosus.

The effect on plant growth of pre-inoculation of Pinus sylvestris with the ectomycorrhizal (ECM) edible basidiomycete Lactarius deliciosus (isolate D45) under controlled conditions, and the development on roots of this basidiomycete, were investigated in gamma-irradiated and unsterilized containers containing different forest soil cores or a perlite-vermiculite mixture. Five months after planting, L. deliciosus mycorrhizal plants exhibited greater growth than the non-mycorrhizal ones in all soil types, i.e. up to a 325% increase in shoot height in the sterilized soils. The experiment demonstrated the dependency of P. sylvestris seedlings upon ECM symbiosis for their survival in gamma-irradiated, microbiologically disturbed soil samples. Furthermore, in two soils, the growth of L. deliciosus-inoculated seedlings was greater in the sterilized soil samples than in the non-sterilized ones, i.e. 46% and 132% increase in shoot height under sterilized soil conditions. In containers randomly sampled from each soil type, the degree of root colonization by the inoculated isolate, calculated as the number of mycorrhizal root tips divided by the total number of root tips x100, ranged from 80% to 35%. Within the short term, the inoculated isolate developed rapidly on roots, dominated, and hampered ectomycorrhiza formation by various unidentified (but not Lactarius) resident ECM fungi in unsterilized soil types. Results indicate that the ECM species L. deliciosus is worth investigating to ascertain if other isolates benefit pine growth like the isolate D45, and are therefore also attractive candidates for forestry applications in the Mediterranean area.