Plant-mediated partner discrimination in ectomycorrhizal mutualisms.

Although ectomycorrhizal fungi have well-recognized effects on ecological processes ranging from plant community dynamics to carbon cycling rates, it is unclear if plants are able to actively influence the structure of these fungal communities. To address this knowledge gap, we performed two complementary experiments to determine (1) whether ectomycorrhizal plants can discriminate among potential fungal partners, and (2) to what extent the plants might reward better mutualists. In experiment 1, split-root Larix occidentalis seedlings were inoculated with spores from three Suillus species (S. clintonianus, S. grisellus, and S. spectabilis). In experiment 2, we manipulated the symbiotic quality of Suillus brevipes isolates on split-root Pinus muricata seedlings by changing the nitrogen resources available, and used carbon-13 labeling to track host investment in fungi. In experiment 1, we found that hosts can discriminate in multi-species settings. The split-root seedlings inhibited colonization by S. spectabilis whenever another fungus was available, despite similar benefits from all three fungi. In experiment 2, we found that roots and fungi with greater nitrogen supplies received more plant carbon. Our results suggest that plants may be able to regulate this symbiosis at a relatively fine scale, and that this regulation can be integrated across spatially separated portions of a root system.

Disentangling above- and below-ground facilitation drivers in arid environments: the role of soil microorganisms, soil properties and microhabitat.

Nurse plants promote establishment of other plant species by buffering climate extremes and improving soil properties. Soil biota plays an important role, but an analysis to disentangle the effects of soil microorganisms, soil properties and microclimate on facilitation is lacking. In three microhabitats (gaps, small and large Retama shrubs), we placed six microcosms with sterilized soil, two per soil origin (i.e. from each microhabitat). One in every pair received an alive, and the other a sterile, inoculum from its own soil. Seeds of annual plants were sown into the microcosms. Germination, survival and biomass were monitored. Soil bacterial communities were characterized by pyrosequencing. Germination in living Retama inoculum was nearly double that of germination in sterile inoculum. Germination was greater under Retama canopies than in gaps. Biomass was up to three times higher in nurse than in gap soils. Soil microorganisms, soil properties and microclimate showed a range of positive to negative effects on understory plants depending on species identity and life stage. Nurse soil microorganisms promoted germination, but the effect was smaller than the positive effects of soil properties and microclimate under nurses. Nurse below-ground environment (soil properties and microorganisms) promoted plant growth and survival more than nurse microhabitat.

Intraspecific variability of Lactarius deliciosus isolates: colonization ability and survival after cold storage.

Intraspecific variability in root colonization, extraradical growth pattern, and survival after cold storage of Lactarius deliciosus isolates was determined in pure culture conditions using Pinus pinaster as a host plant. The ectomycorrhizal ability of L. deliciosus at 30, 45, and 60 days from inoculation was highly variable among isolates and was negatively correlated to the age of the culture (time elapsed from isolation). The formation of rhizomorphs was related to colonization ability, but no relationship was found between colonization and formation of extraradical mycelium. The final colonization achieved at 60 days from inoculation was not related to the tree species under which the sporocarps were collected. However, isolates from sporocarps collected under P. pinaster colonized more rapidly the seedlings than those collected under other pine species. The climatic range of the sporocarps from which the isolates were obtained (maritime vs. continental) was not related to the formation of mycorrhizas at 60 days from inoculation. However, isolates from sporocarps collected from a maritime climate area colonized more rapidly the P. pinaster seedlings than those collected from a continental zone. Tolerance to cold water storage of L. deliciosus was also isolate dependent. Growth revival in agar was obtained from most of the isolates after 28 months of cold storage at 4°C, but only 10 out of 29 isolates showed unaffected growth. The ITS rDNA alignment of all the L. deliciosus isolates showed a low variability with identities over 99%. Most of the variation was detected in the ITS1 region and consisted in single nucleotide changes and/or punctual indel mutations. The number of base differences per sequence from averaging over all sequence pairs was 1.329, which is in the low range when compared with other ectomycorrhizal species. No ITS pattern due to geographical origin of the isolates could be discerned.

Role of plant-fungal nutrient trading and host control in determining the competitive success of ectomycorrhizal fungi.

Multiple ectomycorrhizal fungi (EMF) compete to colonise the roots of a host plant, but it is not known whether their success is under plant or fungal control, or a combination of both. We assessed whether plants control EMF colonisation by preferentially allocating more carbon to more beneficial partners in terms of nitrogen supply or if other factors drive competitive success. We combined stable isotope labelling and RNA-sequencing approaches to characterise nutrient exchange between the plant host Eucalyptus grandis and three Pisolithus isolates when growing alone and when competing either indirectly (with a physical barrier) or directly. Overall, we found that nitrogen provision to the plant does not explain the amount of carbon that an isolate receives nor the number of roots that it colonises. Differences in nutrient exchange among isolates were related to differences in expression of key fungal and plant nitrogen and carbon transporter genes. When given a choice of partners, the plant was able to limit colonisation by the least cooperative isolate. This was not explained by a reduction in allocated carbon. Instead, our results suggest that partner choice in EMF could operate through the upregulation of defence-related genes against those fungi providing fewer nutrients.

Molecular identification of the edible ectomycorrhizal fungus Lactarius deliciosus in the symbiotic and extraradical mycelium stages.

Specific rDNA ITS amplifications, microsatellite-primed PCR and ITS-SSCP analysis were applied to identify and characterize pre-selected isolates of the edible ectomycorrhizal fungus Lactarius deliciosus in different stages of the life cycle. Sampling was performed from pure cultures, mycorrhizas and soil from experimental plots established with nursery-inoculated pine seedlings. A newly-designed reverse primer (LDITS2R) combined with the universal forward ITS1 allowed to perform specific amplifications of L. deliciosus from all the samples. Microsatellite-primed PCR using the (GTG)5 oligonucleotide as a primer showed clear polymorphisms among the different L. deliciosus isolates. The patterns of mycorrhiza samples showed additional bands corresponding to the plant DNA. Single strand conformation polymorphism (SSCP) analysis of the specific rDNA ITS fragment amplified from 18 L. deliciosus isolates showed nine clearly different patterns. Mycorrhiza and soil samples showed coincident patterns with their respective fungal isolates. Specific rDNA ITS amplifications had not been previously used for SSCP analysis of ectomycorrhizas and extraradical mycelium. This relatively simple and inexpensive technique allows tracking L. deliciosus isolates in different stages of the fungus development. Specific ITS-SSCP analysis is promising in studies of the persistence of inoculated L. deliciosus isolates and their competitiveness with native ectomycorrhizal fungi, especially at the extraradical mycelium stage.

Environmental and Geographical Factors Structure Soil Microbial Diversity in New Caledonian Ultramafic Substrates: A Metagenomic Approach.

Soil microorganisms play key roles in ecosystem functioning and are known to be influenced by biotic and abiotic factors, such as plant cover or edaphic parameters. New Caledonia, a biodiversity hotspot located in the southwest Pacific, is one-third covered by ultramafic substrates. These types of soils are notably characterised by low nutrient content and high heavy metal concentrations. Ultramafic outcrops harbour diverse vegetation types and remarkable plant diversity. In this study, we aimed to assess soil bacterial and fungal diversity in New Caledonian ultramafic substrates and to determine whether floristic composition, edaphic parameters and geographical factors affect this microbial diversity. Therefore, four plant formation types at two distinct sites were studied. These formations represent different stages in a potential chronosequence. Soil cores, according to a given sampling procedure, were collected to assess microbial diversity using a metagenomic approach, and to characterise the physico-chemical parameters. A botanical inventory was also performed. Our results indicated that microbial richness, composition and abundance were linked to the plant cover type and the dominant plant species. Furthermore, a large proportion of Ascomycota phylum (fungi), mostly in non-rainforest formations, and Planctomycetes phylum (bacteria) in all formations were observed. Interestingly, such patterns could be indicators of past disturbances that occurred on different time scales. Furthermore, the bacteria and fungi were influenced by diverse edaphic parameters as well as by the interplay between these two soil communities. Another striking finding was the existence of a site effect. Differences in microbial communities between geographical locations may be explained by dispersal limitation in the context of the biogeographical island theory. In conclusion, each plant formation at each site possesses is own microbial community resulting from multiple interactions between abiotic and biotic factors.

Field persistence of the edible ectomycorrhizal fungus Lactarius deliciosus: effects of inoculation strain, initial colonization level, and site characteristics.

Pinus pinea plants were inoculated with different strains of the edible ectomycorrhizal fungus Lactarius deliciosus. The inoculated plants were established in six experimental plantations in two sites located in the Mediterranean area to determine the effect of the initial colonization level and the inoculated strain on fungal persistence in the field. Ectomycorrhizal root colonization was determined at transplantation time and monitored at different times from uprooted plants. Extraradical soil mycelium biomass was determined from soil samples by TaqMan(R) real-time polymerase chain reaction (PCR). The results obtained indicate that the field site played a decisive role in the persistence of L. deliciosus after outplanting. The initial colonization level and the selection of the suitable strain were also significant factors but their effect on the persistence and spread of L. deliciosus was conditioned by the physical-chemical and biotic characteristics of the plantation soil and, possibly, by their influence in root growth. Molecular techniques based on real-time PCR allowed a precise quantification of extraradical mycelium of L. deliciosus in the field. The technique is promising for non-destructive assessment of fungal persistence since soil mycelium may be a good indicator of root colonization. However, the accuracy of the technique will ultimately depend on the development of appropriate soil sampling methods because of the high variability observed.

Tracking mycorrhizas and extraradical mycelium of the edible fungus Lactarius deliciosus under field competition with Rhizopogon spp.

The objective of this study is to evaluate the field persistence of the edible ectomycorrhizal fungus Lactarius deliciosus in competition with two ubiquitous soil fungi. Couples of plants inoculated with either L. deliciosus, Rhizopogon roseolus, or R. luteolus were transplanted, 10 cm apart, in two different sites at the following combinations: L. deliciosus-R. roseolus, L. deliciosus-R. luteolus, L. deliciosus-control (non-inoculated), control-R. roseolus, control-R. luteolus, and control-control. Eight months after transplantation, root colonization and extraradical soil mycelium for each fungal species were quantified. For mycelium quantification, soil cores equidistant to the two plants in each couple were taken, and total deoxyribonucleic acid (DNA) was extracted. Real-time polymerase chain reaction analysis was performed using specific primers and TaqMan Minor groove binding (MGB) probes designed in the ribosomal DNA internal transcribed spacer region of each fungal species. Field site significantly influenced persistence of both mycorrhizas and extraradical mycelium of L. deliciosus. Extraradical mycelium quantity was positively correlated with the final percentage of ectomycorrhizas for the three fungal species. Different competitive pressure between the two Rhizopogon species on L. deliciosus persistence was observed, with R. luteolus having no effect on L. deliciosus survival. Negative correlation between the final percentage of mycorrhizas of L. deliciosus and R. roseolus was observed. However, no relationship was determined between extraradical mycelia of both fungal species. The results obtained suggest that competition between L. deliciosus and R. roseolus takes place in the root system, for ectomycorrhiza formation in available roots, rather than in the extraradical phase.

Determining the outcome of field-based competition between two Rhizopogon species using real-time PCR.

Interest in the ecology of ectomycorrhizal (ECM) fungi has increased considerably, but little is known about interspecific interactions among ECM species. We examined competitive interactions between Rhizopogon occidentalis and R. salebrosus at Point Reyes National Seashore, California, USA. At three field sites, species abundances were compared in single- and two-species treatments on Pinus muricata seedlings inoculated with spores. Competition for root tips was assessed using real-time polymerase chain reaction (PCR) of internal transcribed spacer rDNA. In general, we found strong competitive exclusion of R. salebrosus by R. occidentalis, with >or= 75% of the seedlings in the two-species treatment colonized exclusively by R. occidentalis after 5 and 10 months. However, on the seedlings that were co-colonized, we observed no significant difference in the abundances of R. salebrosus and R. occidentalis, suggesting that once R. salebrosus was established, it was no longer competitively inferior. There were no significant differences in survival, growth, or percentage leaf nitrogen of seedlings colonized with either Rhizopogon species, but both growth and percentage leaf nitrogen were significantly higher for ECM than non-ECM seedlings. We also observed strong positive correlations between actual ECM root tip weight and that inferred from real-time PCR for both species, indicating that this method provided an accurate assessment of root tip occupation and hence ECM competitive dynamics. In conjunction with a previous experiment, our results indicate that competition between these two Rhizopogon species occurs similarly in both field and laboratory settings and that when colonizing from spore, timing largely determines the outcome of initial competitive interactions.