Cell wall proteins of the ectomycorrhizal basidiomycete Pisolithus tinctorius: identification, function, and expression in symbiosis.

Specific cell-cell and cell-substrate interactions direct the growth of ectomycorrhizal fungi to their host root targets. These elaborate mechanisms lead to the differentiation of distinct multihyphal structures, the mantle, and the Hartig net. In the ectomycorrhizal basidiomycete Pisolithus tinctorius, the use of two-dimensional gel electrophoresis, immunocytochemical microscopy, and RNA blot analysis has demonstrated the differential expression of cell wall proteins (CWPs), such as hydrophobins, adhesins, and mannoproteins, during symbiotic interaction. In other fungi, these CWPs have been suggested to play a role in hyphae aggregation, intracellular signaling cascades, and cytoskeletal changes. The recent cloning of the genes for several of these CWPs in P. tinctorius allows us to address their function in symbiosis. This review summarizes our knowledge of CWPs in P. tinctorius and considers parallels with other biotrophic fungi as a possible framework for future work.     

Cytoskeleton and mitochondrial morphology of saprotrophs and the pathogen Heterobasidion annosum in the presence of Suillus bovinus metabolites

Ectomycorrhizal fungi are known to synthesize antifungal compounds both in vitro and in symbiosis with the host-plants. Culture filtrates of the ectomycorrhizal fungus Suillus bovinus (at pHs of 2.5–6) showed antifungal activity towards saprotrophs Trichoderma harzianum, and Trichoderma virens and the pathogen Heterobasidion annosum, by significantly suppressing their growth relative to sterile liquid medium at the same pHs. In the presence of the culture filtrates, hyphae of the saprotrophs and the pathogen were characterized by distensions, irregular and frequent branching, tip damage and cytoplasm coagulation. Since hyphal abnormalities may be evoked by disruptions in the cytoskeleton and mitochondria, their structural changes were also examined. Depolymerization of microtubules was confirmed for all of the fungi. Serious damage to mitochondria morphology may cause significant functional impairment. Growth of mycelia was inhibited in the lower pH S. bovinus culture filtrate, and the mitochondrial morphology was altered. This suggests that the activity of antifungal compounds synthesized by ectomycorrhizal fungus is significantly affected by pH.     

Host‐ and stage‐dependent secretome of the arbuscular mycorrhizal fungus Rhizophagus irregularis

Arbuscular mycorrhizal fungi form the most wide‐spread endosymbiosis with plants. There is very little host specificity in this interaction, however host preferences as well as varying symbiotic efficiencies have been observed. We hypothesize that secreted proteins (SPs) may act as fungal effectors to control symbiotic efficiency in a host‐dependent manner. Therefore, we studied whether arbuscular mycorrhizal (AM) fungi adjust their secretome in a host‐ and stage‐dependent manner to contribute to their extremely wide host range. We investigated the expression of SP‐encoding genes of Rhizophagus irregularis in three evolutionary distantly related plant species, Medicago truncatula, Nicotiana benthamiana and Allium schoenoprasum. In addition we used laser microdissection in combination with RNA‐seq to study SP expression at different stages of the interaction in Medicago. Our data indicate that most expressed SPs show roughly equal expression levels in the interaction with all three host plants. In addition, a subset shows significant differential expression depending on the host plant. Furthermore, SP expression is controlled locally in the hyphal network in response to host‐dependent cues. Overall, this study presents a comprehensive analysis of the R. irregularis secretome, which now offers a solid basis to direct functional studies on the role of fungal SPs in AM symbiosis.     

Cytochemical evidence of a fungal cell wall alteration during infection of Eucalyptus roots by the ectomycorrhizal fungus Cenococcum geophilum

When the ectomycorrhizal fungus Cenococcum geophilum changes from a saprophytic to a symbiotic stage, its cell wall structure becomes simplified. The external hyphal wall layer which, in the saprophytic stage, is highly reactive to the Gomori-Swift test becomes poorly reactive and can no longer be distinguished from the internal wall layer in the Hartig net hyphae. The intensely stained external wall layer was also absent from pure cultures of Cenococcum geophilum grown on a medium with a low sugar content. This cell wall alteration could be due to a decrease in the amount of melanin or of melanin plus cystine-containing proteins. This change may be necessary for increased nutrient exchange between symbionts through hyphal walls.     

Hyphal mats formed by two ectomycorrhizal fungi and their association with Douglas-fir seedlings: A case study

The ectomycorrhizal fungi Gautieria monticola and Hysterangium setchellii both form dense hyphal mats in coniferous forest soils of the Pacific Northwest. We recently observed that all Douglas-fir seedlings found under the canopy of a maturing 60–75 year stand were associated with mats formed by ectomycorrhizal fungi. The significance of these mat communities in relation to seedling establishment and survival is discussed.     

Breeding for better symbiosis

The present review gives a critical assessment of the literature dealing with symbiosis between rhizobia and legumes and between AM fungi and most plants. Associative N₂ fixation (even though strictly speaking not a symbiotic relationship) does have some characteristics of symbiosis due to mutualistic dependence and usefulness of the relationship, and is therefore covered in this review. Nodulation in the rhizobia–legume symbiosis may be limited by an insufficient amount of the nod-gene inducers released from seed and/or roots. However, there is genotypic variation in the germplasm of legume species in all components of the signalling pathway, suggesting a prospect for improving nodulation by selecting and/or transforming legume genotypes for increased exudation of flavonoids and other signalling compounds. Deciphering chromosomal location as well as cloning nod, nif and other genes important in nodulation and N₂ fixation will allow manipulation of the presence and expression of these genes to enhance the symbiotic relationship. Increased efficacy of symbiotic N₂ fixation can be achieved by selecting not only the best host genotypes but by selecting the best combination of host genotype and nodule bacteria. As flavonoids exuded by legume seedlings may not only be nod-gene inducers, but also stimulants for hyphal growth of the AM fungi, selecting and/or transforming plants to increase exudation of these flavonoids may result in a double benefit for mycorrhizal legumes. Mutants unable to sustain mycorrhizal colonisation are instrumental in understanding the colonisation process, which may ultimately pay off in breeding for the more effective symbiosis. In conclusion, targeted efforts to breed genotypes for improved N₂ fixation and mycorrhizal symbiosis will bring benefits in increased yields of crops under a wide range of environmental conditions and will contribute toward sustainability of agricultural ecosystems in which soil-plant-microbe interactions will be better exploited.     

Arbuscular mycorrhizas and ectomycorrhizas of <Emphasis Type="Italic">Uapaca bojeri</Emphasis> L. (Euphorbiaceae): sporophore diversity,patterns of root colonization,and effects on seedling growth and soil microbial catabolic diversity

The main objectives of this study were (1) to describe the diversity of mycorrhizal fungal communities associated with Uapaca bojeri, an endemic Euphorbiaceae of Madagascar, and (2) to determine the potential benefits of inoculation with mycorrhizal fungi [ectomycorrhizal and/or arbuscular mycorrhizal (AM) fungi] on the growth of this tree species and on the functional diversity of soil microflora. Ninety-four sporophores were collected from three survey sites. They were identified as belonging to the ectomycorrhizal genera Afroboletus, Amanita, Boletus, Cantharellus, Lactarius, Leccinum, Rubinoboletus, Scleroderma, Tricholoma, and Xerocomus. Russula was the most frequent ectomycorrhizal genus recorded under U. bojeri. AM structures (vesicles and hyphae) were detected from the roots in all surveyed sites. In addition, this study showed that this tree species is highly dependent on both types of mycorrhiza, and controlled ectomycorrhization of this Uapaca species strongly influences soil microbial catabolic diversity. These results showed that the complex symbiotic status of U. bojeri could be managed to optimize its development in degraded areas. The use of selected mycorrhizal fungi such the Scleroderma Sc1 isolate in nursery conditions could be of great interest as (1) this fungal strain is very competitive against native symbiotic microflora, and (2) the fungal inoculation improves the catabolic potentialities of the soil microflora.     

Arbuscular mycorrhizal fungi: a specialised niche for rhizospheric and endocellular bacteria

Arbuscular mycorrhizal (AM) fungi produce an extensive hyphal network which develops in the soil, producing a specialised niche for bacteria. The aim of this paper is to review briefly the interactions shown by these symbiotic fungi with two bacterial groups: (i) the plant-growth promoting rhizobacteria (PGPRs) which are usually associated with fungal surfaces in the rhizosphere, and (ii) a group of endocellular bacteria, previously identified as being related to Burkholderia on the basis of their ribosomal sequence strains. The endobacteria have been found in the cytoplasm of some isolates of AM fungi belonging to Gigasporaceae and offer a rare example of bacteria living in symbiosis with fungi. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nitrogen Uptake by Trees and Mycorrhizal Fungi in a Successional Northern Temperate Forest: Insights from Multiple Isotopic Methods

Forest succession may cause changes in nitrogen (N) availability, vegetation and fungal community composition that affect N uptake by trees and their mycorrhizal symbionts. Understanding how these changes affect the functioning of the mycorrhizal symbiosis is of interest to ecosystem ecology because of the fundamental roles mycorrhizae play in providing nutrition to trees and structuring forest ecosystems. We investigated changes in tree and mycorrhizal fungal community composition, the availability and uptake of N by trees and mycorrhizal fungi in a forest undergoing a successional transition (age-related loss of early successional tree taxa). In this system, 82–96% of mycorrhizal hyphae were ectomycorrhizal (EM). As biomass production of arbuscular mycorrhizal (AM) trees increased, AM hyphae comprised a significantly greater proportion of total fungal hyphae, and the EM contribution to the N requirement of EM-associated tree taxa declined from greater than 75% to less than 60%. Increasing N availability was associated with lower EM hyphal foraging and ¹⁵N tracer uptake, yet the EM-associated later-successional species Quercus rubra was nonetheless a stronger competitor for ¹⁵N than AM-associated Acer rubrum, likely due to the more extensive nature of the persistent EM hyphal network. These results indicate that successional increases in N availability and co-dominance by AM-associated trees have increased the importance of AM fungi in the mycorrhizal community, while down-regulating EM N acquisition and transfer processes. This work advances understanding of linkages between tree and fungal community composition, and indicates that successional changes in N availability may affect competition between tree taxa with divergent resource acquisition strategies.     

Dual colonization of Eucalyptus urophylla S.T. Blake by arbuscular and ectomycorrhizal fungi affects levels of insect herbivore attack

Abstract 1 Eucalypts are an important part of plantation forestry in Asia but, in south China, productivity is very low. This is due to infertile soils and lack of indigenous symbiotic mycorrhizal fungi. The genus Eucalyptus is unusual because it forms both arbuscular (AM) and ectomycorrhizal (ECM) associations. 2 Eucalyptus urophylla saplings were grown with and without AM (Glomus caledonium) and ECM (Laccaria laccata) fungi in a factorial design. Two experiments were performed: one to simulate nursery conditions and the other to simulate the early stages of plantation establishment. Plant growth was measured over 18 weeks and levels of insect attack were recorded. 3 The AM fungus reduced tree growth in the early stages, but the effect appeared to be transient. No effects of ECM were detected on tree growth, but the ectomycorrhiza reduced colonization by the arbuscular mycorrhiza. AM fungi appear to be rapid invaders of the root system, gradually being replaced by ECM. 4 Both fungal types affected levels of damage by insect herbivores. Most importantly, herbivory by the pest insects Anomala cupripes (Coleoptera) and Strepsicrates spp. (Lepidoptera) was decreased by ECM. 5 It is suggested that mycorrhizal effects on eucalypt insects may be determined by carbon allocation within the plant. Future studies of eucalypt mycorrhizas need to take into account the effects of the fungi on foliar‐feeding insects and also the effects of insect herbivory on mycorrhizal establishment.     

First evidences that the ectomycorrhizal fungus Paxillus involutus mobilizes nitrogen and carbon from saprotrophic fungus necromass

Fungal succession in rotting wood shows a surprising abundance of ectomycorrhizal (EM) fungi during the late decomposition stages. To better understand the links between EM fungi and saprotrophic fungi, we investigated the potential capacities of the EM fungus Paxillus involutus to mobilize nutrients from necromass of Postia placenta, a wood rot fungus, and to transfer these elements to its host tree. In this aim, we used pure cultures of P. involutus in the presence of labelled Postia necromass (¹⁵N/¹³C) as nutrient source, and a monoxenic mycorrhized pine experiment composed of labelled Postia necromass and P. involutus culture in interaction with pine seedlings. The isotopic labelling was measured in both experiments. In pure culture, P. involutus was able to mobilize N, but C as well, from the Postia necromass. In the symbiotic interaction experiment, we measured high ¹⁵N enrichments in all plant and fungal compartments. Interestingly, ¹³C remains mainly in the mycelium and mycorrhizas, demonstrating that the EM fungus transferred essentially N from the necromass to the tree. These observations reveal that fungal organic matter could represent a significant N source for EM fungi and trees, but also a C source for mycorrhizal fungi, including in symbiotic lifestyle.     

Different soil moisture conditions change the outcome of the ectomycorrhizal symbiosis between Rhizopogon species and Pinus muricata

The outcome of species interactions often depends on the environmental conditions under which they occur. In this study, we tested how different soil moisture conditions affected the outcome of the ectomycorrhizal symbiosis between three Rhizopogon species and Pinus muricata in a factorial growth chamber experiment. We found that when grown in 7% soil moisture conditions, ectomycorrhizal plants had similar biomass, photosynthesis, conductance, and total leaf nitrogen as non-mycorrhizal plants. However, when grown at 13% soil moisture, ectomycorrhizal plants had significantly greater shoot biomass, higher photosynthetic and conductance rates, and higher total leaf nitrogen than non-mycorrhizal plants. The differences in plant response by mycorrhizal status in the two soil moisture treatments corresponded with evidence of water limitation experienced by the fungi, which had much lower colonization at 7% compared to 13% soil moisture. Our results suggest that the outcome of the ectomycorrhizal symbiosis can be context-dependent and that fluctuating environmental conditions may strongly affect the way plants and fungi interact. Peter G. Kennedy and Kabir G. Peay contributed equally to this work and order was determined by a coin toss.     

Changes in hyphal morphology and activity of phenoloxidases during interactions between selected ectomycorrhizal fungi and two species of <Emphasis Type="Italic">Trichoderma</Emphasis>

Patterns of phenoloxidase activity can be used to characterize fungi of different life styles, and changes in phenoloxidase synthesis were suspected to play a role in the interaction between ectomycorrhizal and two species of Trichoderma. Confrontation between the ectomycorrhizal fungi Amanita muscaria and Laccaria laccata with species of Trichoderma resulted in induction of laccase synthesis, and the laccase enzyme was bound to mycelia of ectomycorrhizal fungi. Tyrosinase release was noted only during interaction of L. laccata strains with Trichoderma harzianum and T. virens. Ectomycorrhizal fungi, especially strains of Suillus bovinus and S. luteus, inhibited growth of Trichoderma species and caused morphological changes in its colonies in the zone of interaction. In contrast, hyphal changes occurred less often in the ectomycorrhizal fungi tested. Species of Suillus are suggested to present a different mechanism in their interaction with other fungi than A. muscaria and L. laccata.     

T‐DNA insertion,plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor

Ectomycorrhiza is a mutualistic symbiosis formed between fine roots of trees and the mycelium of soil fungi. This symbiosis plays a key role in forest ecosystems for the mineral nutrition of trees and the biology of the fungal communities associated. The characterization of genes involved in developmental and metabolic processes is important to understand the complex interactions that control the ectomycorrhizal symbiosis. Agrobacterium‐mediated gene transfer (AMT) in fungi is currently opening a new era for fungal research. As whole genome sequences of several fungi are being released studies about T‐DNA integration patterns are needed in order to understand the integration mechanisms involved and to evaluate the AMT as an insertional mutagenesis tool for different fungal species. The first genome sequence of a mycorrhizal fungus, the basidiomycete Laccaria bicolor, became public in July 2006. Release of Laccaria genome sequence and the availability of AMT makes this fungus an excellent model for functional genomic studies in ectomycorrhizal research. No data on the integration pattern in Laccaria genome were available, thus we optimized a plasmid rescue approach for this fungus. To this end the transformation vector (pHg/pBSk) was constructed allowing the rescue of the T‐DNA right border (RB)–genomic DNA junctions in Escherichia coli. Fifty‐one Agrobacterium‐transformed fungal strains, picked up at random from a larger collection of T‐DNA tagged strains (about 500), were analysed. Sixty‐nine per cent were successfully rescued for the RB of which 87% were resolved for genomic integration sequences. Our results demonstrate that the plasmid rescue approach can be used for resolving T‐DNA integration sites in Laccaria. The RB was well conserved during transformation of this fungus and the integration analysis showed no clear sequence homology between different genomic sites. Neither obvious sequence similarities were found between these sites and the T‐DNA borders indicating non‐homologous integration of the transgenes. Majority (75%) of the integrations were located in predicted genes. Agrobacterium‐mediated gene transfer is a powerful tool that can be used for functional gene studies in Laccaria and will be helpful along with plasmid rescue in searching for relevant fungal genes involved in the symbiotic process.