Induced defence responses limit Hartig net formation in ectomycorrhizal birch roots

Roots of clonal birches ( Betula pendula ) were inoculated with the ectomycorrhizal fungi Paxillus involutus (isolates P0 and Mi) and Hebeloma cylindrosporum (strains D1 and D105). These fungi showed different rates of mycorrhiza formation in vitro . Mature mycorrhizas were obtained after only 2–4 d with H. cylindrosporum , whereas 6–8 d were necessary with P. involutus isolate P0, and P. involutus isolate Mi was not able to form mature mycorrhiza during the 10 d of the experiment. Temporal changes in PAL activity and the expression of genes encoding intracellular pathogenesis-related proteins were followed after inoculating birch roots with these fungi. Transient increase of PAL activity, and transient induction of expression of the wound-inducible Bet v1-SC1 gene, were observed in roots challenged with both H. cylindrosporum strains and the P. involutus isolate P0. These changes were found to coincide with hyphal penetration between root cells during Hartig net formation, and were never observed in roots inoculated with the poorly aggressive P. involutus isolate Mi. Examination of mycorrhizal root sections under u.v. light indicated the presence of phenolic compounds in the host cell walls at the vicinity of the Hartig net. These results strongly suggest that hyphal penetration between the root cells triggers a transient defence response which, in turn, could limit Hartig net formation to the outer layer of the root cortex.     

The cellular structure of the Hartig net: coenocytic and transfer cell-like organization

The organization of the Hartig net in the mature state was studied in mycorrhizas of Amanita muscaria (Pers. ex. Fries) Hook. Picea abies (L.) Karst. grown in vitro. The tips of the fan-like branched hyphae contain dense cytoplasm with a large number of mitochondria and rER frequently stretched in the direction of the hyphal growth, indicating that active transfer of solutes between host and fungus is localized here. Lack of septation of the hyphae and their intimate juxtaposition, leaving no space in between, result in a coenocytic, transfer cell-like structure of the Hartig net. The multinucleate status of the cells is shown. The advantage of this organization for nutrient exchange between host and fungus and for nutrient transport within the hyphal system is discussed.     

Extracellular complexation of Cd in the Hartig net and cytosolic Zn sequestration in the fungal mantle of Picea abies – Hebeloma crustuliniforme ectomycorrhizas

Compartmentation of heavy metals on or within mycorrhizal fungi may serve as a protective function for the roots of forest trees growing in soils containing elevated concentrations of metals such as Cd and Zn. In this paper we present the first quantitative measurements by X‐ray microanalysis of heavy metals in high‐pressure frozen and cryosectioned ectomycorrhizal fungal hyphae. We used this technique to analyse the main sites of Cd and Zn in fungal cells of mantle and Hartig net hyphae and in cortical root cells of symbiotic Picea abies – Hebeloma crustuliniforme associations to gain new insights into the mechanisms of detoxification of these two metals in Norway spruce seedlings. The mycorrhizal seedlings were exposed in growth pouches to either 1 mM Cd or 2 mM Zn for 5 weeks. The microanalytical data revealed that two distinct Cd‐ and Zn‐binding mechanisms are involved in cellular compartmentation of Cd and Zn in the mycobiont. Whereas extracellular complexation of Cd occurred predominantly in the Hartig net hyphae, both extracellular complexation and cytosolic sequestration of Zn occurred in the fungal tissue. The vacuoles were presumed not to be a significant pool for Cd and Zn storage. Cadmium was almost exclusively localized in the cell walls of the Hartig net (up to 161 mmol kg ^{? 1} DW) compared with significantly lower concentrations in the cell walls of mantle hyphae (22 mmol kg ^{? 1} DW) and in the cell walls of cortical cells (15 mmol kg ^{? 1} DW). This suggests that the apoplast of the Hartig net is a primary accumulation site for Cd. Zinc accumulated mainly in the cell walls of the mantle hyphae (111 mmol kg ^{? 1} DW), the Hartig net hyphae (130 mmol kg ^{? 1} DW) and the cortical cells (152 mmol kg ^{? 1} DW). In addition, Zn occurred in high concentrations in the cytoplasm of the fungal mantle hyphae (up to 164 mmol kg ^{? 1} DW) suggesting that both the cell walls and the cytoplasm of fungal tissue are the main accumulation sites for Zn in P. abies resulting in decreased Zn transfer from the fungus to the root.     

Comparative structure of vesicular-arbuscular mycorrhizas and ectomycorrhizas

During the establishment of vesicular-arbuscular mycorrhizas, fungal hyphae contact the root surface, form appressoria and initiate the internal colonization phase. Structural changes occur in the cell wall, the cytoplasm and the nucleus as the fungus progresses from a presymbiotic to a symbiotic phase. Nuclei in spores are in G₁ whereas in intraradical hyphae they are in G₁ and G₂. Changes in nuclear organization are evident in various stages in the colonization process. Dramatic changes in both symbionts occur as the nutrient exchange interface is established between arbuscules and root cortical cells. An interfacial matrix, consisting of molecules common to the primary wall of the cortical cell, separates the cortical cell plasma membrane from the fungal cell wall. Ectomycorrhizas are characterized structurally by the presence of a mantle of fungal hyphae enclosing the root and usually an Hartig net of intercellular hyphae characterized by labyrinthine branching. As hyphae contact the root surface, they may respond by increasing their diameter and switching from apical growth to precocious branching. The site of initial contact of hyphae may be either the root cap or the ‘mycorrhiza infection zone’. The mantle varies considerably in structure depending on both the plant and fungus genome. In some ectomycorrhizas, the mantle may be a barrier to apoplastic transport, and in most it may store polyphosphate, glycogen, lipids and perhaps protein.     

Laccaria bicolor aquaporin LbAQP1 is required for Hartig net development in trembling aspen (Populus tremuloides)

The development of ectomycorrhizal associations is crucial for growth of many forest trees. However, the signals that are exchanged between the fungus and the host plant during the colonization process are still poorly understood. In this study, we have identified the relationship between expression patterns of Laccaria bicolor aquaporin LbAQP1 and the development of ectomycorrhizal structures in trembling aspen (Populus tremuloides) seedlings. The peak expression of LbAQP1 was 700‐fold higher in the hyphae within the root than in the free‐living mycelium after 24 h of direct interaction with the roots. Moreover, in LbAQP1 knock‐down strains, a non‐mycorrhizal phenotype was developed without the Hartig net and the expression of the mycorrhizal effector protein MiSSP7 quickly declined after an initial peak on day 5 of interaction of the fungal hyphae with the roots. The increase in the expression of LbAQP1 required a direct contact of the fungus with the root and it modulated the expression of MiSSP7. We have also determined that LbAQP1 facilitated NO, H₂O₂ and CO₂ transport when heterologously expressed in yeast. The report demonstrates that the L. bicolor aquaporin LbAQP1 acts as a molecular signalling channel, which is fundamental for the development of Hartig net in root tips of P. tremuloides.     

Metal toxicity and ectomycorrhizas

Metal toxicity (Al and heavy metals) is a major constraint affecting root growth in a number of natural or managed ecosystems. Fine roots of the majority of plant species are associated with mycorrhizal fungi, which may modify the sensitivity of roots to metal stress. In this review, we summarise the available evidence demonstrating beneficial effects of ectomycorrhizas in alleviation of metal toxicity in forest tree seedlings. We identify experimental shortcomings of past research (e.g. the use of shoot metal concentrations as a measure of metal uptake, use of microanalytical techniques biased by element redistribution) that may confound major conclusions drawn from these experiments. Although there is no doubt that in many cases ectomycorrhizal fungi indeed ameliorate metal stress in their host plants, the mechanism(s) involved remain(s) unclear. The role of metal sorption on fungal tissues thought to reduce metal exposure of the host plant is critically reviewed. As direct evidence (both under artificial and soil conditions) supporting a unique role of fungal immobilisation of metals is lacking so far, there is an urgent need to also test alternative tolerance mechanisms such as the release of metal chelating substances, or nutritional and hormonal effects mediated by mycorrhizal fungi.     

Comparative investigations on the differentiation of the endodermis and the development of the Hartig net in mycorrhizae of Picea abies and Larix decidua

Summary The differentiation of the endodermis of mycorrhizal roots of Picea abies and Larix decidua was investigated by means of light and transmission electron microscopy and with fluorescence techniques. The initiation and differentiation of the Hartig net were recorded. Differences between the two tree species were found, as were differences between the two tree species and angiosperms. The Casparian band developed immediately after the origin of endodermal cells from the meristem in mycorrhizae of both tree species. In L. decidua only the primary endodermis was present in most mycorrhizal laterals. The secondary structure of the endodermis was restricted to main roots and proximal parts of larch mycorrhizae. In P. abies mycorrhizae, however, the secondary stage of the endodermis developed soon after the primary endodermis and was characterized by regular alternation of short, active passage cells and elongated, rapidly degenerating cells, the inner surface of which was covered by a thick suberin layer. Hartig net development started in P. abies short roots only after the differentiation of endodermis into the secondary stage, whereas in L. decidua, the Hartig net was already initiated at the primary endodermal stage. Differences were specific for tree species.     

Terminal axons ensheathed in smooth muscle cells of the vas deferens

Summary The ultrastructure of axon profiles which were completely ensheathed in smooth muscle cells has been described in the guinea pig, mouse and rat vas deferens. The axon profiles contained both small (500 Å) and large (1,000 Å) vesicles, neurotubules and mitochondria. Adrenergic axons were clearly identified within smooth muscle cells after treatment of the tissue with 5-or 6-hydroxydopamine, drugs which cause specific ultrastructural changes in adrenergic axons. The ensheathed axons were separated from the surrounding muscle cells by narrow, regular gaps, usually about 100–300 Å wide. Schwann cells seldom accompanied the ensheathed axons. Axons often penetrated the muscle cells in the nuclear region and profiles were sometimes observed among the perinuclear organelles.     

Soil fumigation and phosphorus supply affect the formation of Pisolithus-Eucalyptus urophylla ectomycorrhizas in two acid Philippine soils

To examine the effects of microbial populations and external phosphorus supply of two Philippine soils on mycorrhizal formation, Eucalyptus urophylla seedlings were inoculated with two Pisolithus isolates and grown in fumigated, reinfested and unfumigated soil fertilized with four rates of phosphorus. The Pisolithus isolates used were collected from under eucalypts in Australia and in the Philippines. Soils were infertile acid silty loams collected from field sites in Pangasinan, Luzon and Surigao, Mindanao.Significant interaction was observed between inoculation, soil fumigation and phosphorus supply on mycorrhizal formation by the Australian isolate in Surigao soil but not in Pangasinan soil. Soil fumigation enhanced mycorrhizal formation by the Australian isolate but did not affect root colonization by the Philippine isolate. Root colonization by the Australian isolate was highest in the reinfested soil while for the Philippine isolate it was highest in the unfumigated soil. The Australian isolate was more effective than the Philippine isolate in promoting growth and P uptake of E. urophylla seedlings in both soils. Total dry weight and P uptake of E. urophylla seedlings inoculated with the Australian isolate were maximum in fumigated and in the reinfested Pangasinan and Surigao soils supplied with 8 mg P kg^-1 soil. In the unfumigated soil, growth of seedlings inoculated with the Australian isolate was significantly reduced. Seedlings inoculated with the Philippine isolate had the largest dry weights and P contents in unfumigated Pangasinan and Surigao soils supplied with 8 mg P kg^-1 soil.These results indicate that the performance of the Australian Pisolithus isolate was markedly affected by biological factors in unfumigated soil. Thus, its potential use in the Philippines needs to be thoroughly tested in a variety of unfumigated soils before its widespread use in any inoculation programme.     

Carbon and nitrogen metabolism in ectomycorrhizal fungi and ectomycorrhizas

The literature concerning the metabolism of carbon and nitrogen compounds in ectomycorrhizal associations of trees is reviewed. The absorption and translocation of mineral ions by the mycelia require an energy source and a reductant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid and carbohydrate syntheses during the growth of the mycelia. Competition for photosynthates occurs between the fungal cells and the various vegetative sinks in the host tree. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolites between the various sites of utilization are only poorly understood. Both ascomycetous and basidiomycetous ectomycorrhizal fungi synthesize and some, if not all, accumulate mannitol, trehalose and triglycerides. The fungal strains employ the Embden--Meyerhof pathway of glucose catabolism and the key enzymes of the pentose phosphate pathway (6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, transaldolase and transketolase). Anaplerotic CO2 fixation, via pyruvate carboxylase and/or phosphoenolpyruvate carboxykinase, provides high pools of amino acids. This process could be important in the recapture and assimilation of respired CO2 in the rhizosphere. The ectomycorrhizas are thought to contain the Embden--Meyerhof pathway, the pentose phosphate pathway and the tricarboxylic acid cycle, which provide the carbon skeletons for the assimilation of ammonia into amino acids. The main route of assimilation of ammonia appears to be through the glutamine synthetase-glutamate synthase cycle in the ectomycorrhizas. Glutamate dehydrogenase plays a minor role in this process. Glutamate dehydrogenase and glutamine synthetase are present in free-living ectomycorrhizal fungi and they participate in the assimilation of ammonia and the synthesis of amino acids through the glutamate dehydrogenase/glutamine synthetase sequence. In both in vitro cultures of fungi and ectomycorrhizas, the assimilated nitrogen accumulates in glutamine. Glutamine, but also ammonia, are thought to be exported from the fungal tissues to the host cells. Studies on the metabolism of ectomycorrhizas and ectomycorrhizal fungi have focused on the metabolic pathways and compounds which accumulate in the symbiotic tissues. Studies on regulation of the overall process, and the control of enzyme activity in particular, are still fragmentary.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphological-anatomical characterization and identification of Tomentella ectomycorrhizas

Over the last two decades, much information has been gathered on the ectomycorrhizal fungus community composition of plant associations of boreal, temperate, and tropical regions. Worldwide, Tomentella ectomycorrhizas (ECM) are often common and dominant in the mycorrhizosphere of coniferous and deciduous forests. They are present under different environmental conditions and associate with diverse plant hosts. Tomentella sporocarps, however, are rarely found aboveground, so Tomentella species are often missing from fungus community studies based on fruit-body presence. Tomentella is a resupinate genus of Thelephoraceae (Basidiomycota) forming black-brown, brown, yellow, or ochre ECM on the roots of gymnosperm and angiosperm trees, distinguished by typical morphological-anatomical characteristics (clamped hyphae, angular mantle, surface network, special rhizomorphs and cystidia). In this paper, we review the taxonomic position and morphological-anatomical characteristics of Tomentella ECM. A short summary of the microscopic features used for distinguishing tomentelloids during morphotyping and identification is presented in order to support molecular and ecological studies of ectomycorrhizal fungus communities.     

An update on nutrient transport processes in ectomycorrhizas

Nutrient transport, namely absorption from the soil solution as well as nutrient transfer from fungus to plant and carbon movement from plant to fungus are key features of mycorrhizal symbiosis. This review summarizes our current understanding of nutrient transport processes in ectomycorrhizal fungi and ectomycorrhizas. The identification of nutrient uptake mechanisms is a key issue in understanding nutrition of ectomycorrhizal plants. With the ongoing functional analysis of nutrient transporters, identified during sequencing of fungal and tree genomes, a picture of individual transport systems should be soon available, with their molecular functions assessed by functional characterization in, e.g., yeast mutant strains or Xenopus oocytes. Beyond the molecular function, systematic searches for knockout mutants will allow us to obtain a full understanding of the role of the individual transporter genes in the physiology of the symbionts. The mechanisms by which fungal and plant cells obtain, process and integrate information regarding nutrient levels in the external environment and the plant demand will be analyzed.     

Fine-scale distribution of pine ectomycorrhizas and their extramatrical mycelium

In order to clarify the functional role of individual ectomycorrhizal (EcM) fungal species in the field, we need to relate their abundance and distribution as mycorrhizas to their abundance and distribution as extramatrical mycelium (EMM). We divided each of four 20 cm x 20 cm x 2 cm slices of pine forest soil into 100 cubes of 2 cm x 2 cm. For each cube, ectomycorrhizas were identified and the presence of EMM of the EcM fungi recorded as ectomycorrhizas was determined by terminal restriction fragment length polymorphism (T-RFLP) analysis of ITS rDNA. Ectomycorrhizas and EMM of seven EcM species were mapped. Spatial segregation of mycorrhizas and EMM was evident and some species produced their EMM in different soil layers from their mycorrhizas. The spatial relationship between mycorrhizas and their EMM generally conformed to their reported exploration types, but EMM of smooth types (e.g. Lactarius rufus) was more frequent than expected. Different EcM fungi foraged at different spatial scales.