Ectomycorrhizal Development Research Articles

Differential Strategies of Ectomycorrhizal Development between Suillus luteus and Pinus massoniana in Response to Nutrient Changes.

Ectomycorrhizal fungi employ different strategies for mycelial growth and host colonization under varying nutrient conditions. However, key genes associated with mycorrhizal interaction should be influenced solely by the inoculation treatment and not by nutrient variations. To utilize subtle nutrient differences and rapidly screen for key genes related to the interaction between Suillus luteus and Pinus massoniana, we performed an inoculation experiment using culture bottles containing high- and low-nutrient media. Interestingly, S. luteus LS88 promoted the growth of P. massoniana seedlings without mature ectomycorrhiza, and the impact of LS88 inoculation on P. massoniana roots was greater than that of nutrient changes. In this study, the resequenced genome of the LS88 strain was utilized for transcriptome analysis of the strain. The analysis indicated that a unique gene encoding glutathione S-transferase (GST) in LS88 is likely involved in colonizing P. massoniana roots. In this study, the GST gene expression was independent of nutrient levels. It was probably induced by P. massoniana and could be used as a marker for S. luteus colonization degree.

Populus MYC2 orchestrates root transcriptional reprogramming of defence pathway to impair Laccaria bicolor ectomycorrhizal development.

The jasmonic acid (JA) signalling pathway plays an important role in the establishment of the ectomycorrhizal symbiosis. The Laccaria bicolor effector MiSSP7 stabilizes JA corepressor JAZ6, thereby inhibiting the activity of Populus MYC2 transcription factors. Although the role of MYC2 in orchestrating plant defences against pathogens is well established, its exact contribution to ECM symbiosis remains unclear. This information is crucial for understanding the balance between plant immunity and symbiotic relationships. Transgenic poplars overexpressing or silencing for the two paralogues of MYC2 transcription factor (MYC2s) were produced, and their ability to establish ectomycorrhiza was assessed. Transcriptomics and DNA affinity purification sequencing were performed. MYC2s overexpression led to a decrease in fungal colonization, whereas its silencing increased it. The enrichment of terpene synthase genes in the MYC2-regulated gene set suggests a complex interplay between the host monoterpenes and fungal growth. Severalroot monoterpenes have been identified as inhibitors of fungal growth and ECM symbiosis. Our results highlight the significance of poplar MYC2s and terpenes in mutualistic symbiosis by controlling root fungal colonization. We identified poplar genes which direct or indirect control by MYC2 is required for ECM establishment. These findings deepen our understanding of the molecular mechanisms underlying ECM symbiosis.

The Role of Cryptogams in Soil Property Regulation and Vascular Plant Regeneration: A Review

Despite their small size, cryptogams (lichen, liverwort, and moss) are important for ecosystem stability. Due to their strong stress resistance, cryptogams often cover extreme environments uninhabitable for vascular plants, which has an important impact on the material cycle and energy flow of various terrestrial ecosystems. In this article, we review and discuss the effects of cryptogams on soil properties (moisture and fertility) and vascular plant regeneration over the past two decades. Cryptogams strongly affect soil water content by influencing precipitation infiltration, non-rainfall water input, soil evaporation, soil water holding capacity, and soil permeability, ultimately helping to reduce soil water content in areas with low annual precipitation (<500 mm). However, in areas with high annual precipitation (>600 mm) or where the soil has other water sources, the presence of cryptograms is conducive to soil water accumulation. Cryptogam plants can increase soil fertility and the availability of soil nutrients (TOC, TN, TP, TK, and micronutrients) in harsh environments, but their effects in mild environments have not been sufficiently investigated. Cryptogam plants exert complex effects on vascular plant regeneration in different environments. The primary influence pathways include the physical barrier of seed distribution, shading, allelopathy, competition, influences on ectomycorrhizal development and individual reproduction, and the regulation of soil water content, temperature, and nutrients.

Transcriptome Profiling Reveals Differential Gene Expression of Secreted Proteases and Highly Specific Gene Repertoires Involved in Lactarius-Pinus Symbioses.

Ectomycorrhizal fungi establish a mutualistic symbiosis in roots of most woody plants. The molecular underpinning of ectomycorrhizal development was only explored in a few lineages. Here, we characterized the symbiotic transcriptomes of several milkcap species (Lactarius, Russulales) in association with different pine hosts. A time-course study of changes in gene expression during the development of L. deliciosus–Pinus taeda symbiosis identified 6 to 594 differentially expressed fungal genes at various developmental stages. Up- or down-regulated genes are involved in signaling pathways, nutrient transport, cell wall modifications, and plant defenses. A high number of genes coding for secreted proteases, especially sedolisins, were induced during root colonization. In contrast, only a few genes encoding mycorrhiza-induced small secreted proteins were identified. This feature was confirmed in several other Lactarius species in association with various pines. Further comparison among all these species revealed that each Lactarius species encodes a highly specific symbiotic gene repertoire, a feature possibly related to their host-specificity. This study provides insights on the genetic basis of symbiosis in an ectomycorrhizal order, the Russulales, which was not investigated so far.

Granular Calcite Stimulates Natural Mycorrhization and Growth of White Spruce Seedlings in Peat-Based Substrates in Forest Nursery.

The acidity of peat-based substrates used in forest nurseries limits seedling mineral nutrition and growth as well as the activity of microorganisms. To our knowledge, no study has yet evaluated the use of granular calcite as a covering material to increase pH, calcium and CO2 concentrations in the rhizosphere and ectomycorrhizal development. The objective is to compare different covering treatments on early colonization of the roots by ectomycorrhizal fungi, as well as the growth and calcium nutrition of white spruce seedlings in the forest nursery. Three treatments were used to cover the plant cavities (Silica (29 g/cavity; control treatment), Calcite (24 g/cavity) and calcite+ (31 g/cavity)) and were distributed randomly inside each of the five complete blocks of the experimental design. The results show that calcite stimulates natural mycorrhization. Seedlings grown with calcite have significant gains for several growth and physiological variables, and that the periphery of their root plugs are more colonized by the extramatrical phase of ectomycorrhizal fungi, thus improving root-plug cohesion. The authors discuss the operational scope of the results in relation to the tolerance of seedlings to environmental stress and the improvement of their quality, both in the nursery and in reforestation sites.

An ectomycorrhizal fungus alters sensitivity to jasmonate, salicylate, gibberellin, and ethylene in host roots.

The phytohormones jasmonate, gibberellin, salicylate, and ethylene regulate an interconnected reprogramming network integrating root development with plant responses against microbes. The establishment of mutualistic ectomycorrhizal symbiosis requires the suppression of plant defense responses against fungi as well as the modification of root architecture and cortical cell wall properties. Here, we investigated the contribution of phytohormones and their crosstalk to the ontogenesis of ectomycorrhizae (ECM) between grey poplar (Populus tremula x alba) roots and the fungus Laccaria bicolor. To obtain the hormonal blueprint of developing ECM, we quantified the concentrations of jasmonates, gibberellins, and salicylate via liquid chromatography-tandem mass spectrometry. Subsequently, we assessed root architecture, mycorrhizal morphology, and gene expression levels (RNA sequencing) in phytohormone-treated poplar lateral roots in the presence or absence of L. bicolor. Salicylic acid accumulated in mid-stage ECM. Exogenous phytohormone treatment affected the fungal colonization rate and/or frequency of Hartig net formation. Colonized lateral roots displayed diminished responsiveness to jasmonate but regulated some genes, implicated in defense and cell wall remodelling, that were specifically differentially expressed after jasmonate treatment. Responses to salicylate, gibberellin, and ethylene were enhanced in ECM. The dynamics of phytohormone accumulation and response suggest that jasmonate, gibberellin, salicylate, and ethylene signalling play multifaceted roles in poplar L. bicolor ectomycorrhizal development.

The European delicacy Tuber melanosporum forms mycorrhizae with some indigenous Chinese Quercus species and promotes growth of the oak seedlings.

We aimed to test whether Tuber melanosporum and native Chinese oak species could form stable mycorrhizal symbioses. Six oak species were all either inoculated or not, with spores of the Périgord black truffle in the greenhouse. Ectomycorrhizal development was monitored for up to 32 months. Seedling growth was assessed 2 years after inoculation. From 6 months after inoculation, Tuber melanosporum ectomycorrhizae were successfully produced on five Quercus species endemic to China, as shown by morphological, anatomical, and molecular analyses. Quercus mongolica and Q. longispica showed high receptivity to mycorrhization by T. melanosporum. The symbioses obtained with these two species and with Quercus senescens were stable for at least 32 months. Averaged over all three oak species, mycorrhization by T. melanosporum significantly enhanced canopy diameter, number of leaves, and mean leaf dimension. In spring 2019, mycorrhization by T. melanosporum accelerated budbreak in Q. mongolica. Quercus fabrei and Q. variabilis formed ectomycorrhizae up to 9 months after inoculation but seedlings died 3 months later, probably because of damage by grazing insects. Quercus pseudosemecarpifolia failed to form ectomycorrhizae. Results suggest that T. melanosporum-mycorrhized Q. mongolica and Q. longispica seedlings could be tested for ascocarp production and increased performance in the field.

Lichens Contribute to Open Woodland Stability in the Boreal Forest Through Detrimental Effects on Pine Growth and Root Ectomycorrhizal Development

In the boreal forest, open lichen woodlands have been described as an alternative stable state to closed-crown feather moss forest. In this study, we addressed the role of terricolous lichens in stabilizing open woodlands by hindering tree regeneration and/or growth. Based on field and greenhouse experiments, we compared germination and growth of jack pine (Pinus banksiana) on feather mosses (primarily Pleurozium schreberi) and lichens (primarily Cladonia stellaris), using bare mineral soil as a control. Drivers were investigated by (1) manipulating nutrient supply, (2) simulating shade of a closed canopy on the ground layer with the assumption this would mitigate lichen influence on pine growth, and (3) examining pine root ectomycorrhizal colonization and diversity as indicators of pine ability to take up nutrients. Total growth of 6-month-old greenhouse and 2–3-year-old field seedlings, as well as belowground growth of 2-year-old greenhouse seedlings, was significantly greater in moss than in lichen. Seed germination was not affected by ground cover type. Although field phosphorus and base cation availability was greater in mosses than in lichens, fertilization did not entirely compensate for the negative effects of lichens on pine growth in the greenhouse. Ground layer shading had no impact on pine growth. Lichens were associated with reduced abundance and modified composition of the root ectomycorrhizal community. By suggesting that terricolous lichens constitute a less favorable growth substrate than mosses for pine, our results support the hypothesis that lichens contribute to open woodland stability in the potentially closed-crown feather moss forest.

A nonnative and a native fungal plant pathogen similarly stimulate ectomycorrhizal development but are perceived differently by a fungal symbiont.

The effects of plant symbionts on host defence responses against pathogens have been extensively documented, but little is known about the impact of pathogens on the symbiosis and if such an impact may differ for nonnative and native pathogens. Here, this issue was addressed in a study of the model system comprising Pinus pinea, its ectomycorrhizal symbiont Tuber borchii, and the nonnative and native pathogens Heterobasidion irregulare and Heterobasidion annosum, respectively. In a 6-month inoculation experiment and using both in planta and gene expression analyses, we tested the hypothesis that H.irregulare has greater effects on the symbiosis than H.annosum. Although the two pathogens induced the same morphological reaction in the plant-symbiont complex, with mycorrhizal density increasing exponentially with pathogen colonization of the host, the number of target genes regulated in T.borchii in plants inoculated with the native pathogen (i.e. 67% of tested genes) was more than twice that in plants inoculated with the nonnative pathogen (i.e. 27% of genes). Although the two fungal pathogens did not differentially affect the amount of ectomycorrhizas, the fungal symbiont perceived their presence differently. The results may suggest that the symbiont has the ability to recognize a self/native and a nonself/nonnative pathogen, probably through host plant-mediated signal transduction.

Effect of nitrogen deposition on growth of birch seedlings and ectomycorrhizal development with/without phosphorous treatments

Effect of nitrogen deposition on growth of birch seedlings and ectomycorrhizal development with/without phosphorous treatments

Polyphosphate (phytate) formation in Quercus acutissima-Scleroderma verrucosum ectomycorrhizae supplied with phosphate

Quercus acutissima seedlings were cultivated in growth pouches, inoculated with Scleroderma verrucosum and supplied with inorganic phosphorus (Pi) to evaluate the change in P attributable to mycorrhization and to determine the anatomical localization of polyphosphate (phytate) and polysaccharide. Semithin sections were stained with metachromatic toluidine blue O (TBO) and fluoresced with acriflavine-HCl. The total P content was analyzed during the ectomycorrhizal (ECM) development stages and spherical electron-dense opaque granules were analyzed using TEM-EDS. The spherical polyphosphates (phytate) were precipitated and stained with TBO as dark variable sized granules in the vacuoles of the mantle hyphae, Hartig net and the epidermal and apical meristem cells of the ECM root. Higher levels of P and cations K, Mg, Ca, Na which indicated polyphosphate (phytate), were detected in the spherical electron-dense opaque granules in the mantle hyphae vacuoles, Hartig net and epidermal cells. The increase in phytate granules was correlated with the ECM development stages in the ECM root tip tissue. The total P content of the root tips also increased during each developmental stage, because of ECM development. The polysaccharide granules fluoresced in the apical meristem cells during the mature mantle stage. Wall ingrowth was observed in epidermal cells adjacent to Hartig net hyphae. Thus, the P acquired from the external Pi supply by the mantle hyphae was converted to polyphosphates, which were transferred initially to the epidermal cells in the host root tips that interfaced with mantle hyphae during the early mantle stage before Hartig net formation.

Ectomycorrhizal inoculation with Pisolithus tinctorius increases the performance of Quercus suber L. (cork oak) nursery and field seedlings

Mediterranean ecosystems are characterized by large arid areas where the patchy distribution of trees offers little protection against harsh climate conditions for seedling establishment. Climate change is predicted to result in an increase in these arid regions, with pronounced effects on vegetation. Production of seedlings with developed ectomycorrhizas is a promising strategy for minimizing the initial transplant shock, thereby increasing plant survival and growth during the first, most critical years of a plantation. One important species in the Mediterranean basin is Quercus suber (cork oak), which occurs, together with other evergreen oak species, in an agro-silvo-pastoral system that represents an example of sustainable land use in Europe. In this study, a Pisolithus tinctorius isolate was used for ectomycorrhizal colonization of cork oak nursery seedlings, and the effects on aboveground plant growth and leaf structural and physiological parameters were investigated. Ectomycorrhizal development resulted in a significant increase in leaf area, dry weight, nitrogen content, and photosynthetic pigments, and mycorrhizal plants showed a higher photosynthetic capacity and water use efficiency. Nursery-inoculated plants established in the field showed increased survival and growth during the first year after transplant. These results indicate a potential for further enhancing the use of mycorrhizal inoculation as a cultivation practice in forest nurseries. Considering the difficulty of soil restoration under limiting environmental conditions, nursery inoculation with ectomycorrhizal fungi can be an important advantage for improving the quality of seedling stock and its performance after out-planting in the field, benefiting the regeneration of arid regions and the reintroduction of inocula of ectomycorrhizal fungi into these areas.

Do Arbuscular Mycorrhizal Fungi Affect Metallothionein Mt2 Expression In Brassica Napus L. Roots?

Arbuscular mycorrhizal fungi are the most widespread root fungal symbionts, forming associations with the vast majority of plant species. Ectomycorrhizal development alters gene expression in plant symbionts. In this work we examined the effect of arbuscular mycorrhizal fungi spores on the growth and development of Brassica and on the expression of BnMT2 in winter rape. In a pot experiment, rape seedlings growing on different types of sterile and nonsterile soils were inoculated simultaneously with mycorrhizal fungi spores of Acaulospora longula, Glomus geosporum, Glomus mosseae and Scutellospora calospora. As compared with control plants growing in the absence of spores, ten-week-old seedlings of Brassica napus L. in sterile soil inoculated with arbuscular spores had longer shoots and higher fresh biomass of above-ground plant parts. In other types of substrates enriched with mycorrhizal fungi spores, the plants were smaller than non-inoculated plants. The presence of AMF spores stimulated the elongation growth of hypocotyls in both analyzed substrates. BnMT2 expression was highest in plants growing on the sterile substrate. Generally, the presence of mycorrhizal fungi spores appeared to have an adverse effect on the growth of rape plants.

Identification of quantitative trait loci affecting ectomycorrhizal symbiosis in an interspecific F1 poplar cross and differential expression of genes in ectomycorrhizas of the two parents: Populus deltoides and Populus trichocarpa

A Populus deltoides × Populus trichocarpa F1 pedigree was analyzed for quantitative trait loci (QTLs) affecting ectomycorrhizal development and for microarray characterization of gene networks involved in this symbiosis. A 300 genotype progeny set was evaluated for its ability to form ectomycorrhiza with the basidiomycete Laccaria bicolor. The percentage of mycorrhizal root tips was determined on the root systems of all 300 progeny and their two parents. QTL analysis identified four significant QTLs, one on the P. deltoides and three on the P. trichocarpa genetic maps. These QTLs were aligned to the P. trichocarpa genome and each contained several megabases and encompass numerous genes. NimbleGen whole-genome microarray, using cDNA from RNA extracts of ectomycorrhizal root tips from the parental genotypes P. trichocarpa and P. deltoides, was used to narrow the candidate gene list. Among the 1,543 differentially expressed genes (p value ≤ 0.05; ≥5.0-fold change in transcript level) having different transcript levels in mycorrhiza of the two parents, 41 transcripts were located in the QTL intervals: 20 in Myc_d1, 14 in Myc_t1, and seven in Myc_t2, while no significant differences among transcripts were found in Myc_t3. Among these 41 transcripts, 25 were overrepresented in P. deltoides relative to P. trichocarpa; 16 were overrepresented in P. trichocarpa. The transcript showing the highest overrepresentation in P. trichocarpa mycorrhiza libraries compared to P. deltoides mycorrhiza codes for an ethylene-sensitive EREBP-4 protein which may repress defense mechanisms in P. trichocarpa while the highest overrepresented transcripts in P. deltoides code for proteins/genes typically associated with pathogen resistance.

Prof. Dr Gopi Krishna Podila, 1957–2010

Prof. Dr Gopi Krishna Podila, 1957–2010

Seed germination and seedling physiology of Larix kaempferi and Pinus densiflora in seedbeds with charcoal and elevated CO2

We investigated the effect of ectomycorrhizal colonization, charcoal and CO2 levels on the germination of seeds of Larix kaempferi and Pinus densiflora, and also their subsequent physiological activity and growth. The seeds were sown in brown forest soil or brown forest soil mixed with charcoal, at ambient CO2 (360 μmol mol−1) or elevated CO2 (720 μmol mol−1), with or without ectomycorrhiza. The proportions of both conifer seeds that germinated in forest soil mixed with charcoal were significantly greater than for seeds sown in forest soil grown at each CO2 level (P < 0.05; t-test). However, the ectomycorrhizal colonization rate of each species grown in brown forest soil mixed with charcoal was significantly lower than in forest soil at each CO2 treatment [CO2] (P < 0.01; t-test). The phosphorus concentrations in needles of each seedling colonized with ectomycorrhiza and grown in forest soil were greater than in nonectomycorrhizal seedlings at each CO2 level, especially for L. kaempferi seedlings (P < 0.05; t-test), but the concentrations in seedlings grown in brown forest soil mixed with charcoal were not increased at any CO2 level. Moreover, the maximum net photosynthetic rate of each seedling for light and CO2 saturation (P max) increased when the seedlings were grown with ectomycorrhiza at 720 μmol mol−1 [CO2]. Ectomycorrhizal colonization led to an increase in the stem diameter of each species grown in each soil treatment at each CO2 level. However, charcoal slowed the initial growth of both species of seedling, constraining ectomycorrhizal development. These results indicate that charcoal strongly assists seed germination and physiological activity.

Growth and physiological activity in Larix kaempferi seedlings inoculated with ectomycorrhizae as affected by soil acidification

To evaluate the effect of ectomycorrhizal colonization on growth and physiological activity of Larix kaempferi seedlings grown under soil acidification, we grew L. kaempferi seedlings with three types of ectomycorrhizae for 180 days in acidified brown forest soil derived from granite. The soil had been treated with an acid solution (0 (control), 10, 30, 60, and 90 mmol H+ kg−1). The water-soluble concentrations of Ca, Mg, K, Al, and Mn increased with increasing amounts of H+ added to the soil. Ectomycorrhizal development significantly increased in soil treated with 10 and 30 mmol H+ kg−1 but was significantly reduced in soil treated with 60 and 90 mmol H+ kg−1. The concentrations of Al and Mn in needles or roots increased with increasing H+ added to the soil. The total N in seedlings significantly increased with increasing H+ in soil and colonization with ectomycorrhiza. The maximum net photosynthetic rate at light and CO2 saturation (Pmax) was greater in soil treated with 10 mmol H+ kg−1 than in controls, and was less is soils treated with greater than with 30 mmol H+ kg−1, especially with 60 and 90 mmol H+ kg−1. However, colonization with ectomycorrhiza significantly reduced the concentration of Al and Mn in needles or roots and increased the values of Pmax and total dry mass (TDM). The relative TDM of L. kaempferi seedlings was approximately 40% at a (BC, base cation)/Al ratio of 1.0. However, ectomycorrhizal seedlings had a 100–120% greater TDM at a BC/Al ratio of 1.0 than non-ectomycorrhizal seedlings, even though the acid treatment reduced their overall growth.

Effect of Ectomycorrhizal Development on Growth in Pine Seedlings

Effect of Ectomycorrhizal Development on Growth in Pine Seedlings

Ectomycorrhizal development and pine seedlings growth in response to different physical factors

An impact of temperature, light and relative humidity were studied on root colonisation by mycorrhizal fungi and on pine seedling growth. Moderate and high light intensities favoured mycorrhizal colonisation and seedling growth. Pine seedlings inoculated with Pisolithus tinctorius compared to other fungi attained maximum growth. Survival of pine seedlings was higher under moderate light intensity than low and high light intensity. Seedling growth and mycorrhizal colonisation was better at 25°C than 10°C. Variation in humidity did not show much difference in mycorrhizal colonisation and seedling growth. However, seedling survival was greater at high than at low humidity. Pine seedlings showed best survival with 0.5 P level (46.153 mg P/kg soil) of phosphorus at 25 °C temperature and under moderate light intensity. Among the mycorrhizal fungi used P. tinctorius was the most effective endophyte and was followed by Laccaria laccata, Rhizopogon luteolus and Collybia radicata under various physical factors.

Synthesis and establishment of Tuber melanosporum Vitt. ectomycorrhizae on two Nothofagus species in Chile

Axenically germinated seedlings of two species of Southern beech (Nothofagus obliqua, N. glauca) from Chile were inoculated with spores of the Périgord black truffle (Tuber melanosporum). Ectomycorrhizal development was monitored for 6 months in the greenhouse and compared to the performance of the natural host species Quercus ilex and Quercus robur. Seedling survival and mycorrhization showed major differences in both Nothofagus species: T. melanosporum readily formed ectomycorrhizae with seedlings of N. obliqua, although at a lower rate than with Q. ilex but at a proportion very similar to Q. robur; survival and colonization rates were high, and seedling growth was not visibly affected by the high soil pH required by T. melanosporum. In contrast, more than 50% of N. glauca seedlings died after inoculation, and mycorrhiza formation was very sparse. In both species, no colonization by adventive ectomycorrhizal fungi could be observed, whereas both species of Quercus showed minor colonization by another fungus, probably Inocybe or Hebeloma. Our results show that it is possible to infect N. obliqua with the Périgord black truffle under greenhouse conditions, which opens up the possibility of cultivating this truffle as a secondary crop during reforestation with N. obliqua in Chile.