Ectomycorrhizal Fungus Pisolithus Tinctorius Research Articles

Nitrogen Acquisition and Transport in the Ectomycorrhizal Symbiosis-Insights from the Interaction between an Oak Tree and Pisolithus tinctorius.

In temperate forests, the roots of various tree species are colonized by ectomycorrhizal fungi, which have a key role in the nitrogen nutrition of their hosts. However, not much is known about the molecular mechanisms related to nitrogen metabolism in ectomycorrhizal plants. This study aimed to evaluate the nitrogen metabolic response of oak plants when inoculated with the ectomycorrhizal fungus Pisolithus tinctorius. The expression of candidate genes encoding proteins involved in nitrogen uptake and assimilation was investigated in ectomycorrhizal roots. We found that three oak ammonium transporters were over-expressed in root tissues after inoculation, while the expression of amino acid transporters was not modified, suggesting that inorganic nitrogen is the main form of nitrogen transferred by the symbiotic fungus into the roots of the host plant. Analysis by heterologous complementation of a yeast mutant defective in ammonium uptake and GFP subcellular protein localization clearly confirmed that two of these genes encode functional ammonium transporters. Structural similarities between the proteins encoded by these ectomycorrhizal upregulated ammonium transporters, and a well-characterized ammonium transporter from E. coli, suggest a similar transport mechanism, involving deprotonation of NH4+, followed by diffusion of uncharged NH3 into the cytosol. This view is supported by the lack of induction of NH4+ detoxifying mechanisms, such as the GS/GOGAT pathway, in the oak mycorrhizal roots.

Metabolomics and transcriptomics to decipher molecular mechanisms underlying ectomycorrhizal root colonization of an oak tree

Mycorrhizas are known to have a positive impact on plant growth and ability to resist major biotic and abiotic stresses. However, the metabolic alterations underlying mycorrhizal symbiosis are still understudied. By using metabolomics and transcriptomics approaches, cork oak roots colonized by the ectomycorrhizal fungus Pisolithus tinctorius were compared with non-colonized roots. Results show that compounds putatively corresponding to carbohydrates, organic acids, tannins, long-chain fatty acids and monoacylglycerols, were depleted in ectomycorrhizal cork oak colonized roots. Conversely, non-proteogenic amino acids, such as gamma-aminobutyric acid (GABA), and several putative defense-related compounds, including oxylipin-family compounds, terpenoids and B6 vitamers were induced in mycorrhizal roots. Transcriptomic analysis suggests the involvement of GABA in ectomycorrhizal symbiosis through increased synthesis and inhibition of degradation in mycorrhizal roots. Results from this global metabolomics analysis suggest decreases in root metabolites which are common components of exudates, and in compounds related to root external protective layers which could facilitate plant-fungal contact and enhance symbiosis. Root metabolic pathways involved in defense against stress were induced in ectomycorrhizal roots that could be involved in a plant mechanism to avoid uncontrolled growth of the fungal symbiont in the root apoplast. Several of the identified symbiosis-specific metabolites, such as GABA, may help to understand how ectomycorrhizal fungi such as P. tinctorius benefit their host plants.

In vitro interactions between the ectomycorrhizal Pisolithus tinctorius and the saprotroph Hypholoma fasciculare fungi: morphological aspects and volatile production

ABSTRACT Ectomycorrhizal fungi are crucial for forests sustainability. For Castanea sativa, ectomycorrhizal fungus Pisolithus tinctorius is an important mutualist partner. Saprotrophic fungi Hypholoma fasciculare, although used for biocontrol of Armillaria root disease, it negatively affected the interaction between the P. tinctorius and plant host roots, by compromise the formation of P. tinctorius-C. sativa mycorrhizae. In this work, fungal morphology during inhibition of H. fasciculare against P. tinctorius was elucidated. P. tinctorius growth was strongly affected by H. fasciculare, which was significantly reduced after six days of co-culture and become even more significant through time. During this period, P. tinctorius developed vesicles and calcium oxalate crystals, which were described as mechanisms to stress adaption by fungi. H. fasciculare produced different volatile organic compounds in co-cultures over time and differ between single or in dual-species. H. fasciculare highly produced sesquiterpenes (namely, α-muurolene) and nitrogen-containing compounds, which are recognised as having antimicrobial activity.

Ectomycorrhizal fungi inoculation alleviates simulated acid rain effects on soil ammonia oxidizers and denitrifiers in Masson pine forest.

Acid rain can cause severe effects on soil biota and nutrient biogeochemical cycles in the forest ecosystem, but how plant-symbiotic ectomycorrhizal fungi will modulate the effects remains unknown. Here, we conducted a full factorial field experiment in a Masson pine forest by simultaneously controlling the acidity of the simulated rain (pH 5.6 vs. pH 3.5) and the ectomycorrhizal fungi Pisolithus tinctorius inoculation (non-inoculation vs. inoculation), to investigate the effects on ammonia oxidizers and denitrifiers. After 10 months, compared with the control (rain pH 5.6, and non-inoculation), simulated acid rain (pH 3.5) reduced soil nutrient content, decreased archaeal amoA gene abundance and inhibited denitrification enzyme activity. Also, simulated acid rain altered the community compositions of all the examined functional genes (archaeal amoA, bacterial amoA, nirK, nirS and nosZ). However, inoculation with ectomycorrhizal fungi under acid rain stress recovered soil nutrient content, archaeal amoA gene abundance and denitrification enzyme activity to levels comparable to the control, suggesting that ectomycorrhizal fungi inoculation ameliorates simulated acid rain effects. Taken together, ectomycorrhizal fungi inoculation - potentially through improving soil substrate availability - could alleviate the deleterious effects of acid rain on nitrogen cycling microbes in forest soils.

Ectomycorrhizal inoculation with Pisolithus tinctorius reduces stress induced by drought in cork oak.

We investigated whether the performance of cork oak under drought could be improved by colonization with the ectomycorrhizal fungus Pisolithus tinctorius. Results show that inoculation alone had a positive effect on plant height, shoot biomass, shoot basal diameter, and root growth. Under drought, root growth of mycorrhizal plants was significantly increased showing that inoculation was effective in increasing tolerance to drought. In accordance, mycorrhizal plants subjected to drought showed less symptoms of stress when compared to non-mycorrhizal plants, such as lower concentration of soluble sugars and starch, increased ability to maintain fatty acid content and composition, and increased unsaturation level of membrane lipids. After testing some of the mechanisms suggested to contribute to the enhanced tolerance of mycorrhizal plants to drought, we could not find any by which Pisolithus tinctorius could benefit cork oak, at least under the drought conditions imposed in our experiment. Inoculation did not increase photosynthesis under drought, suggesting no effect in sustaining stomatal opening at low soil water content. Similarly, plant water status was not affected by inoculation suggesting that P. tinctorius does not contribute to an increased plant water uptake during drought. Inoculation did increase nitrogen concentration in plants but it was independent of the water status. Furthermore, no significant mycorrhizal effect on drought-induced ROS production or osmotic adjustment was detected, suggesting that these factors are not important for the improved drought tolerance triggered by P. tinctorius.

Biochemical and ecophysiological responses to manganese stress by ectomycorrhizal fungus Pisolithus tinctorius and in association with Eucalyptus grandis.

At relatively low concentrations, the element manganese (Mn) is essential for plant metabolism, especially for photosynthesis and as an enzyme antioxidant cofactor. However, industrial and agricultural activities have greatly increased Mn concentrations, and thereby contamination, in soils. We tested whether and how growth of Pisolithus tinctorius is influenced by Mn and glucose and compare the activities of oxidative stress enzymes as biochemical markers of Mn stress. We also compared nutrient accumulation, ecophysiology, and biochemical responses in Eucalyptus grandis which had been colonized by the ectomycorrhizal Pisolithus tinctorius with those which had not, when both were exposed to increasing Mn concentrations. In vitro experiments comprised six concentrations of Mn in three concentrations of glucose. In vivo experiments used plants colonized by Pisolithus tinctorius, or not colonized, grown with three concentrations of Mn (0, 200, and 1000μM). We found that fungal growth and glucose concentration were correlated, but these were not influenced by Mn levels in the medium. The anti-oxidative enzymes catalase and glutathione S-transferase were both activated when the fungus was exposed to Mn. Also, mycorrhizal plants grew more and faster than non-mycorrhizal plants, whatever Mn exposure. Photosynthesis rate, intrinsic water use efficiency, and carboxylation efficiency were all inversely correlated with Mn concentration. Thus, we originally show that the ectomycorrhizal fungus provides protection for its host plants against varying and potentially toxic concentrations of Mn.

Role of Plant Genotype and Soil Conditions in Symbiotic Plant-Microbe Interactions for Adaptation of Plants to Cadmium-Polluted Soils

We highlighted some of the key problems associated with the use of beneficial microorganisms for improving adaptation of plants to soils, polluted with heavy metals (HMs), especially Cd. Inoculation of pea line SGE and its Cd-tolerant mutant SGECdt with nodule bacteria Rhizobium leguminosarum bv. viciae demonstrated that nodulation process may be disturbed at Cd concentrations below threshold toxicity levels for each partner and the plant genotype plays a major role in nodulation under Cd stress. A comparative mathematical analysis of available information about Cd tolerance, accumulation of HMs (Cd, Cr, Cu, Ni, Pb, Sr and Zn), response to mycorrhizal fungus Glomus sp. and 15 phenotypic traits of 99 pea varieties revealed that (1) the Cd-sensitive varieties were more efficient in exploring the protective potential of symbiosis to compensate their deficit in Cd tolerance and (2) correlations between the studied traits exist and can be helpful for selection of plant-microbe systems adapted to polluted soils. In pot experiment with 11 varieties of Indian mustard, the plant growth-promoting effect of rhizobacterium Variovorax paradoxus 5C-2 negatively correlated with Cd tolerance and shoot Cd concentration of the plants grown in Cd-supplemented soil. In an outdoor pot experiment, inoculation of willow with the ectomycorrhizal fungus Pisolithus tinctorius and a cocktail of rhizobacteria stimulated root exudation, decreased soil pH and increased Cd mobilization in soil and Cd uptake by plants, but decreased plant growth at a moderate contamination level (25 mg Cd kg−1). Opposite effects were observed in highly contaminated soil (77 mg Cd kg−1). We propose a preliminary systematic framework of interactions between these factors that determine the success of microbial inoculation aimed at improving crop performance on HM-polluted soils or enhancing phytoremediation.

Effect of inoculation and fertilization with phosphorus in the survival and growth of Pinus pseudostrobus, in eroded acrisols

Loss of soils and the consequent damage at local and regional scales is one of the most severe and extensive environmental problems. When the soil structure, and even the soil itself, is lost, revegetation faces difficult obstacles, not least the availability of nutrients for plants. As part of a project to develop revegetation strategies in the gullies formed in acrisols, a revegetation assay was established with Pinus pseudostrobus, in which the effect of inoculation with the ectomycorrhizal fungus Pisolithus tinctorius and the addition of phosphorous (KH2PO4) was tested. Five years after initial plantation, the inoculated and non-inoculated trees presented 86% and 65% survival, respectively, representing a difference of 21% between both groups (X2=10.17; P=0.001). Differences in growth were also correlated with inoculation; inoculated trees reached 158±5.4cm in height and 4.8±0.16cm in diameter, while non-inoculated trees reached 134±6.1cm in height and 3.8±0.14cm in diameter (P<0.05 for both variables). These results indicate that inoculation allows this sensitive species to prosper in severely degraded soils and that phosphorus fertilization is not an effective measure.

New microsatellite markers for the ectomycorrhizal fungus Pisolithus tinctorius sensu stricto reveal the genetic structure of US and Puerto Rican populations

Abstract The ectomycorrhizal fungus Pisolithus tinctorius has been introduced to many areas around the world as a source of inoculum for pine plantations. However, little is known about the genetic structure of fungal populations in their introduced habitats. To study the genetics of exotic P. tinctorius populations, we developed and employed seven new microsatellite markers and compared samples from Puerto Rico (exotic range) and the eastern United States (native range). Bayesian cluster analysis, neighbor joining analysis and FST values all strongly separated Puerto Rican populations from North American populations. Consistent with a founder effect, populations from Puerto Rico had reduced allelic richness when compared to samples from the United States. The genetic structure of P. tinctorius populations in Puerto Rico is weak to modest and is not correlated with geographic distance reflecting anthropogenic movement of inoculum with forestry practices.

Potential of ectomycorrhizal fungus Pisolithus tinctorius to tolerate and to degrade trifluoroacetate into fluoroform

Potential of ectomycorrhizal fungus Pisolithus tinctorius to tolerate and to degrade trifluoroacetate into fluoroform

Co-metabolic degradation of mono-fluorophenols by the ectomycorrhizal fungi Pisolithus tinctorius

The release of fluorinated organic compounds from fire retardants or agrochemical products may have a significant negative effect on soil ecosystems. In this study, the ability of Pisolithus tinctorius to tolerate and degrade mono-fluorophenols (FP) was assessed. In vitro studies showed fungal growth in the presence of 0.45mM of 2-FP and 3-FP, but not in the presence of 4-FP. P. tinctorius was able to degrade up to 79% and 92% of 1mM 2-FP and 3-FP, respectively, in glucose supplemented liquid medium, suggesting that 2- and 3-FP degradation occurred in co-metabolism with glucose consumption. 3-Fluorocatechol (FC) and 4-FC were identified as metabolic intermediates using HPLC and LC–MS. Liberation of fluoride was not detected suggesting that a fluorinated dead-end product was formed. In extracts of cells collected at the end of cultures supplemented with the mono-FPs, a metabolic intermediate compatible with a mass corresponding to a fluoromuconate compound, according to LC–MS data, was recovered. The results further suggest that ectomycorrhizal fungi may be able to degrade mono-FP in pure culture while using glucose as a carbon source, through a similar pathway as that found in bacteria. To our knowledge, this is the first time that degradation of mono-FPs by an ectomycorrhizal fungus is reported.

Isolation and characterization of a mycorrhiza helper bacterium from rhizosphere soils of poplar stands

If mycorrhizal formation could be enhanced by co-inoculation with mycorrhiza helper bacteria (MHB) which promote rapid root colonization by specific ectomycorrhizal fungi, this would be of advantage to the poplar forest industry. A number of poplar rhizobacterial strains were isolated from 11 regions of the eastern China. Four of the isolates, SY15, DZ18, HLJ4, and PY10, were characterized as MHB potential strains based on their positive effect on growth of ectomycorrhizal fungi Pisolithus tinctorius (Pers.) and Lactarius insulsus (Fr.). Under greenhouse conditions, one of the bacterial isolate, DZ18, significantly promoted the poplar trees growth and ectomycorrhizal colonization of P. tinctorius and L. insulsus on Populus deltoides Marsh. In contrast, the other three isolates SY15, HLJ4, and PY10 promoted fungal growth in vitro experiments but did not enhance ectomycorrhizal (ECM) formation in the greenhouse experiment. Therefore, it was concluded that DZ18 can be considered as an MHB strain. DZ18 was identified as Bacillus sp. based on morphological, physiological, and biochemical analyses in combination with analysis of 16S rDNA gene sequences.

Effects of Pseudomonas fluorescens on the Water Parameters of Mycorrhizal and Non-Mycorrhizal Seedlings of Pinus halepensis

Inoculation of forest seedlings with mycorrhizal fungi and rhizobacteria can improve the morphological and physiological qualities of plants, especially those used for regeneration of arid areas. In this paper, under standard nursery conditions, Aleppo pine seedlings were inoculated with Pseudomonas fluorescens CECT 5281 rhizobacteria. Some of these seedlings were also inoculated with the ectomycorrhizal fungus Pisolithus tinctorius. Five months after the inoculations, we examined the growth, water parameters (osmotic potential at full turgor [Ψπfull], osmotic potential at zero turgor [Ψπ0], and the tissue modulus of elasticity near full turgor [Emax]), mycorrhizal colonisation, and concentration of macronutrients (N, P, K, Ca and Mg) in the seedlings. Subsequently, a trial was conducted to assess the root growth potential. P. fluorescens CECT 5281 decreased the cellular osmotic potential of P. halepensis seedlings but increased its elasticity. P. tinctorius + P. fluorescens caused osmotic adjustment at zero turgor and increased tissue elasticity, which improved tolerance to water stress. All inoculations improved the growth and nutrition of the seedlings but caused non-significant effects on root growth potential. The co-inoculation Pisolithus tinctorius + Pseudomonas fluorescens at the nursery may be a suitable technique for producing improved seedling material for restoration purposes.

Effect of diflubenzuron on the development of Pinus pinaster seedlings inoculated with the ectomycorrhizal fungus Pisolithus tinctorius

Diflubenzuron (DFB) is an insecticide commonly used to control forest pests. The objectives of this study were to assess the effect of diflubenzuron on the development of Pinus pinaster seedlings and Pisolithus tinctorius under laboratory conditions and to study the possible protective role of this ectomycorrhizal fungus against the effects of diflubenzuron. In vitro experiments revealed that diflubenzuron inhibited fungal growth at all tested concentrations (0.01, 0.1, 1, 10 and 100 mg L(-1)). Root growth was inhibited at the two highest diflubenzuron concentrations. The activity of the antioxidant defence system of non-inoculated P. pinaster increased at 1 and 10 mg DFB kg(-1) substrate, and inoculation increased the threshold to the highest concentration. The protective role of the ectomycorrhizal fungus was seen in the increase of CAT activity. This study revealed that despite causing no mortality, diflubenzuron has the ability to cause sub-lethal damage to P. pinaster. The disproportionate use of this insecticide may lead to higher amounts of its residues in soil and the biosphere, endangering trees, fungi and their symbiosis.

Detection of Forest Ectomycorrhizal Fungi- Pisolithus tinctorius by Nested PCR

For investigating a suitable molecular approach to detect ectomycorrhizal fungi(EMF),the internal transcribed spacer(ITS) region of the EMF Pisolithus tinctorius was amplified with universal primers ITS1-F/ITS4-B and sequenced.Based on the conserved sequence determined by alignment using Clustal W,a pair of specific primers named PtF/PtR were designed by Primer premier 5.0.The specificity of the primer pair was then analyzed in 10 P.tinctorius strains together with 6 reference strains,and a 347 bp product was amplified from all P.tinctorius strains but there was no product from the 6 reference strains.A nested PCR procedure using ITS1-F/ITS4-B as the first-round primers followed by PtF/PtR increased detection sensitivity 1 000-fold to 10 fg DNA template.The target EMF from the mycorrhiza of Pinus massoniana potted seedlings inoculated with P.tinctorius was detected by this technique.The results indicated that the nested PCR assay could be used as a sensitive and accurate way to detect forest EMF P.tinctorius.

Defence responses of Norway spruce seedlings to elicitors of ectomycorrhizal fungus Pisolithus tinctorius and pathogen Heterobasidion annosum are affected by zeatin riboside

Cytokinins are known to attenuate defence responses of plants after elicitor application or inoculation with fungi. To evaluate their role in the regulation of colonisation of Norway spruce (Picea abies) seedlings with mycorrhizal and pathogenic fungus, we analysed the effects of zeatin riboside (ZR) on: i) growth of ectomycorrhizal fungus Pisolithus tinctorius and pathogen Heterobasidion annosum in axenic cultures, ii) colonisation intensity of selected fungi on P. abies seedlings and iii) induction of defence reactions of spruce seedlings following elicitor treatment. Mycorrhizal fungus P. tinctorius showed increased growth at concentrations higherthan 10-2 µM ZR that was accompanied by increased ergosterol concentrations. In contrast, decreased growth of pathogen H. annosum was observed at the highest ZR (10 µM) concentration. ZR treatment also increased colonisation of spruce seedlingswith the mycorrhizal fungus. Application of cell wall preparations of both fungi increased peroxidase (POD) activity in the roots of treated spruce seedlings, whereas only elicitors of H. annosum increased also phenylalanine ammonia-lyase (PAL) activity,levels of soluble phenolics and salicylic acis (SA) concentrations. Application of ZR negated the increased activity of POD that was observed in elicitor treated seedlings, accompanied by increased levels of soluble phenolics in the roots of elicitated seedlings. In contrast, no effects of ZR treatment on PAL activity and SA accumulation could be observed. Our results suggest involvement of ZR in the regulation of cell wall modifications during the fungal colonisation with P. tinctorius and formation of ectomycorrhizae, by affecting the growth of fungal partner and non-specific defence reactions of the plant host.

Effects of the ectomycorrhizal fungus Pisolithus tinctorius and Cd on physiological properties and Cd uptake by hybrid poplar Populus alba × glandulosa

The effects of the ectomycorrhizal fungus Pisolithus tinctorius and cadmium (Cd) on physiological properties and Cd uptake by Populus alba <TEX>${\times}$</TEX> glandulosa was investigated under greenhouse conditions. Cd treatment decreased the photosynthetic rate (<TEX>$P_N$</TEX>) of both non-mycorrhizal (NM) plants (16.3%) and ectomycorrhizal (ECM) plants (11.5%). In addition, the reduction in total dry weight by Cd treatment was greater in ECM plants (24.3%) than that in NM plants (17.6%). Mycorrhizal infection increased the <TEX>$P_N$</TEX> and transpiration rate in both control and Cd-treated plants. Cd treatment increased superoxide dismutase (SOD) activity and decreased glutathione reductase activity, and the increase of SOD activity by Cd treatment was greater in NM plants (40.3%) than that in ECM plants (3.7%). Thiol content increased in both NM and ECM plants treated with Cd solution, and the increase in thiol content in NM plants (43.9%) was greater than that of ECM plants (15.6%). Cd uptake in the leaves, stems, and roots of ECM plants was 69.9%, 167.2% and 72.8%, respectively, higher than in the NM plants. However, the increase in Cd uptake ability of ECM plants resulted in a reduction in dry weight.

Micropropagation of Pinus massoniana and mycorrhiza formation in vitro

A protocol was developed for the micropropagation of Pinus massoniana and mycorrhiza formation on rooted microshoots. Seedling explants were first cultured on Gresshoff and Doy (GD) medium supplemented with 6-benzyladenine (BA) alone or in combination with α-napthaleneacetic acid (NAA) to stimulate the formation of intercotyledonary axillary buds. The frequency of axillary bud induction was up to 97% on medium supplemented with 4.0 mg l−1 BA and 0. 2 mg l−1 NAA, and the average number of buds per explant reached up to 5.5 on medium with 4.0 mg l−1 BA and 0.1 mg l−1 NAA. Axillary buds elongated rapidly after being transferred to half-strength GD medium containing activated charcoal (0.1% w/v). Shoot proliferation was achieved by cutting elongated shoots into stem segments and subculturing on GD medium containing 2 mg l−1 BA and 0.2 mg l−1 NAA. Root primordia were induced in 82% of shoots when transferred to half-strength GD medium containing 0.2 mg l−1 NAA. Root elongation was achieved in a hormone-free GD agar medium or a perlite substrate. Rooted plantlets were inoculated with the mycelium of ectomycorrhizal fungus Pisolithus tinctorius and the formation of ectomycorrhiza-like structures was achieved in vitro.

Growth response of Acacia mangium Willd. seedlings to arbuscular mycorrhizal fungi and four isolates of the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch

Some Acacia mangium Willd. plantations in Asia grow poorly due to low soil fertility and the absence of compatible mycorrhizal fungi. This legume tree can be colonized by arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi but inoculation is not routinely practiced. This study aimed to compare the effectiveness of AM fungi and four isolates of the ECM fungus Pisolithus tinctorius (Pers.) Coker and Couch (Pt) in promoting growth of A. mangium seedlings under glasshouse conditions. AM inoculants were: Glomus etunicatum, G. fasciculatum, G. macrocarpum and Gigaspora margarita and mixed species extracted from rhizosphere soil of a Populus stand in Suwon (AMKFRI), and Carex (AMM6) and Populus (AMM7) growing in mine tailings in Korea. Pisolithus isolates were from Philippines (PtPhil) and Korea (PtKFRI, PtMKACC, PtKACC). Generally, ECM fungi promoted height and diameter growth of A. mangium more than the AM inoculants. The Korean Pisolithus increased plant dry weight by 122–145%, mixed AM inoculants by 61–97%, and Glomus and Gigaspora by 45–72% over the control. PtKACC gave the highest root colonization and promoted the highest growth and concentration of most nutrients. Mycorrhizal root colonization was positively correlated with plant dry weight, Na, Fe and Cu concentrations and N, P, K, Ca, Na, Fe and Cu contents. In conclusion, the results provide strong evidence for benefits of mycorrhizal inoculation on A. mangium seedlings under glasshouse conditions. The Korean Pisolithus isolates (particularly PtKACC), and two AM fungi (AMKFRI and AMM6) are potential mycorrhizal inoculants but their effectiveness and persistence should be determined on degraded lands in tropical countries where A. mangium is being planted for rehabilitation.

Ectomycorrhizal fungi enhance nitrogen and phosphorus nutrition of Nothofagus dombeyi under drought conditions by regulating assimilative enzyme activities

Drought stress conditions (DC) reduce plant growth and nutrition, restraining the sustainable reestablishment of Nothofagus dombeyi in temperate south Chilean forest ecosystems. Ectomycorrhizal symbioses have been documented to enhance plant nitrogen (N) and phosphorus (P) uptake under drought, but the regulation of involved assimilative enzymes remains unclear. We studied 1-year-old N. dombeyi (Mirb.) Oerst. plants in association with the ectomycorrhizal fungi Pisolithus tinctorius (Pers.) Coker & Couch. and Descolea antartica Sing. In greenhouse experiments, shoot and root dry weights, mycorrhizal colonization, foliar N and P concentrations, and root enzyme activities [glutamate synthase (glutamine oxoglutarate aminotransferase (GOGAT), EC 1.4.1.13-14), glutamine synthetase (GS, EC 6.3.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), nitrate reductase (NR, EC 1.6.6.1), and acid phosphomonoesterase (PME, EC 3.1.3.1-2)] were determined as a function of soil-water content. Inoculation of N. dombeyi with P. tinctorius and D. antartica significantly stimulated plant growth and increased plant foliar N and P concentrations, especially under DC. Ectomycorrhizal inoculation increased the activity of all studied enzymes relative to non-mycorrhizal plants under drought. We speculate that GDH is a key enzyme involved in the enhancement of ectomycorrhizal carbon (C) availability by fuelling the tricarboxylic acid (TCA) cycle under conditions of drought-induced carbon deficit. All studied assimilative enzymes of the ectomycorrhizal associations, involved in C, N, and P transfers, are closely interlinked and interdependent. The up-regulation of assimilative enzyme activities by ectomycorrhizal fungal root colonizers acts as a functional mechanism to increase seedling endurance to drought. We insist upon incorporating ectomycorrhizal inoculation in existing Chilean afforestation programs.