Culture of mycorrhizal tree seedlings under controlled conditions: Effects of nitrogen and aluminium

A sand culture system was developed for growth of mycorrhizal seedlings under monoxenic conditions, with frequently renewed nutrient solution The composition of the nutrient solution resembled that of a forest soil solution, based on long-term measurements from forest sites at Soiling, northern Germany. Seedlings of Picea abies (L.) Karst. inoculated with Lactarius rufus (Scop.) Fr. were grown in this culture system. Plants developed rapidly, having almost totally mycorrhizal root systems. Nitrate at 2.7 mM in the nutrient solution and applied over a 13 week period had no negative effect on mycorrhizal development. Ammonium at 2.7 mM reduced the degree of mycorrhizal infection slightly, in such a way that the degree of mycorrhizal infection was reduced to a much less extent than the total number of root lips. Hence, the impact of NH⁺₄ may be primarily on root development and not on mycorrhizal fungal colonization. When the concentrations of NO-₃ and NH⁺₄ used in the present study are compared to those found in forest soil solutions, NO-₃ and NH₄⁺ would not appear to influence mycorrhizal development negatively under natural conditions. Aluminium at 0.8 mM and applied over a 13 week period reduced Mg uptake into roots and needles by 52 and 64%, respectively, resulting in needle chlorosis and strongly reduced photosynthetic activity. From a comparison of this study with others, no major difference in physiological response to aluminium exposure between non-mycorrhizal seedlings and seedlings colonized with Lactarius rufus was found.     

Comparison of biomass allocation in ectomycorrhizal and nonmycorrhizal Douglas fir seedlings of similar nutrition and overall size

Biomass allocation in 6-month-old ectomycorrhizal Douglas fir seedlings was compared to that in nonmycorrhizal seedlings of the same age, nutrient status and total biomass. Seedlings colonized by Rhizopogon vinicolor had the same distribution of biomass between roots, stems and needles, but only 56% of the total length of roots (including mycorrhizal branches) compared to nonmycorrhizal seedlings. Laccaria laccata had no effect on distribution of biomass or root length of seedlings. The results for Rhizopogon provide direct evidence that the process of ectomycorrhizal colonization can significantly affect plant biomass allocation by one or more mechanisms not directly related to altered nutrition or overall plant size.     

Discovering the role of Patagonian birds in the dispersal of truffles and other mycorrhizal fungi

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The last common ancestor of Sarcolaenaceae and Asian dipterocarp trees was ectomycorrhizal before the India-Madagascar separation, about 88 million years ago

Phylogenetic studies comparing the Dipterocarpaceae and the Sarcolaenaceae, a tree family endemic to Madagascar, have shown that the Sarcolaenaceae share a common ancestor with Asian dipterocarps. This suggests that Asian dipterocarps drifted away from Madagascar with the India-Seychelles landmass and then dispersed through Asia. Although all dipterocarps examined so far have been found to be ectomycorrhizal, the ectomycorrhizal status of Sarcolaenaceae had not been investigated. Here we establish the ectomycorrhizal status of Sarcolaenaceae using histological and molecular methods. This indicates that the common ancestor of the Sarcolaenaceae and Asian dipterocarps was ectomycorrhizal, at least before the separation of the Madagascar-India landmass, 88 million years ago.     

The high-affinity poplar ammonium importer PttAMT1.2 and its role in ectomycorrhizal symbiosis

One way to elucidate whether ammonium could act as a nitrogen (N) source delivered by the fungus in ectomycorrhizal symbiosis is to investigate plant ammonium importers. Expression analysis of a high-affinity ammonium importer from Populus tremulax tremuloides (PttAMT1.2) and of known members of the AMT1 gene family from Populus trichocarpa was performed. In addition, PttAMT1.2 function was studied in detail by heterologous expression in yeast. PttAMT1.2 expression proved to be root-specific, affected by N nutrition, and strongly increased in a N-independent manner upon ectomycorrhiza formation. The corresponding protein had a K(M) value for ammonium of c. 52 microm. From the seven members of the AMT1 gene family, one gene was exclusively expressed in roots while four genes were detectable in all poplar organs but with varying degrees of expression. Ectomycorrhiza formation resulted in a strong upregulation of three of these genes. Our results indicate an increased ammonium uptake capacity of mycorrhized poplar roots and suggest, together with the expression of putative ammonium exporter genes in the ectomycorrhizal fungus Amanita muscaria, that ammonium could be a major N source delivered from the fungus towards the plant in symbiosis.     

Identification of symbiosis-regulated genes in Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza by differential hybridization of arrayed cDNAs

Ectomycorrhiza development alters gene expression in the fungal and plant symbionts. The identification of a large number of genes expressed exclusively or predominantly in the symbiosis will contribute greatly to the understanding of the development of the ectomycorrhizal symbiosis. We have constructed a cDNA library of 4-day-old Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza and sequenced 850 cDNAs cloned randomly or obtained through suppression subtractive hybridization (SSH). Based on the absence of a database match, 43% of the ectomycorrhiza ESTs are coding for novel genes. At the developmental stage analysed (fungal sheath formation), the majority of the identified sequences represented 'housekeeping' proteins, i.e. proteins involved in gene/protein expression, cell-wall proteins, metabolic enzymes, and components of signalling systems. We screened arrayed cDNAs to identify symbiosis-regulated genes by using differential hybridization. Comparisons of signals from free-living partners and symbiotic tissues revealed significant differences in expression levels (differential expression ratio >2.5) for 17% of the genes analysed. No ectomycorrhiza-specific gene was detected. The results successfully demonstrate the use of the cDNA array and SSH systems as general approaches for dissecting symbiosis development, and provide the first global picture of the cellular functions operating in ectomycorrhiza.     

Effects of inorganic nitrogen and phosphorus supply on nitrogen aquisition from alanine by birch seedlings in symbiosis with Paxillus involutus

Northern forests are exposed to relatively high ammonia inputs due to high atmospheric deposition and the common practise of forest fertilization. It is not known how increased soil NH₄ ⁺concentrations affect acquisition of symbiosis-mediated N from organic sources. We examined the effect of inorganic N and P availability on N acquisition from alanine by 43 weeks old birch (Betula pendula) seedlings in symbiosis with the ectomycorrhizal fungus Paxillus involutus. The seedlings were exposed for 9 weeks to nutrient additions equivalent to 43 kg N and 6.4 kg P ha^-1 (low N and P availability), 250 kg N and 38 kg P ha^-1(high N and P availability) or to 250 kg N and 6.4 kg P ha^-1 (high N and low P availability). Carbon and nitrogen allocation between the symbionts was assessed by exposing the foliage to ¹⁴CO₂ and the mycelium to ¹⁵N-alanine or ¹⁵NH₄ ⁺ simultaneously and measuring the distribution of the isotopic tracers after a three-day chase period. High inorganic N combined with low P availability did not have marked effect on symbiosis-mediated N uptake from alanine, whilst high N and P availability reduced alanine-derived ¹⁵N translocation by the fungus to the plant. Shoot ¹⁵N concentration and concentration of ¹⁴C in the extramatrical mycelium correlated significantly across treatments pointing to controlled reciprocity of transactions between the partners.     

Mycorrhiza formation and elevated CO2 both increase the capacity for sucrose synthesis in source leaves of spruce and aspen

The effects of mycorrhiza formation in combination with elevated CO₂ concentrations on carbon metabolism of Norway spruce ( Picea abies ) seedlings and aspen ( Populus tremula × Populus tremuloides ) plantlets were analysed. Plants were inoculated for 6 wk with the ectomycorrhizal fungi Amanita muscaria and Paxillus involutus (aspen only) in an axenic Petri-dish culture at 350 and 700 μl l⁻¹ CO₂ partial pressure. After mycorrhiza formation, a stimulation of net assimilation rate was accompanied by decreased activities of sucrose synthase, an increased activation state of sucrose-phosphate synthase, decreased fructose-2,6-bisphosphate and starch, and slightly elevated glucose-6-phosphate contents in source leaves of both host species, independent of CO₂ concentration. Exposure to elevated CO₂ generally resulted in higher net assimilation rates, increased starch as well as decreased fructose-2,6-bisphosphate (aspen only) content in source leaves of both mycorrhizal and nonmycorrhizal plants. Our data indicate only slightly improved carbon utilization by mycorrhizal plants at elevated CO₂. They demonstrate however, that both factors which modulate the sink-source properties of plants increase the capacity for sucrose synthesis in source leaves mainly by allosteric enzyme regulation.     

Mycorrhizal weathering: A true case of mineral plant nutrition?

Weatherable minerals in all podzol surface soils andshallow granitic rock under European coniferousforests studied hitherto are criss-crossed bynumerous open, tubular pores, 3–10 µm in width. Wehypothesize that these pores were formed bycomplex-forming, low-molecular weight organic acidsexuded by or formed in association with mycorrhizalfungi. It is well known that ectomycorrhizal myceliumrepresents a greatly extended, and better distributed,surface area for the absorption of nutrients. However, there have been few investigations of how thewhereabouts of individual hypha affect nutrientuptake. The results presented here provide directevidence that the mycelium is able to penetrate, andmost probably create, microsites which areinaccessible to plant roots and isolated from bulksoil solution phenomena. Dissolved products could betranslocated to the host plant roots, bypassing thesoil solution with often toxic concentration ofAl³⁺ from acid rain, and bypassing competitionfor nutrient uptake by other organisms. Furthermore,there is strong evidence that ``rock-eating'mycorrhizal fungi play a role in the formation ofpodzol E horizons. The partly speculativeinterpretations presented here challenge conventionalideas about (1) the importance of nutrient uptakefrom the bulk soil solution (2) criteria for criticalloads of acid atmospheric deposition for forests, and(3) the process of podzolization.