Divergence in gene expression related to variation in host specificity of an ectomycorrhizal fungus

Ectomycorrhizae are formed by mutualistic interactions between fungi and the roots of woody plants. During symbiosis the two organisms exchange carbon and nutrients in a specific tissue that is formed at the contact between a compatible fungus and plant. There is considerable variation in the degree of host specificity among species and strains of ectomycorrhizal fungi. In this study, we have for the first time shown that this variation is associated with quantitative differences in gene expression, and with divergence in nucleotide sequences of symbiosis-regulated genes. Gene expression and sequence evolution were compared in different strains of the ectomycorrhizal fungus Paxillus involutus; the strains included Nau, which is not compatible with birch and poplar, and the two compatible strains Maj and ATCC200175. On a genomic level, Nau and Maj were very similar. The sequence identity was 98.9% in the 16 loci analysed, and only three out of 1075 genes analysed by microarray-based hybridizations had signals indicating differences in gene copy numbers. In contrast, 66 out of the 1075 genes were differentially expressed in Maj compared to Nau after contact with birch roots. Thirty-seven of these symbiosis-regulated genes were also differentially expressed in the ATCC strain. Comparative analysis of DNA sequences of the symbiosis-regulated genes in different strains showed that two of them have evolved at an enhanced rate in Nau. The sequence divergence can be explained by a decreased selection pressure, which in turn is determined by lower functional constraints on these proteins in Nau as compared to the compatible strains

Effects of liming on ectomycorrhizal fungi infecting Pinus sylvestris L .1. Mycorrhizal infection in limed humus in the laboratory and isolation of fungi from mycorrhizal roots

Pinus sylvestris L. seedlings were grown in the laboratory in a forest humus which had been treated with different levels of CaO to give a soil pH gradient from 4 to 7. After 12 and 15 wk the resulting ectomycorrhizal infection was classified according to macroscopical morphology. The uniformity of the infection types distinguished was evaluated by isolating the fungi from the root tips and infecting seedlings with the isolates obtained. The isolation method was improved by using a medium containing benomyl and chlortetracyclin and by isolating from very small particles. For some mycorrhizal types the fungus was successfully isolated from 80% of the plated root particles. The number of root tips that became mycorrhizal increased from 70% at pH 4 to nearly 100% around pH 5. Thereafter it decreased linearly to reach a minimum of slightly less than 40% at pH 7.5. Five different mycorrhizal types could be distinguished. They occurred with different abundance, distribution and pH optima. They were present throughout the entire pH range, except for a yellow type, identified as Piloderma croceum Erikss. & Hjortst., which was not found at values higher than 6.2. There was no great differences between infections on plants harvested after 12 and 15 wk. Seedling growth was constant to pH 5 then it increased up to about pH 7, at higher pH values growth declined

Effects of lime and ash treatments on ectomycorrhizal infection of Pinus sylvestris L. Seedlings planted in a pine forest

Plots in a 40-yr-old Scots pine (Pinus sylvestris) stand in S. Sweden were treated with 5 tons tonnes? lime/ha, 7.5 tons wood-ash/ha, or left untreated. One-yr-old P. sylvestris seedlings were planted in each of the plots in early June, 12 months after ash treatment and 18 months after lime treatment. The pH of the limed, ash treated and untreated plots were 5.2, 6.4 and 3.8, respectively. Four months later 6 different ectomycorrhizal types had infected the seedlings in all the treatments. A mycorrhizal type designated 'pink' was more than twice as common in the lime treatments as in the control and ash treatments. Piloderma croceum was significantly more abundant in limed soil than in ash treated soil. The results were compared with those from a previously published bioassay performed in the laboratory, where P. sylvestris seedlings had been grown in soil from the same forest. Similar soil Ph values in the 2 studies resulted in different relative infection rates of the mycorrhizal types found. All but one mycorrhizal type, designated 'white', were found in the laboratory experiment. This difference suggests that mycelial connections to the mature host plants may significantly alter the ability of different fungi to colonize host plant roots in competition with each other in comparison with situations in which the fungi infect from propagules in the soil

Effects of liming on ectomycorrhizal fungi infecting Pinus-sylvestris L. 3. Saprophytic growth and host plant infection at different ph values in unsterile humus

Five ectomycorrhizal fungi were studied in relation to the effects of applications of lime and wood ash to the growth substrate. Growth and survival of inoculated fungi in unsterile humus and relative root colonization frequency by inoculated and indigenous ectomycorrhizal fungi were measured. Growth of the fungi was tested in Petri dish systems with humus taken from a field site treated with lime and wood ash. The infection potential of the fungi was tested by introducing Pinus sylvestris L. seedlings into the growth systems. Paxillus involutus (Fr.) Fr. was the only fungus affected both by the pH increase, and by the different treatments applied, in all aspects of its ecology tested (growth and survival in humus, infection potential and competitive ability). No other fungus grew saprophytically, but they showed the similar changes in infection potential in response to pH whether lime or ash had been used

Isolation of fluorescein diacetate stained hyphae from soil by micromanipulation

Stained fluorescent hyphae were picked out from soil using a Skerman micromanipulator. About 30% of the plated out hyphae produced visible colonies, at least half of which could be considered to be derived from hyphae, The dominating genera were Penicillium, Oidiodendron and Mortierella. Non-sporulating fungi were rather few (4'5%). Results were compared with those obtained using the dilution plate and soil washing techniques

Ectomycorrhizal mycelial species composition in apatite amended and non-amended mesh bags buried in a phosphorus-poor spruce forest.

We studied the effect of apatite amendment on ectomycorrhizal (EM) mycelial biomass production and species composition in a phosphorus-poor spruce forest using sand-filled mesh bags. Control and apatite-amended bags were buried in pairs in the lower part of the organic horizon for one growth season. DNA extraction, PCR of the ITS region, cloning and random sequencing were used to examine the fungal species composition in each bag. Sequences were identified by comparison with the UNITE database and GenBank. Our study confirmed previous results that the major fungal ingrowth in mesh bags was of EM origin. On average 13 species were detected in each bag. Tylospora fibrillosa, Amphinema sp., Tomentellopsis submollis, and Xerocomus badius made up almost 80% of the EM sequences. High biomass was related to increased dominance of specific species. There were no statistically significant differences in biomass production estimated from PLFA 18:2omega6, 9, or between fungal communities of apatite-amended and control bags estimated from DNA after one growth season. The potential of the mesh bag method in studies of functional diversity of EM mycelia in the field is discussed

Effects of liming on ectomycorrhizal fungi infecting Pinus sylvestris L. 2. Growth-rates in pure culture at different ph values compared to growth-rates in symbiosis with the host plant

Growth rates of ectomycorrhizal fungi were measured in pure culture at pH 3-8 on MMN-agar and sterilized peat, with and without nutrients added. Mycorrhizas were synthesized with Pinus sylvestris L. seedlings and the growth rate of the external mycelium was measured in peat at pH 3.8 and 7.3. The fungi were three isolates of Piloderma croceum Erikss. & Hjorts., two isolates of an unidentified (pink) mycorrhizal type and one isolate of another unidentified (white long) mycorrhizal type, all isolated from P. sylvestris roots. All three fungi showed much higher (2-5 times) growth rates when grown as symbionts than when grown in pure culture. The P. croceum isolates also grew as symbionts at pH 7.3, where no growth occurred in pure culture. These findings emphasize the danger of generalizing from data obtained in pure culture studies to explain what may happen when fungi grow in symbiosis with host plants. Generally, growth rates in pure culture were higher and the pH tolerance levels were wider on agar than on peat. The exceptions were one P. croceum isolate, which had the fastest pure culture growth rate on peat with MMN, and the pink isolates, which grew at higher pH on peat than on agar. There were no significant differences between growth on peat with and without MMN

The effects of liming on mycelial colonization and carbon allocation in ectomycorrhizal mycelia attached to Pinus silvestris plants

The effects of liming on ectomycorrhizal mycelial colonization and carbon allocation were assessed using Pinus silvestris plants grown in association with an ectomycorrhizal fungus. Plants were placed in perspex growth chambers divided by a central partition so that soils of different pH could be used on either side of the chambers. Differences between the growth form and growth rates of the fungi and allocation of plant assimilate were determined at different pH values