Sari Timonen

Immunofluorescence microscopy of the cytoskeleton in filamentous fungi after quick-freezing and low-temperature fixation

In filamentous fungi a well-preserved hyphal cytoskeleton was obtained for indirect immunofluorescence (IIF) microscopy by quick-freezing in Freon 22 cooled with liquid nitrogen, followed by fixation with glutaraldehyde in ethanol or formaldehyde in methanol at −75°C. Several factors previously claimed to alter the structure of the cytoskeleton during the fixation process were totally or partially avoided. No microtubule stabilizing buffers were used, detergent treatment of hyphae was unnecessary when the fixative was formaldehyde in methanol, and the visualization of the microtubule cytoskeleton was possible without enzymatic treatment of hyphal walls. The procedure confirmed the picture of the structure of the microtubule cytoskeleton in the hyphae earlier achieved by more conventional fixation methods, and made possible the resolution of some new details. The visualization of actin was also improved.

Nested PCR detection of Archaea in defined compartments of pine mycorrhizospheres developed in boreal forest humus microcosms

Archaea colonising defined compartments of Scots pine Suillus bovinus or Paxillus involutus mycorrhizospheres developed in forest humus-containing microcosms were investigated by nested polymerase chain reaction (PCR), cloning, restriction fragment length polymorphism (RFLP) and sequencing. Archaea representing six RFLP groups were detected in the system. Sequence analysis of clones representing the different RFLP types confirmed the presence of novel Finnish forest soil Crenarchaeota. Archaeal sequences were identified from mycorrhizas of both P. involutus and S. bovinus, at the margins of the external mycelium and in uncolonised humus but not from non-mycorrhizal short roots. Fungal and compartment-specific crenarchaeal occupation of mycorrhizospheres is discussed in relation to bacterial community distribution in similar systems.

Archaea in dry soil environments

Archaea belong to the least well known major group of soil inhabiting microbes as the concept of the very existence of the archaea was introduced only in 1977 and the domain of Archaea established in 1990. The first reports of finding these organisms in soils were published even later. This paper will review the research carried out of the archaea in dry moderate soil environments. It will particularly consider the specific habitats where the archaea live in soils, as well as their associations with other organisms. There is thus far relatively little knowledge about the metabolism of the soil archaea, but the knowledge about their exact habitats and associations as well as their genetic potential point the way to discovering more about the different soil archaeal functions.

Distribution of cren- and euryarchaeota in scots pine mycorrhizospheres and boreal forest humus.

Archaeal 16S rRNA gene sequences have been found in a variety of moderate-temperature habitats including soil and rhizospheres. In this study, the differences of archaeal communities associated with Scots pine (Pinus sylvestris L.) short roots, different types of mycorrhizospheric compartments, and uncolonized boreal forest humus were tested by direct DNA extraction, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE), and sequencing. The results indicated that mycorrhizal colonization of Scots pine roots substantially influence the archaeal community of pine rhizospheres. Colonization of short roots by most mycorrhizal fungi tested increased both archaeal frequency and diversity. Most of the archaeal sequences encountered in mycorrhizas belonged to the phylum Euryarchaeota, order of Halobacteriales. The difference in archaeal diversity between the mycorrhizospheric compartments and humus was profound. Most compartments with fungal components contained euryarchaeotal 16S rRNA gene sequences, whereas a high diversity of crenarchaeotal sequences and no euryarchaeotal sequences were found in forest humus outside mycorrhizospheres.

Effect of Tree Species and Mycorrhizal Colonization on the Archaeal Population of Boreal Forest Rhizospheres

ABSTRACT Group 1.1c Crenarchaeota are the predominating archaeal group in acidic boreal forest soils. In this study, we show that the detection frequency of 1.1c crenarchaeotal 16S rRNA genes in the rhizospheres of the boreal forest trees increased following colonization by the ectomycorrhizal fungus Paxillus involutus. This effect was very clear in the fine roots of Pinus sylvestris, Picea abies, and Betula pendula, the most common forest trees in Finland. The nonmycorrhizal fine roots had a clearly different composition of archaeal 16S rRNA genes in comparison to the mycorrhizal fine roots. In the phylogenetic analysis, the 1.1c crenarchaeotal 16S rRNA gene sequences obtained from the fine roots formed a well-defined cluster separate from the mycorrhizal ones. Alnus glutinosa differed from the other trees by having high diversity and detection levels of Crenarchaeota both on fine roots and on mycorrhizas as well as by harboring a distinct archaeal flora. The similarity of the archaeal populations in rhizospheres of the different tree species was increased upon colonization by the ectomycorrhizal fungus. A minority of the sequences obtained from the mycorrhizas belonged to Euryarchaeota (order Halobacteriales).

Tolerance and biodegradation of m‐toluate by Scots pine, a mycorrhizal fungus and fluorescent pseudomonads individually and under associative conditions

The tolerance to, and degradation of m‐toluate by Scots pine (Pinus sylvestris), a symbiotic mycorrhizal fungus (Suillus bovinus) and Pseudomonas fluorescens strains, with or without m‐toluate‐degrading capacity, was determined individually and in all symbiotic/associative plant‐microbe combinations. Fungal survival on medium with m‐toluate was increased in co‐culture with the degradative bacterial strains on agar plates (up to 0·02%, w/v). When fungi were grown in mycorrhizal association with Scots pine seedlings in test‐tube microcosms containing expanded clay pellets and growth media, the fungus was able to withstand m‐toluate concentrations up to 2·0%, w/v in all treatments. The seedling tolerance remained unaltered regardless of the presence or absence of mycorrhizal fungi or biodegradative bacteria. Reduction in m‐toluate levels was only detected in treatments inoculated with bacterial strains harbouring TOL catabolic plasmids. The plant and fungus, alone or in mycorrhizal symbiosis, were unable to cleave m‐toluate. The presence of easily available plant‐derived carbon sources did not impede m‐toluate degradation by the bacteria in the mycorrhizosphere.

Identification of nitrogen mineralization enzymes, l-amino acid oxidases, from the ectomycorrhizal fungi Hebeloma spp. and Laccaria bicolor

Amino acids are major nitrogen sources in soils and they harbour a central position in the nitrogen metabolism of cells. We determined whether Hebeloma spp. and Laccaria bicolor expressed the enzyme L-amino acid oxidase (LAO), which catalyses the oxidative deamination of the alpha-amino group of L-amino acids. We measured LAO activities from the mycelial extracts of seven laboratory-grown fungal strains with three methods, and we measured how LAO activities were expressed in one Hebeloma sp. strain grown on four nitrogen sources. Hebeloma spp. and L. bicolor converted L-phenylalanine, but not D-phenylalanine, to hydrogen peroxide, 2-oxoacid, and ammonia, suggesting that they expressed LAO enzymes. The enzymes utilized five out of seven tested L-amino acids as substrates. LAO activities were maximal at pH 8, where Michaelis constant (Km) values were 2-5mm. The LAO of Hebeloma sp. was expressed on every nitrogen source analysed, and the activities were the highest in mycelia grown in nitrogen-rich conditions. We suggest that LAO is a mechanism for cellular amino acid catabolism in Hebeloma spp. and L. bicolor. Many soil bacteria and fungi also express LAO enzymes that have broad substrate specificities. Therefore, LAO is a potential candidate for a mechanism that catalyses nitrogen mineralization from amino acids at the ecosystem level.

ITS rDNA sequence-based phylogenetic analysis of Tomentellopsis species from boreal and temperate forests, and the identification of pink-type ectomycorrhizas

Fungi in the genus Tomentellopsis were subjected to molecular phylogenetic analyses in order to clarify species-level relationships and mycorrhiza-forming ability with coniferous and deciduous trees. Fungal nucleotide sequence data from the internal transcribed spacer of nuclear ribosomal DNA (ITS rDNA) region were obtained from fruitbodies, ectomycorrhiza and pure cultures. Maximum parsimony (MP), distance (neighbor joining, NJ) and maximum likelihood (ML) analyses of aligned ITS sequences highlighted three clades designated T. echinospora, T. submollis and T. bresadoliana. Sporocarp tissue and ectomycorrhizas, or isolated mycelia, previously described as Pinirhiza rosea, Piceirhiza rosea or Pink were clearly identified in a strongly supported T. submollis clade. Host-linked population variation in this clade was also noted that may reflect active speciation activity. A fungus isolated from Beige-type mycorrhizas formed on Scots pine seedlings appeared in the paraphyletic T. echinospora clade which included sequences showing greater divergence. The data provide a primary classification of Tomentellopsis species that is urgently needed in assessment of the importance of resupinate thelephoroid fungi in mycorrhizal communities associated with trees in coniferous and deciduous forest ecosystems.

Cytoskeleton in mycorrhizal symbiosis

An understanding of the role played by the cytoskeleton in formation and function of mycorrhizas has been hampered by the technical difficulty of working with mycorrhizal material. Recently, however, improved labelling techniques suitable for both plant and fungal symbionts in combination with either epifluorescence microscopy or laser scanning confocal microscopy have resulted in new information. As well, molecular methods have made it possible to monitor changes of cytoskeletal elements during mycorrhiza development. Currently we know that the cytoskeletal systems of both plant and fungal partners undergo changes during both ecto- and endomycorrhizal symbiosis. However, little information is available concerning the regulatory factors or the cause and effect relationship of cytoskeletal changes and cellular events. In this article, research involving the cytoskeleton of mycorrhizas is reviewed in detail, whereas basic information of the cytoskeleton of plant and fungal cells is only briefly discussed as background. A brief comparison is also made between the information on mycorrhizas with that of biotrophic pathogenic fungi and the Rhizobium–legume symbiosis.

Effects of sugar beet chitinase IV on root-associated fungal community of transgenic silver birch in a field trial

Heterogenous chitinases have been introduced in many plant species with the aim to increase the resistance of plants to fungal diseases. We studied the effects of the heterologous expression of sugar beet chitinase IV on the intensity of ectomycorrhizal (ECM) colonization and the structure of fungal communities in the field trial of 15 transgenic and 8 wild-type silver birch (Betula pendula Roth) genotypes. Fungal sequences were separated in denaturing gradient gel electrophoresis and identified by sequencing the ITS1 region to reveal the operational taxonomic units. ECM colonization was less intense in 7 out of 15 transgenic lines than in the corresponding non-transgenic control plants, but the slight decrease in overall ECM colonization in transgenic lines could not be related to sugar beet chitinase IV expression or total endochitinase activity. One transgenic line showing fairly weak sugar beet chitinase IV expression without significantly increased total endochitinase activity differed significantly from the non-transgenic controls in the structure of fungal community. Five sequences belonging to three different fungal genera (Hebeloma, Inocybe, Laccaria) were indicative of wild-type genotypes, and one sequence (Lactarius) indicated one transgenic line. In cluster analysis, the non-transgenic control grouped together with the transgenic lines indicating that genotype was a more important factor determining the structure of fungal communities than the transgenic status of the plants. With the tested birch lines, no clear evidence for the effect of the heterologous expression of sugar beet chitinase IV on ECM colonization or the structure of fungal community was found.