Taina Pennanen

MICROBIAL COMMUNITY STRUCTURE AND pH RESPONSE IN RELATION TO SOIL ORGANIC MATTER QUALITY IN WOOD-ASH FERTILIZED, CLEAR-CUT OR BURNED CONIFEROUS FOREST SOILS

Humus phospholipid fatty acid (PLFA) analysis was used in clear-cut, wood-ash fertilized (amounts applied: 1000, 2500, and 5000 kg ha−1), or prescribed burned (both in standing and clear-cut) coniferous forests to study the effects of treatments on microbial biomass and community structure. The microbial biomass (total PLFAs) decreased significantly due to the highest rate of wood-ash fertilization, clear-cutting, and the two different fire treatments when compared to control amounts. Fungi appeared more seriously reduced by these treatments than bacteria, as revealed by a decreased index of fungal:bacterial PLFAs when compared to the controls. The community structure was evaluated using the PLFA pattern. The largest treatment effect was due to burning in both areas studied, which resulted in increases in 16:1ω5 and proportional decreases in 18:2ω6. Clear-cutting and the different amounts of ash application resulted in similar changes in the PLFA pattern to the burning treatments, but these were less pronounced. Attempts to correlate the changes in the PLFA pattern to soil pH, bacterial pH response patterns (measured using thymidine incorporation), or substrate quality (measured using IR spectroscopy) were only partly successful. Instead, we hypothesize that the changes in the PLFA pattern of the soil organisms were related to an altered substrate quantity, that is the availability of substrates after the treatments.

Fungal community analysis by high-throughput sequencing of amplified markers – a user's guide

Novel high-throughput sequencing methods outperform earlier approaches in terms of resolution and magnitude. They enable identification and relative quantification of community members and offer new insights into fungal community ecology. These methods are currently taking over as the primary tool to assess fungal communities of plant-associated endophytes, pathogens, and mycorrhizal symbionts, as well as free-living saprotrophs. Taking advantage of the collective experience of six research groups, we here review the different stages involved in fungal community analysis, from field sampling via laboratory procedures to bioinformatics and data interpretation. We discuss potential pitfalls, alternatives, and solutions. Highlighted topics are challenges involved in: obtaining representative DNA/RNA samples and replicates that encompass the targeted variation in community composition, selection of marker regions and primers, options for amplification and multiplexing, handling of sequencing errors, and taxonomic identification. Without awareness of methodological biases, limitations of markers, and bioinformatics challenges, large-scale sequencing projects risk yielding artificial results and misleading conclusions.

Structure of the Microbial Communities in Coniferous Forest Soils in Relation to Site Fertility and Stand Development Stage.

A bstractThe structure, biomass, and activity of the microbial community in the humus layer of boreal coniferous forest stands of different fertility were studied. The Scots pine dominated CT (Calluna vulgaris type) represented the lowest fertility, while VT (Vaccinium vitis-idaéa type), MT (Vaccinium myrtillus type), and OMT (Oxalis acetocella–Vaccinium myrtillus type) following this order, were more fertile types. The microbial community was studied more closely by sampling a succession gradient (from a treeless area to a 180-years-old Norway spruce stand) at the MT type site. The phospholipid fatty acid (PLFA) analysis revealed a gradual shift in the structure of the microbial community along the fertility gradient even though the total microbial biomass and respiration rate remained unchanged. The relative abundance of fungi decreased and that of bacteria increased with increasing fertility. The structure of the bacterial community also changed along the fertility gradient. Irrespective of a decrease in fungal biomass and change in bacterial community structure after clear-cutting, the PLFA analysis did not show strong differences in the microbial communities in the stands of different age growing on the MT type site. The spatial variation in the structure of the microbial community was studied at a MT type site. Semivariograms indicated that the bacterial biomass, the ratio between the fungal and bacterial biomasses, and the relative amount of PLFA 16:1ω5 were spatially autocorrelated within distances around 3 to 4 m. The total microbial and fungal biomasses were autocorrelated only up to 1 m. The spatial distribution of the humus microbial community was correlated mainly with the location of the trees, and consequently, with the forest floor vegetation.

Ecosystem properties and microbial community changes in primary succession on a glacier forefront

Abstract We studied microbial community composition in a primary successional chronosequence on the forefront of Lyman Glacier, Washington, United States. We sampled microbial communities in soil from nonvegetated areas and under the canopies of mycorrhizal and nonmycorrhizal plants from 20- to 80-year-old zones along the successional gradient. Three independent measures of microbial biomass were used: substrate-induced respiration (SIR), phospholipid fatty acid (PLFA) analysis, and direct microscopic counts. All methods indicated that biomass increased over successional time in the nonvegetated soil. PLFA analysis indicated that the microbial biomass was greater under the plant canopies than in the nonvegetated soils; the microbial community composition was clearly different between these two types of soils. Over the successional gradient, the microbial community shifted from bacterial-dominated to fungal-dominated. Microbial respiration increased while specific activity (respiration per unit biomass) decreased in nonvegetated soils over the successional gradient. We proposed and evaluated new parameters for estimating the C use efficiency of the soil microbial community: “Max” indicates the maximal respiration rate and “Acc” the total C released from the sample after a standard amount of substrate is added. These, as well as the corresponding specific activities (calculated as Max and Acc per unit biomass), decreased sharply over the successional gradient. Our study suggests that during the early stages of succession the microbial community cannot incorporate all the added substrate into its biomass, but rapidly increases its respiration. The later-stage microbial community cannot reach as high a rate of respiration per unit biomass but remains in an “energy-saving state,” accumulating C to its biomass.

Microbial community structure and characteristics of the organic matter in soils under Pinus sylvestris, Picea abies and Betula pendula at two forest sites

Abstract Microbial biomass C (Cmic), C mineralization rate, phospholipid fatty acid (PLFA) profiles and community level physiological profiles (CLPPs) using Biolog were determined from the humus and mineral soil layers in adjacent stands of Scots pine (Pinus sylvestris L.), Norway spruce [Picea abies (L.) Karst.] and silver birch (Betula pendula Roth) at two forest sites of different fertility. In addition, the Fourier-transformed infrared (FTIR) spectra were run on the samples for characterization of the organic matter. Cmic and C mineralization rate tended to be lowest under spruce and highest under birch, at the fertile site in all soil layers and at the less fertile site in the humus layer. There were also differences in microbial community structure in soils under different tree species. In the humus layer the PLFAs separated all tree species and in the mineral soil spruce was distinct from pine and birch. CLPPs did not distinguish microbial communities from the different tree species. The FTIR spectra did not separate the tree species, but clearly separated the two sites.

Microbial communities in boreal coniferous forest humus exposed to heavy metals and changes in soil pH : a summary of the use of phospholipid fatty acids, Biolog® and 3H-thymidine incorporation methods in field studies

Abstract This paper summarises our recent field studies on the microbial communities of boreal coniferous forest humus exposed to environmental stress, heavy metals and changes in humus pH. The microbial community was measured using the phospholipid fatty acid (PLFA) and Biolog® analyses, and the actual factor in the environment exerting the selective pressure on the bacterial community was estimated with the help of tolerance determinations using the 3 H-thymidine incorporation technique. The field experiments showed that the structure of the microbial community inhabiting the boreal coniferous forest humus was influenced by changes in humus pH and heavy metal concentrations at levels where no, or only small, effects on the microbial biomass or carbon mineralization rate were seen. The alterations in the humus PLFA patterns were related to the abundance of the major groups of microorganisms, bacteria and fungi. Changes in the relative proportions of Gram-negative and Gram-positive bacteria, including actinomycetes, were also shown. With the help of the 3 H-thymidine incorporation technique, it was demonstrated that forest humus bacterial communities exposed to heavy metals or alterations in humus pH were able to adapt to the environmental disturbance in question. When combining the results from the PLFA and 3 H-thymidine analyses, it was revealed that the increased tolerance of the humus bacterial community to heavy metals or to altered pH resulted at least partly from a change in microbial species composition. Coniferous forest humus seemed to contain a bacterial group, consisting mainly of Gram-positive bacteria, which were adapted more easily to the acidifying environment and a group of bacteria, mainly Gram-negative ones, which were more easily adapted to the humus with a higher pH. The Biolog® technique, which determines the community level physiological profile (CLPP) of the bacterial community was less sensitive and less suitable than the PLFA analysis to detect the characteristics of the forest humus microbial community. The 3 H-thymidine incorporation technique was the most sensitive of the techniques used in this study to detect the influence of environmental disturbances on the microbial community. In addition, a gradient of coniferous forest stands having naturally different humus pH because of the different site properties was studied to compare these natural microbial communities with the communities subjected to anthropogenic change in humus pH. In order to reveal the similarity of the humus samples with respect to their community structure, the PLFA patterns from all the field studies were subjected to multivariate cluster analysis. The structure of the forest humus microbial community was shown to be strongly influenced by the indigenous fertility of the coniferous forest site type, which was in turn related to humus nutritional status, pH, moisture, tree species and ground vegetation. Thus, a prerequisite for successful determination of the impacts of environmental stress on forest humus microbial community is the homogeneity of the forest site types between the experimental plots.

Fungal community dynamics in relation to substrate quality of decaying Norway spruce (Picea abies [L.] Karst.) logs in boreal forests

Decaying wood plays an important role in forest biodiversity, nutrient cycling and carbon balance. Community structure of wood-inhabiting fungi changes with mass loss of wood, but the relationship between substrate quality and decomposers is poorly understood. This limits the extent to which these ecosystem services can be effectively managed. We studied the fungal community and physico-chemical quality (stage of decay, dimensions, density, moisture, C : N ratio, lignin and water or ethanol extractives) of 543 Norway spruce logs in five unmanaged boreal forest sites of southern Finland. Fungi were identified using denaturing gradient gel electrophoresis and sequencing of DNA extracted directly from wood samples. Macroscopic fruiting bodies were also recorded. Results showed a fungal community succession with decreasing wood density and C : N ratio, and increasing moisture and lignin content. Fungal diversity peaked in the most decayed substrates. Ascomycetes typically colonized recently fallen wood. Brown-rot fungi preferred the intermediate decay stages. White-rot fungi represented approximately one-fifth of sequenced species in all decay phases excluding the final phase, where ectomycorrhizal (ECM) fungi became dominant. Lignin content of logs with white-rot fungi was low, and ECM fungi were associated with substrates containing abundant nitrogen. Macroscopic fruiting bodies were observed for only a small number of species detected with molecular techniques.

Rapid PCR-based method for the direct analysis of fungal communities in complex environmental samples

Abstract Fungal community analysis using 18S rDNA primer pairs and denaturing gradient gel electrophoresis of PCR products (Vainio, E.J., Hantula, J., 2000. Mycological Research 104, 927–936) was applied to field studies of the forest ecosystem. We report a DNA extraction method producing high quality DNA allowing successful PCR amplification from problematic samples without use of nested polymerase chain reaction (PCR) procedures. The analysis was found to be applicable for samples from environments of varying fungal diversities and high organic matter content: wood samples from fallen branches of trees, laboratory mini-ecosystems and forest humus samples. When the method was tested using replicate forest soil samples, it was shown to be highly reproducible.

Towards standardization of the description and publication of next‐generation sequencing datasets of fungal communities

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Belowground interspecific competition in mixed boreal forests: fine root and ectomycorrhiza characteristics along stand developmental stage and soil fertility gradients

We studied fine roots and ectomycorrhizas in relation to aboveground tree and stand characteristics in five mixed Betula pendula Roth, Picea abies (L.) H. Karst., and Pinus sylvestris L. stands in Southern Finland. The stands formed gradients of developmental stage (15-, 30-, and 50-year-old stands) in the stands of medium fertility, and of site fertility in the young stands (30-year-old fertile, medium fertile, and least fertile stands). The biomass of the external hyphae of ectomycorrhizas (ECM) was the highest, and the diversity of the fungal community the lowest, in the most fertile stand. The vertical distributions of fine roots of the three tree species were mostly overlapping, indicating high inter-specific belowground competition in the stands. We did not find any clear trends in the fine root biomass (FRB) or length across the stand developmental stages. The FRB of the conifers varied with site fertility, whereas in B. pendula it was almost constant. In contrast to the conifers, the specific root length (SRL) of B. pendula clearly increased from the most fertile to the least fertile stand. This indicates differences in the primary nutrient acquisition strategy between conifers and B. pendula.