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- Materials and methods
There are a number of studies where plant growth response to natural soil inoculum has been compared to that on sterile soil. However, we lack experimental evidence highlighting differential plant species-related growth patterns in natural soils that are likely to support dissimilar AMF communities, as the bulk of experimental studies have involved inoculations with a limited number of easily cultured AM fungi which are often poorly represented in the natural root-colonizing AMF communities (Read 2002). The only exceptions known to us are pot experiments showing differential plant growth in the presence of natural soil or root inocula of different origin (Johnson 1993; Kiers et al. 2000; Corkidi et al. 2002; Frank et al. 2003). However, the relative contribution of native AMF communities to the observed plant growth responses cannot be assessed in these studies because the AM fungi colonizing plant roots were not identified.
In order to overcome the questionable ecological relevance of experiments using a few easily cultured AMF (Read 2002), experiments coupling naturally co-occurring plant species and AMF communities are needed. At the same time, to understand the effects of native AMF communities, it is still not enough to use soil inocula either completely ‘blind’ or containing known spore communities, as long as we lack information on the actual composition of AMF communities that colonize and symbiotically interact with plant roots after exposure to soil inocula. To our knowledge, the experiment reported here is one of the first to couple these two aspects: a contrasted natural soil inoculation experiment to determine species-specific plant performance that also includes high-resolution identification of root-colonizing AMF communities of the plants investigated.
Our goal was to determine whether seedling establishment and growth of native plant species differs on natural soil inoculum originating from different sites. If a differential response was identified, a valid assumption worth investigation is that distribution of a plant species may depend, in addition to other factors, on the composition of local root symbiotic AMF communities. As a relevant model, we chose a pair of plant species with similar morphology, ecology and phylogeny: Pulsatilla patens and Pulsatilla pratensis[both (L.) Mill. Ranunculaceae]. The former is now rare with a limited distribution, while the latter is common in Estonia (Pilt & Kukk 2002). One of the major reasons for differences in local and regional abundance of plants might be related to their differential responses to the presence of (particular) soil microbes (Klironomos 2002, 2003). Seedlings of both Pulsatilla species investigated here are seldom encountered in established vegetation, as establishment takes place almost exclusively in disturbed gaps or areas of moderate disturbance (Uotila 1996; Pilt & Kukk 2002). Therefore it is probable that AMF spores are the main infection source for Pulsatilla spp. seedlings, while root colonization via functioning hyphal networks is less significant due to the lack of established vegetation in disturbed sites. Such an infection by spores may be relatively more beneficial for seedlings than inoculation via intact mycelial network, as in the latter case competitive interactions may overwhelm benefits (Kytöviita, Vestberg & Tuomi 2003).
We aimed to investigate whether different soil AM fungal communities contribute to differential establishment and growth of two Pulsatilla species in conditions simulating gapped environments. In a seedling-establishment experiment of factorial design, we used soil inoculum from two sites with distinct vegetation characteristics: a boreal forest stand, and dry grassland. Both rare and common species coexist at the forest site, but only the common P. pratensis is found in the grassland site. Unlike many earlier studies, we possess background data on AMF present in roots of the experimental Pulsatilla plants from the phylogenetic analysis of amplified and cloned small-subunit ribosomal RNA gene (SSU rDNA) sequences (Öpik et al. 2003).
Materials and methods
- Top of page
- Materials and methods
Soil inoculum was collected from two divergent sites in Estonia: grassland and forest. The first is a 0·5 ha remnant patch of dry perennial grassland at Pangodi in central Estonia (58°10′ N, 26°34′ E), used as pasture until agricultural activity ceased 15 years ago. There are some single, mature Scots Pines (Pinus sylvestris L.) in the area. The plant community consists of 35 species of perennial grasses and forbs, 33 of which are potentially arbuscular mycorrhizal. Helictotrichon pratense (L.) Besser, Galium verum L. and Festuca rubra L. represent the predominant species. The second site is a sparse, mature, boreal Scots Pine-dominated forest at Soomaa National Park, south-western Estonia (58°24′ N, 25°19′ E). The ectomycorrhizal Scots Pine covers 40–50% of the area; in the understorey ericoid mycorrhizal Calluna vulgaris L. predominates in the shrub layer and Hylocomium splendens (Hedw.) B., S. and G. and Pleurozium schreberi (Brid.) Mitt. in the moss layer. There are five AM herbaceous plant species in the community, with a cover of less than 5%. Among these, Festuca ovina L. is the most abundant. The distance between the sites is ≈90 km. Soils in both sites are dry arenosols with weakly differentiated horizons and rather similar properties (Table 1). Topsoil samples (3–10 cm) were collected from 10 randomly chosen sites in both target ecosystems in the second half of August 1999 and stored in the dark at 10 °C. Samples from each location were sieved to remove roots and then pooled for the experiment. Soil pH, P, N and C contents were determined according to Moore & Chapman (1986).
Table 1. Characteristics of natural soils (data from Pilt & Kukk 2002 for 30 cm topsoil layer), and of soil mixtures used in the current experiment: forest inoculum – forest soil mixed with sterilized grassland soil; grassland inoculum – grassland soil mixed with sterilized forest soil; sterile soil – both soils sterilized and mixed
|Soil||pH (H2O)||Total N (%)||Mobile P (mg per 100 g soil)||Total C (%)|
|Soomaa forest||8·6||0·109|| 9·8||ND|
|1 : 1 Soil mixture used in the current experiment (n = 5, mean ± SE)|
|Forest inoculum||6·9 (± 0·01)||0·08 (± 0·001)|| 9·0 (± 0·07)||2·3 (± 0·12)|
|Grassland inoculum||6·8 (± 0·01)||0·07 (± 0·001)|| 8·9 (± 0·08)||2·21 (± 0·08)|
|Sterile soil||6·8 (± 0·01)||0·07 (± 0·000)|| 9·1 (± 0·04)||2·17 (± 0·07)|
Pulsatilla patens and P. pratensis are perennial forbs that grow in dry grasslands and boreal forests. Pulsatilla patens is now restricted to 27 isolated populations in Estonia (Pilt & Kukk 2002). In contrast, P. pratensis is relatively abundant. Mature seeds of both species were collected in summer 1999 from three local populations. Seeds were pooled, visually examined and selected to avoid those damaged by herbivores or pathogenic fungi.
Plant seeds were sown on 8 February 2000 onto a 1 : 1 mixture of the two natural soils, with one of the soils being autoclaved (40 min at 121 °C). We did not detect any significant differences in soil characteristics between soil mixtures where either the soil from grassland, soil from forest, or both soils were autoclaved (Table 1). A mixture of both autoclaved soils served as a non-mycorrhizal control treatment. Hereafter, the soil inoculum treatments are referred to as grassland inoculum (mixed with sterilized forest soil); forest inoculum (mixed with sterilized grassland soil); and sterile soil (both soils sterilized). All pots received 2 ml filtered washing of respective mixed soil inoculum to correct for possible differences in soil bacterial and fungal communities (Koide & Li 1989).
The experiment was conducted in the greenhouse of the Viikki Biocentre at Helsinki University. Seeds were sown at a uniform density (1·2 seeds cm−2) into pots (9 × 12 cm, depth × diameter). Pots were carefully watered with tap water as required. Each treatment was replicated 10 times. Plants were grown in full daylight (daylength 16 h) for 14 weeks. At both harvests the plants were still in at the juvenile stage, and there were no major differences in plant phenology between the two harvests.
Plant establishment rate was recorded daily until no new germlings were detected, then plants were thinned to three individuals per pot (at maximum equidistant positions to avoid competition). Two seedlings from each pot were harvested carefully 5 weeks later (10 for biomass analysis; five for root staining; five for molecular analysis) and the third was allowed to grow on further for 9 weeks. The 14-week-old plants were harvested to prevent plants from becoming root-bound in pots. Shoots and roots were separated, dried at 85 °C for 24 h, and weighed. A correction in root biomass values at second harvest was made using differences between fresh and dry weight of root sample (for AMF determination) per treatment. Phosphorus and nitrogen concentrations in 14-week-old plants were determined following Kjeldahl digestions of dried plant tissues. Due to the limited plant material available, the P concentration of plants in sterile soil treatment could not be determined.
The percentage AMF colonization (root length colonized, %) was estimated on the basis of the full root system of five seedlings (first harvest), or 1–2 g (FW) randomly selected root segments (second harvest). Root samples were stained (Koske & Gemma 1989) and the percentage of colonization was determined (Rajapakse & Miller 1992).
The AMF community composition in the roots of four to five seedlings from each treatment was determined from randomly selected root samples (total length 5 cm), or the entire root system if under this length, as described for a related study by Öpik et al. (2003). Briefly, a ≈550 bp fragment located in the middle section of the SSU gene was amplified with the AMF-specific primer pair NS31/AM1 (Simon, Lalonde & Bruns 1992; Helgason et al. 1998). The PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and individual bands excised, cloned and sequenced. The fungal sequence groups identified in the seedlings roots of this study comprise part of a larger AMF community survey of native plants and trap seedlings of P. patens and P. pratensis in various Estonian locations (Öpik et al. 2003).
For analysis of the plant establishment rate, a repeated-measures anova was conducted, with plant species and soil inoculum as fixed factors and time as the repeated-measures factor. Biomass, root AMF colonization rate, and plant P and N concentrations (presented as percentage of plant dry biomass) data were subjected to anova. Biomass and percentage AMF colonization data were, respectively, log- and arcsine-transformed prior to statistical analysis. All analyses were conducted with the Windows version of statistica (StatSoft, Inc., 2000, Tulsa, OK, USA). Similarities of AM fungal communities, calculated on the basis of the fungal sequence groups’ presence/absence in a root system, were analysed by multivariate cluster analysis (Ward's linkage method and Euclidean distance measure) implemented in pc-ord ver. 4·01 (McCune & Mefford 1999).