Appendix B. Additional methodological details for experiments 1 and 2.
We grew plant communities of varying diversity in large outdoor mesocosms. All species were herbaceous biennials or perennials, except Erigeron annus, which was an herbaceous annual (see Appendix D). To each mesocosm, we added either: (1) 500 mL of sterile distilled water for the sterile control; (2) 500 mL of sterile distilled water containing 5000 field-extracted arbuscular mycorrhizal fungal (AMF) spores (mixed species); or (3) 500 mL of sterile distilled water containing 1 × 109 field-collected microbial cells (including pathogens, parasites, and saprobes) that passed through a 20 µm filter (AMF do not typically pass through this filter size, which we confirmed by assessing plants for mycorrhizal fungal colonization at the end of the experiment; Table 1). The fourth experimental treatment was 90 kg of a 1:1 mixture of silica sand and untreated field soil, which contained soil microbes and fauna. Each mesocosm was then showered with 1500 seeds from randomly selected grassland plant species comprising one of the four levels of species richness. Each treatment combination was replicated five times, for a total of 80 experimental units.
The plant communities were a mix of grasses, legumes, and forbes (Reich et al. 2001), and were similar in composition to the plant community found at the University of Guelph and used in experiment 1. We selected six perennial herbaceous target species, including two C-4 grasses: Andropogon gerardii and Schizachyrium scoparium; two forbs: Anemone cylindrica and Solidago rigida; and two legumes: Lespedeza capitata and Lupinus perennis. We were unable to use the same species in both experiments, but using a different group of species and finding consistent results suggests that our findings are a robust test of a general mechanism that should extend to other common species.
In each plot, we extracted soil at three locations using a cylindrical soil core (5.08 cm diameter wide by 60 cm deep) and then gently homogenized the three samples by hand. Seeds were germinated by incubating them at 4°C for four weeks in paper towels moistened with distilled water, after which we planted germinated seeds of the target species in soils of conspecifics and heterospecifics, and across the diversity gradient (1, 4, or 16 species). Because diversity levels also influence soil nitrogen (Tilman et al. 1997), we also used ongoing N mineralization measurements to test whether differences in growth resulted from other factors besides the soil biota influenced by diversity. Soil net N mineralization rates were measured in situ each year in each plot for the five years preceding the soil extraction using a semi-open core one-month incubation beginning in late June (Reich et al. 2001). The initial soil sampling prior to the incubation was used to measure in situ soil solution N concentration.
For all treatments, we grew each plant individually in a cone-shaped pot (3.8 cm diameter × 21 cm depth; 164 mL volume) in a temperature-controlled greenhouse under summer-like conditions with respect to day-length (14 hours per day) and temperature (~25 ºC). After three months, we harvested the plants and visually quantified the incidence of disease (DI; the number of lesions per root) and the disease severity index (DSI; the proportional area of root disease). Immediately after quantifying disease, we oven-dried the roots, shoots, and leaves of each plant at 60°C for at least 48 hours and then weighed them to determine total plant productivity. Although visual disease estimates may underestimate disease effects on plant growth, it provides a consistent estimate of fungal damage among treatments (Mitchell 2003), and thus enabled us to attribute our results to pathogens and parasites, rather than saprobes or AMF.
Mitchell, C. E. 2003. Trophic control of grassland production and biomass by pathogens. Ecological Letters 6:147155.
Reich, P. B., J. M. H. Knops, D. Tilman, J. Craine, D. Ellsworth, M. Tjoelker, T. Lee, S. Naeem, D. Wedin, D. Bahauddin, G. Hendrey, S. Jose, K. Wrage, J. Goth, and W. Bengston. 2001. Plant diversity enhances ecosystem responses to elevated CO2 and nitrogen deposition. Nature 410:809812.
Tilman, D., J. M. H. Knops, D. Wedin, P. B. Reich, M. Ritchie, and E. Siemann. 1997 The influence of functional diversity and composition on ecosystem processes. Science 277:13001302.