Methods
The work described here was conducted at the Cedar Creek Ecosystem Science Reserve (CDR), East Bethel, Minnesota, part of the U.S. Long Term Ecological Research (LTER) Network (Latitude 45.4o N, 93.2 o W). CDR is situated on a sandy outwash plain formed during Wisconsin glaciation 11,000 ybp. These infertile have very low nitrogen and support low productivity grasslands (Fay et al. 2015). Mean annual precipitation and mean annual temperature at the site are 750 mm and 6°C respectively (Borer et al. 2015). The heterotroph manipulation experiment was conducted within an existing plant diversity experiment (Tilman et al. 2001). Below, we describe the experiment in detail.
The heterotroph manipulation experiment was nested within a long-term grassland plant diversity experiment initiated in 1994 (Tilman et al. 2001). Heterotroph manipulation treatments were established in 2008 in 33 of the 9 m x 9 m experimental plots, with planted richness of 1 (n=15), 4 (n= 9) and 16 species (n=9). In each of the 33 experimental plots, 5 treatments (control, insecticide, foliar fungicide, soil fungicide, and all pesticides combined) were randomly assigned to subplots (1 per treatment per plot) of 1.5 m x 2 m for a total of 165 experimental subplots (33 diversity plots x 5 treatments). Subplots were separated by 0.5 m wide buffer strips to prevent drift. The experiment was fenced to exclude deer.
All pesticides were applied regularly throughout the growing season from mid-April to end of August each year. The treatments were maintained as follows: foliar fungicide as biweekly application of Quilt (Syngenta Crop Protection, Inc., Greensboro, NC), a combination of Azoxystrobin (7.5%) and Propiconazole (12.5%); soil fungicide as monthly applications of Ridomil Gold SL (Syngenta Crop Protection, Inc., Greensboro, NC), a soil drench fungicide containing Mefenoxam (45.3%); and insecticide as biweekly applications of Marathon II (OHP, Inc., Mainland, PA; 21.4% Imidacloprid). Once or twice each season, Malathion was applied instead of Marathon II to reduce the possibility of insecticide adaptation by the local insect populations. Previous work from this experiment has shown that these heterotroph removal treatments lower foliar damage by arthropods and by foliar fungi by approximately 50-67% (Borer et al. 2015). Results from a greenhouse experiment showed that none of the pesticides had any direct effects on plant growth in the absence of heterotrophs (Seabloom et al. 2017).