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).