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Methods for Experiment 244 -

Above Ground Biomass Sampling

Biomass was collected from a clip strip that is 1m x 10cm. A handheld clipper was used to cut as close to the ground as possible and collect the biomass. Clip strips were sorted to species, dried and weighed. This is done for every plot.

Below Ground Biomass Sampling

A 2 inch diameter root core was used to collect three 30cm deep root cores from each clipped strip after the above ground harvest. The soil is washed away from the roots. The roots are collected, dried and weighed. This is done for every plot.

Ceptometer Sampling

Ceptometer readings are taken once a month in the Percent Cover areas of each plot throughout the growing season. One measurement is taken above canopy and 3 measurements are taken below the canopy. The machine averages the 3 below readings and gives an estimate of LAI.

Cropscan Radiometry Sampling

Cropscan Radiometry readings are taken once a week throughout the growing season above a permanently-established point within each plot.

Percent Cover Sampling

The percent cover area is in the middle of the plot and is 1m x 0.5m. The percent of the ground that is covered by each individual species that is rooted within the percent cover area for every plot is measured. The total for the vegetation and non-vegetation cannot be more than 100%.

Pesticide Applications

There are 5 treatments: 1. Soil drench fungicide 2. Foliar fungicide 3. Insecticide 4. All pesticides 5. Control (no pesticides) Pesticides are applied once every 2 weeks to the plot throughout the growing season, weather permitting. They are applied by a licensed applicator (Jon Anderson). The foliar fungicide treatment is composed of Quilt (Syngenta Crop Protection, Inc., Greensboro, NC, USA), a combination of Azoxystrobin (7.5%) and Propiconazole (12.5%), applied every two weeks. The soil fungicide treatment is composed of monthly applications of Ridomil Gold SL (Syngenta Crop Protection, Inc., Greensboro, NC, USA), a soil drench fungicide containing Mefenoxam (45.3%). The insecticide treatment is composed of Marathon II (OHP, Inc., Mainland, PA, USA; 21.4% Imidacloprid) applied ever two weeks. One to two times each growing season, Malathion is applied instead of Marathon II to reduce the possibility for insecticide adaptation by the local insect populations.

Photographs

Photographs are taken from the east edge of every plot. A tripod is used so the picture is from the same location every time.

Plot Description

Plots are 1.5m x 2m and nested in 3 E120 plots and 38 BAC plots (n = 41 total). The plots were established in 2007. There are 5 treatments nested in each larger plot, individual plots run along the east edge of the whole plot. Treatments are numbered from south to north and they are also colored to indicate the treatment.

adte244 - Leaf carbon, nitrogen and phosphorus

Leaf sampling and analysis

Percent carbon by dry weight. Leaves and petioles from each species were dried and homogenized by plot. A subsample of the dried and homogenized tissue of each plant species was assayed for %C and %N using dry combustion GC analysis (Ecosystems Analysis Laboratory at University of Nebraska, using a COSTECH ESC 4010 Element Analyzer). Percent nitrogen by dry weight. Leaves and petioles from each species were dried and homogenized by plot. A subsample of the dried and homogenized tissue of each plant species was assayed for %C and %N using dry combustion GC analysis (Ecosystems Analysis Laboratory at University of Nebraska, using a COSTECH ESC 4010 Element Analyzer). Percent Phosphorus - Leaves and petioles from each species were dried and homogenized by plot. A subsample of the dried and homogenized tissue of each plant species from each plot was ashed (following Miller 1998) and assayed for %P via sulfuric acid digestion followed by spectrophotometric analysis (see Clesceri et al. 1998 for methods details). REFS Clesceri, L. S., A. E. Greenberg, and A. D. Eaton. 1998. Standard methods for the examination of water and wastewater. American Public Health Association., New York. Miller, R. O. 1998. High-temperature oxidation: dry ashing. Handbook of Reference Methods for Plan Analysis. CRC Press, Boca Raton, FL.

aete244 - Multispectral Radiometry percent reflectance

Instrumentation

Multispectral Radiometer Cropscanner MSR5, a 5-band LANDSAT Thematic Mapper compatible model (460-1750nm). CROPSCAN, Inc. Rochester, MN

Radiometry Sampling Protocol

The Multispectral Radiometer Cropscanner is a machine used to measure the reflectance of 5 different wavelengths. Before Taking Readings: Be sure Date, Time, GMT (Greenwich Mean Time) Difference and Location (latitude and longitude) are set correctly for sampling Location. Attach sensors and cables and a do real-time VIEW mode readings test in the sunlight to verify the millivolt readings correspond to changes in irradiance intensity. To see if the readings will change, place something over the top of the sensor head. The IRR values should decrease. When you place the sensor head back in full sun, the readings will increase. Checklist in the field: Attach the sensor head to the pole and assemble the equipment. Before taking the first plot scan, change the position of the top of the radiometer relative to the sun to see if the IRR (I) reading changes accordingly. Check that the sensor head is level. Take the readings facing south from the north part of the plot. This is to keep shadows from interfering with the readings. Take 1 reading per plot over the percent cover area. When Taking Readings: The best readings are obtained under clear skies or lightly cloudy days with IRR readings above 400 Watts per square meter. If there are cumulus clouds (large puffy), let them pass out of the way of the sun before taking readings. Wear grays or darker colored clothing to reduce the amount of reflectance from your clothes onto the sample area. Try to position the radiometer front (serial number side) toward the sun when taking readings. This eliminates your shadow from the sampling area and also yields the best cosine response correction of the top or up-facing sensors. After positioning and leveling the radiometer and initiating a scan, you may move to the next plot as soon as the double asterisks appear on the screen or you hear the double beep from the DLC.

agae244 - Greenhouse test of pesticide effects on plant growth

Greenhouse methods

It is possible that the pesticides used in E244 cause direct effects on plant growth even in the absence of the target fungi and arthropods. Therefore, we performed a greenhouse experiment to test for the effects of the insecticide, foliar fungicide and soil fungicide on plant growth. A soil mix composed of 80 percent soil sourced from the field site (Cedar Creek Ecosystem Science Reserve) and 20 percent vermiculite was used for potting. Soil was coarsely sieved and the soil mix was homogenized and autoclaved. The experiment was conducted using two C4 grasses (Andropogon gerardii and Schizichyrium scoparium) and two legumes (Lespedeza capitata and Lupinus perennis). These four species are common at Cedar Creek and represent a wide range of the functional trait variability present in the pool of species in the diversity experiment (Craine et al. 2002; Borer et al. 2015). A total of 60 pots were seeded with one of the four species (total 240 pots) with seeds obtained from Prairie Restoration Inc. Ten pots of each species were randomly assigned to each of six treatments (Foliar Fungicide, Insecticide, Soil Drench fungicide, All Treatments, Foliar-Water Control and Soil-Water Control). All pots were watered every other day and treatments were applied once every 2 weeks. Leaf-water and soil-water controls were treated with water at the time of pesticide application. Pesticide application rates were calculated based on the surface area of the pots to match the field experiment, and the pesticides used in the experiment were the same as those used in the field experiment. Plants were allowed to grow for a total of 15 weeks. At the end of this period, the above and belowground parts of each individual were harvested, cleaned, oven-dried (at 70 degrees C for 48 hours) and weighed to the nearest 0.01 gram. Papers cited in these methods: Craine, J.M., Tilman, D., Wedin, D., Reich, P., Tjoelker, M. & Knops, J. (2002). Functional traits, productivity and effects on nitrogen cycling of 33 grassland species. Funct. Ecol., 16, 563?574. Borer, E.T., Lind, E.M., Ogdahl, E.J., Seabloom, E.W., Tilman, D., Montgomery, R.A. et al. (2015). Food-web composition and plant diversity control foliar nutrient content and stoichiometry. J. Ecol., 103, 1432?1441. Results from these data are published in: Seabloom, E. W., L. Kinkel, E. T. Borer, Y. Hautier, R. A. Montgomery, D. Tilman and B. Casper (2017). "Food webs obscure the strength of plant diversity effects on primary productivity." Ecology Letters 20(4) doi: 10.1111/ele.12754