Ecological Archives E093-139-A1

Nicholas A. Barber, Lynn S. Adler, Nina Theis, Ruth V. Hazzard, and E. Toby Kiers. 2012. Herbivory reduces plant interactions with above- and belowground antagonists and mutualists. Ecology 93:1560–1570.

Appendix A. Methodological details of plant propagation, field site, cucurbitacin analyses, and mycorrhizal staining.

Plant propagation and site

On 20 May 2009, we planted Cucumis sativus seeds (Marketmore 76, Johnny's Selected Seeds, Winslow, Maine, USA) in organic soil mix (Fort Lite Soil mix, Vermont Compost Company, Montpelier, Vermont) in 72-cell flats. Flats were watered, and seeds germinated in a greenhouse with natural light. Plant were transplanted at the one-leaf stage, and immediately prior to transplanting were watered with organic fish emulsion fertilizer (Neptune's Harvest, Gloucester, Massachusetts) diluted in water to approximately 8 mL/L.

The experimental site at the University of Massachusetts Center for Agriculture (South Deerfield, Massachusetts, USA) was a 0.4 ha agricultural field with fine sandy loam soil that had been under organic management practices for 4 years prior to this study. In previous years, cover crops incorporated in the spring and fall included red clover (Trifolium pretense), sorghum sudangrass (Sorghum bicolor), rye (Secale cereal), and oats (Avena sativa). Seedlings were arranged in 31 rows of 8 plants each and spaced 3 m apart, and extra plants were planted along plot margins for use as pollen donors. Throughout the study weeds were controlled using a cultivator, by hoeing, and by hand.

Cucurbitacin extraction and quantification

Leaves collected on 23–24 July were dried in an oven at 45 °C, ground in a Wiley mill, and then stored in the same oven. Roots from plants harvested for larval counts between 22 and 31 July were dried, ground, and stored in the same manner as leaves. Samples were extracted in methanol; 3 mL methanol were added to 0.3 g leaf powder, vortexed for 30 s, and sonicated for 10 min. These samples were centrifuged and the supernatant was poured off, measured, and diluted 1:1 with distilled water. The diluted supernatant was filtered (13 mm diameter, 0.45 μm nylon syringe filter) and analyzed on a Perkin-Elmer (Shelton, Connecticut, USA) series 200 quaternary pump HPLC system and diode array detector at 254 nm using an Altima C18 column (250 mm × 4.6 mm, Grace Davison, Deerfield, IL, USA). The eluent was 55% acetonitrile/45% H2O at 1 ml/min. Because we did not have access to a cucurbitacin C standard, we used peak area/mg as a proxy for cucurbitacin C content.

Mycorrhizal staining

Root fragments were cleared in 2.5% potassium hydroxide. Cleared roots were rinsed and stained in trypan blue in a 90 °C water bath for 5 min. Stained roots were rinsed and stored in destaining solution (2:1:1, lactic acid:glycerol:water) until mounting on microscope slides.

When quantifying colonization, we only counted hyphae in which there was a visible connection to the root to exclude other types of non-mycorrhizal hyphae.

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