Ecological Archives E094-184-A1

Marie Duhamel, Roel Pel, Astra Ooms, Heike Bücking, Jan Jansa, Jacintha Ellers, Nico M. van Straalen, Tjalf Wouda, Philippe Vandenkoornhuyse, E. Toby Kiers. 2013. Do fungivores trigger the transfer of protective metabolites from host plants to arbuscular mycorrhizal hyphae?. Ecology 94:2019–2029.

Appendix A. Methodological details of plant propagation/set up, nutrient regime, and hyphal chemical analysis.

Plant propagation

Before seedlings were planted in bags, the mesh bags were washed with demineralized water, and sterilized with 70% ethanol. Each mesh bag contained 300 g of a sterilized 9:1 mixture of quartz sand mixed with organic dune sand (see Verbruggen et al., 2012 for soil description), resulting in 0.8% of organic matter per bag (15% humidity). All pots were covered with black plastic foil to avoid evaporation.

Nutrient solution

The nutrient solution was injected below the quartz sand surface (over a depth of 1–8 cm) at 6 equidistant positions around the meshbag to achieve an even extraradical mycelium (ERM) development, and to minimize the growth of algae on the sand surface of the pots.

Hyphal Harvest

To measure the hyphal mass, blocks of sand were removed from the pot. The sand portion on the sieve was partly submerged in demineralised water and gently shaken to allow the sand to pass, leaving the intact ERM with quartz grains still attaching to the hyphal surface. By applying a gentle flow of demineralized water a portion of these attached grains could be removed.

To extract the hyphae from in-vitro medium, the fungal compartment of the medium was cut in 5–6 pieces and transferred in a 50 mL tube together with 25 mL 10mM sodium citrate buffer (pH 6, 37 °C). The solution was vortexed for 30 minutes at 100 rpm and sieved with nylon mesh. The washing steps were repeated until all medium was washed away. Roots were removed by hand using a stereomicroscope and tweezers. Hyphae were put in an eppendorf tube, freezed it in liquid nitrogen and stored in a -80 °C freezer until analysis.

Chemical analysis

Quantitative HPLC analysis was conducted using an Ultimate 3000 system (Dionex, USA) consisting of a dual pump module, autosampler, column compartment and photodiode array detector. A LiChroCART 125-4 LiChrospher 100RP-18 (5 µm) (Merck, Darmstadt, Germany) was used as analytical column. The mobile phase consisted of (A) phosphate buffer (2.5 mM KH2PO4; pH= 4.0 with phosphoric acid) and (B) acetonitrile. The gradient elution had the following profile: 0–0.2 min, 0% B; 0.2–10.5 min, 0–6% B; 10.5–24.5 min, 6–50% B; 24.5–26.5 min, 50–0% B; 26.5–29 min, 0% B. The column flow rate was 1 mL/min at 20 ºC, and the injection volume was 10 µl. The absorbance of catalpol and aucubin were measured at 204 nm, and verbascoside was measured at 215 nm. SM peaks in the chromatograms were identified by comparison of retention time of authentic iridoid and verbascoside standards (Carl Roth GmbH, Germany): catalpol (purity 99%) at 4.8 min, aucubin (purity 98,5%) at 7.6 min and verbascoside (purity 98%) at 17.9 min. The peak area was integrated using Chromeleon Software Release 6.60 (Dionex Corp.) with external standards. Standard calibration curves were plotted using various concentrations of catalpol, aucubin and verbascoside (range: 10–250 µg/mL for each compound). The detection limit of catalpol and aucubin was 250 ng/mL and 80 ng/mL for verbascoside.

Statistical analysis

The functions used in R to perform the statistical analyses are the following:

- normality and homogeneity of variances: ks.test(); levene.test()

- analyses of plant data, hyphal biomass and secondary metabolite concentrations: glm(); anova(lm()); TukeyHSD()

- molecular analyses and data on secondary metabolites in hyphae: t.test(); welch.test()

- power analyses: power.anova.test(); power.t.test()

For GLM analysis, the best possible GLM was selected after calculation of Akaike Information Criterion. In this study, the most appropriate family wise errors were Inverse Gaussian or Gaussian depending on the data. Modalities were included in the GLM one at a time. From the GLM, data were analysed by a one-sample t test.


Fig. A1. Experimental set-up.


Literature cited

Verbruggen, E., C. El Mouden, J. Jansa, G. Akkermans, H. Bücking, S. A. West, and E. T. Kiers. 2012. Spatial structure and inter-specific cooperation: theory and an empirical test using the mycorrhizal mutualism. American Naturalist 179:E133–E146.

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