Appendix B. Influence of enemy exclusion on arbuscular mycorrhizal infection.
We were concerned about the potential for fungicides to disrupt arbuscular mycorrhizae (AM), which aid plants in nutrient acquisition (Smith and Read 1997) and have been shown to facilitate invasions by other grasses (Klironomos 2002). We tested for the effects of our pesticide applications on AM mycorrhizae in May 2007, after both pesticide mixtures had been used. To determine whether or not the pesticide treatments designed to remove enemies also influenced non-target arbuscular mycorrhizal (AM) infection, we measured incidence of AM infection in pesticide treated and control plots at the four invaded sites (Bald Hill, Jasper, Pisgah, and Sweet Home).Methods
We took two samples from each of three pesticide treated plots and three control plots at each of four sites during the first week of May, 2007, after both pesticide mixtures had been applied. The plants in each plot were chosen on opposite sides of the plots (to be sure we sampled the greatest within plot distance), and were all > 2 cm in diameter (to be sure we had enough roots), but were otherwise sampled randomly. Samples were collected by placing a 5.7 cm diameter by 8.5 cm deep the tulip bulber over a Brachypodium sylvaticum plant and pressing down to fill the bulber with roots and soil. Root segments that were attached to the B. sylvaticum stem were washed with water and fixed in 50% ethanol until staining. For staining, the roots were cleared overnight in 10% KOH, bleached in alkaline H2O2, acidified in 1% HCl, and stained in acidic glycerol containing 0.05% trypan blue (Koske and Gemma 1989, Bauer et al. 2003). Stained roots were cut into 1 cm segments and mycorrhizal infection rate was determined using the gridline intersect method (Giovannetti and Mosse 1980) under a dissecting microscope (10–100×). For each plant, 100 grid intersections were scored for the presence or absence of arbuscules, vesicles, and/or hyphae of mycorrhizal fungi.
We used ANOVA to test for differences among sites, treatments, and their interaction. The analysis was done on means of the two samples in each plot. Site and the treatment by site interaction were treated as random effects and treatment as a fixed effect. To improve normality of the residuals, the percentage data was arcsine transformed. Differences among sites were assessed with Tukey’s tests.
There was no significant effect of treatment on AM infection (F2.941,16, P = 0.4079), nor were there differences in the effects of the treatment across sites (no treatment by site interaction, F1.023,16, P = 0.1850). There were, however, significant differences among sites (F10.533,16, P = 0.0422) in AM infection, with infection being significantly higher at Mt. Pisgah than at the Jasper site (Fig. B1).
|FIG. B1. Arbuscular mycorrhizal infection rate of Brachypodium sylvaticum in pesticide treated (×) vs. water sprayed control plots (solid circle) at four sites in the invaded range. Site means ± SE.|
Bauer, C. R., C. H. Kellog, S. D. Bridgham, and G. S. Lamberti. 2003. Mycorrhizal colonization across hydrologic gradients in restored and reference freshwater wetlands. Wetlands 19:775–788.
Giovannetti, M., and B. Mosse. 1980. An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytologist 84:498–500.
Klironomos, J. N. 2002. Feedback with soil biota contributes to plant rarity and invasiveness in communities. Nature 417:67–70.
Koske, R. E., and J. N. Gemma. 1989. A modified procedure for staining roots to detect VA mycorrhizas. Mycorrhizal Research 92:486–488.
Smith, S. E., and D. J. Read. 1997. Mycorrhizal Symbiosis. Academic Press, San Diego, California, USA.