Effects of mercury on ectomycorrhizal fungi Sharron Crane 1,2, John Dighton 1,3 and Tamar Barkay 1,2 1 Graduate Program in Ecology and Evolution, 2 Department of Biochemistry and Microbiology, 3 Rutgers Pinelands Field Station, New Lisbon, New Jersey Cook College, Rutgers University, New Brunswick, NJ for S. Crane: In wooded ecosystems with low buffering capacity soils, atmospherically deposited mercury is highly bioavailable. Dominant tree species of the New Jersey Pine Barrens establish mutualistic relationships with ectomycorrhizal fungi (ECM), which are important regulators of nutrient and metal uptake by woody plants. Because they are their host plant’s direct connection with the soil, ECM may play a critical role in ecosystem response to mercury. It is therefore important to understand ECM growth patterns in response to mercury and their ability to accumulate, transform, and translocate mercury. Introduction Objectives to determine the effect of mercury on the radial expansion of ECM colonies to determine the effect of mercury on mycelial branching patterns of ECM to investigate the ability of ECM to accumulate mercury Materials and Methods ECM cultures were maintained either in modified Melin- Norkrans (MMN) liquid medium or on 1.8% MMN agar. Mercury was added to experimental medium as HgCl 2. Radial growth data were collected by taking the average of eight radial measurements of ECM colonies grown on solid medium in Petri dishes. Hyphal branch angle data were collected from 400x magnified images of ECM colonies using ImageJ image analysis software. Figure 5: Accumulation of Hg by L. laccata cultures grown on solid medium Accumulation of mercury by L. laccata increases proportionately to an increase in mercury concentration. RMANOVA indicates that: a significant effect of both Hg concentration and time x Hg concentration on the growth of all isolates tested (p<0.0001). The effect of Hg concentration on the growth of C. geophilum lessens over time according to profile analysis of its growth curves (data not shown). A concentration of 10 μM Hg has no effect on the growth of P. bicolor and may stimulate growth in S. decipiens Total Hg addedAmount absorbed% absorbed 20 ug 0.88   ug 1.7   2.0 Table 3: Accumulation of Hg by L. laccata relative to total amount added Figure 3: Biomass of L. laccata grown on MMN agar at three different Hg concentrations slope= ± r 2 = p=0.0058

LITERATURE CITED Marx, D. H The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infections. I. Antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology 59:  Mercury is inhibitory to both radial expansion and biomass increase of L. laccata. L. laccata absorbs mercury during growth.  Low concentrations of mercury may stimulate growth in some ECM. This may be an attempt to escape toxic conditions caused by mercury.  S. decipiens absorbs little mercury upon initial exposure. After a marked delay, the amount of mercury absorbed per unit dry weight increases, indicating an active biological component to this activity. A release of mercury after. Hg content was measured using a Leeman Labs Hydra AA Mercury Analyzer. All graphs were generated using GraphPad Prism 4.0. Error bars represent standard error of three or more replicates except where otherwise noted. Statistical analyses were performed using PROC GLM in SAS version 8. Results Begin treatment on day 9 Figure 1: The effect of mercury on radial growth of Laccaria laccata Repeated measures analysis of variance (RMANOVA) indicates a significant effect of both Hg concentration (p=0.0001) and time x Hg concentration (p<0.0001). Table 1: The effect of Hg(II) on branch angles of L. laccata hyphae. Figure 2: 400x magnification of L. laccata hyphae. Arrows mark examples of branch angles. Hg concentrationAverage branch angle (  ) 1 n 0  M90.57  2.57 a  M76.13  2.47 b  M84.48  1.70 a 80 1 Means ± standard error. Superscripts indicate significant differences among means at the 95% confidence level. Conclusions Figure 6: The effect of mercury on radial growth of five ECM: x-axis=days, y-axis=colony radius (mm) n=2 S. decipiens accumulated little mercury during the first 3 hours of incubation. By 20 hours, mercury accumulation by S. decipiens had reached a peak before decreasing 50% between 20 and 24 hours of incubation. Rinsing with nitric acid had no effect on the amount of mercury associated with S. decipiens biomass. Significance Figure 7: A time series study of the accumulation of Hg by Suillus decipiens from a liquid matrix Understanding growth responses of ECM to mercury will facilitate:  the design of recovery models for forests subjected to mercury contamination  better decision-making in regards to inoculation regimes for reforestation of mercury-contaminated areas