Differential analysis of active mychorrizae present among living and deceased Piñon (Piñus edulis) and Juniper (Juniperus monosperma) trees in the Los Piños Mountains New Mexico Differential analysis of active mychorrizae present among living and deceased Piñon (Piñus edulis) and Juniper (Juniperus monosperma) trees in the Los Piños Mountains New Mexico Kimberly F. Elsenbroek

Climactic Influences It is predicted that the region will be greatly affected by climate change and see an increase of temperature by 4 o C (Diffenbaugh et al. 2008; IPPC 2007) and become drier overall (Seager et al. 2007). To predict how future environmental conditions will affect local biomes, we must understand how they function now.

Eddy Covariance Tower United States Forest Service (USFS). Monitors several different biomes Carbon/water exchange with the atmosphere, productivity, evapotranspiration and ecosystem respiration. Little is known about below-ground activities at these sites.

History of the sites Tower Establishment Experimental 2009 Control 2007 Towers are ~5km apart and experience similar environmental conditions Piñon/Juniper(PJ) woodlands are a wide spread and significant biome of the southwest and cover ~30 million Hectares. ¼ of New Mexico is covered in PJ woodlands. They are refuge for wildlife and provided wood-fuel for humans in the past. 4

Site Description biology.unm.edu/litvak/Pinyon%20Juni per/Pinyon%20Juniper.html

Simulating Future Conditions The experimental site has simulated bark beetle attacks on piñon trees during high stress events by girdling the trees. o Bark beetles are a growing epidemic in the southwest. o Fungus o Stress o Continuing impacts of climate change o Future conditions

7 Timeline of tree response Sept 29, 2009 Nov 12, 2009 Apr 9, 2010 July 30, 2010

Fungi Arbuscular mycorrhizal (AMF) and ectomycorrhizal fungi (ECM) play an important role in belowground and plant community functions and form symbiotic relationships. o Increase stomatal conductance o Alter plants response to drought o Alter hormone regulation (Auge 2004)

Fungi interactions/types-of-mycorrhizae.html AMF are associated with Juniper (Root associated fungi [RAF]) ECM are associated with Piñon

Project Description During this project we compared the microbial activity between: Dead piñon Living piñon Living juniper

Research Question/Hypothesis R 1 : Which tree(s) will have the highest rates of microbial activity? H 1 : Microbial activity will be higher under the living piñon trees when compared to the dead piñon trees.

Methods Six transects were established at each site (control and experimental) 5 comparisons at the experimental site for each transect. 3 comparisons at the control site for each transect. Areas selected for transect establishment contain trees to satisfy the Piñon and Juniper comparisons.

13 TOWER Plot 1Plot 2 Plot 3 Plot 4 Plot 5 Plot 6 EXPERIMENTAL SITE

14 LP LJ LP DP LJ DP LJ S S S S S S S S S S S S S S S S= Sample (3cores/sample) DP=Dead piñon LP=Live piñon LJ=Live juniper *This graphic represents 1 plot There are 6 plots per sampling site Experimental Site Sampling PLOT ONE

15 TOWER Plot 1 Plot 2 Plot 3 Plot 4 Plot 5 Plot 6 CONTROL SITE

16 LP LJ LP LJ S S S S S S S S S S= Sample (3cores/sample) LP=Live piñon LJ=Live juniper *This graphic represents 1 plot There are 6 plots per sampling site Control Site Sampling PLOT ONE

Sample Collection Soil samples were taken using a 2 X 10 cm core at a rate of 3 cores from beneath each tree and between the two trees being compared. 3 samples per comparison. ~ 15 samples from each plot. Cores taken underneath and between each tree were combined and represent the average mycorrhizal community.

Sample Processing Soil samples were sieved using a 4 and 2-mm mesh to collect living coarse roots. ~8 mL KOH and MeOH solution was added to a sub-sample (~5mL) of soil in a falcon tube. –Sample was homogenized by shaking.

Water holding capacity and organic matter content Water holding capacity (WHC)was determined gravimetrically. o A subsample of ~ 1g was placed in aluminum pans and weighed to calculate: weight of soil + water content. o Samples were then dried at 60 o C for >/= 1hr and weighed to determine dry weight. This tells us the WHC of each sample Samples were combusted at 500 o C to burn off organic matter (OM) and then weighed again to determine OM content (Stursova et. al 2006). o OM was burned off using a muffle oven.

Extracellular Enzyme Assays Extracellular enzyme assays (EEA) were measured using 1.0 g soil/bicarbonate buffer slurry placed in microplates with substrates.

Ergosterol Extractions Ergosterol occurs in yeast or fungal cell membranes only. The ergosterol solution (KOH and MeOH) will stop all activity except those associated with ergosterol. o We then measure that to find out how much fungi is in each sample. Presence of ergosterol will be measured using techniques from the Treseder Lab Protocol for Ergosterol Extraction.

Statistical Analyses A one-way ANOVA analysis was used to compare the enzyme activity between living and dead piñon trees to living juniper trees. Enzyme activities were analyzed between each comparison. o Analyses were made on activity per hour, per gram of dry weight. 22

Results Junipers tend to have higher rates of microbial activity compared to living and dead Piñon trees for Bgluc and LAP EEA. o Differences were highly significant. Patterns did not follow the same distribution for NAG and AlkP EEA. o Differences were not significant.

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Results

Results 26 P = n.s. P = n.s Comparisons Between Trees

Discussion Enzyme activities found in our study are similar to those found in Sinsabaugh et. al o The study was conducted on the Sevilleta National Wildlife Refuge in a PJ forest. o EEA were related to climactic gradients. o The experimental site in our study simulates predicted future climactic gradients. Climactic treatments at our experimental site had a significant effect on the EEA.

For the future Do pre and post monsoon season comparisons. Sample at both control and experimental sites equally for a better comparison Control site was only sampled once instead of 3X. Finish ergosterol extractions and analyzing all of the data.

The Big Picture The tower estimates belowground activity using data from soil respiration. o This project will allow us to determine the accuracy of those estimations. Data will be pooled with existing ecosystem data: o Soil respiration o Ecosystem productivity Contributes to a larger study in which these analyses are being conducted at each biome/tower site. 29

Acknowledgements Robert Sinsabaugh Jennifer Johnson Daniel Warnock 2011 REU Students 2011 Interns Scott Collins Marcy Litvak Don Natvig Sevilleta REU program

QUESTIONS?