Introduction:Who I am.

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- Differences that exist between the three different types of soils:

- Clear-cut – missing most vegetation due to human degradation of forest floorwhen harvesting trees. High rate of erosion. Deteriorating developed soil.

- Non-serpentine soil – most variety and greater amount of flora present. Soil ismore established, having higher organic matter content than that of the two otherkinds of soil present, and is less likely to be weathered by erosion. Soil profiledevelopment is higher, and soil-moisture holding capacity is higher than other soilspresented.

- Serpentine soil – soil consists of ultramafic parent rock. High in magnesium andiron, the soil conditions are inhospitable to most plants. Some plants present onserpentine soils have become endemic and are only able to persist on serpentinesoil. Lack of organic matter leaves the soil susceptible to higher rates of erosion,and the soil is thin and loose.

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Serpentine soil – loose, thin soil. Ultramafic rocky terrain. Sparsely populated with limited species of flora on soil. Low soil moisture holding capacity. Trees are stunted in growth

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To understand the process, the cycles should be addressed first.

How are the N and C cycles affected by serpentine soil? Carbon derived from atmosphere, Nitrogen primarily derived from detritus (decayed organic matter), but can also be taken in atmospherically if first converted to a useable form by nitrogen fixating bacteria that live in roots and soil

How are endophytes affected by serpentine soil?

Project interdisciplinary due to:

Biogeochemistry is defined as being the process of understanding how the interacting carbon and nitrogen cycles are cycled through the plant/soil system by use of fauna and flora heterotrophs (National Center for Atmospheric Research. (2018). CommunityEarthSystemModel:CLMBiogeochemistry. Retrieved from http://www.cesm.ucar.edu/models/clm/biogeochemistry.html.

Biology being the study of life

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Ecology: how the biotic and abiotic factors of an ecosystem interact with one another

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Isotopes are of the same respective element, having same # of protons, but not of neutrons. Stable isotope ratios measured in this experiment were 15N:14N and 13C:12C

A greater d15N:d14N value in a plant would imply that there may exist a more closed N cycle for the plant than that of what is projected above and mean that there may exist limited microbial involvement and lack of nitrogen sources being available in serpentine soil.

A greater d13C:d12C value in a plant would imply that the plant may be under water stress, and that Rubisco is not discriminating against 13C during some point.

The stomata of a plant open during the day to allow for the capture of atmospheric CO2 to be used during the process of photosynthesis by way of the enzyme Rubisco. Rubisco discriminates against C13 when the plant is not under stress. However, if the plant is subjected to water stress the stomata close, preserving the water that the plant has for internal processes and Rubisco will utilize d13C instead of discriminating against it. By measuring the amount of variation that is present for d13C and d12C in both serpentine and non-serpentine leaf tissue samples, we can compare the sites to ascertain if a plant is under stress due to low water availability.

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If that wasn’t enough, we took it one step farther, and took a look at what is happening to an organism that lives inside the tissues of the leaves of plants that proliferate on both serpentine and non-serpentine soil systems.

Found in every terrestrial plant taxa studied to date, fungal endophytes spend at least part of their life cycle growing between its host’s leaf cells while showing no visible pathogenic symptoms.

Endophytes are subjected to the same macronutrients that the host plant isolates from its surrounding environment

How endophytes are affected by the stressful environments that they are subjected to when present in leaves of flora living in serpentine soils has not been well studied

Evolutionarily, have these organisms become more generalized or speciated in their macronutrient needs? Are they capable of proliferating in many different hosts or are they able to live within only a few host plant species? Are endophytes from both soil systems able to produce the same amount of growth in extracts derived from leaf tissues from the same species of tree that was found on both serpentine and non-serpentine soils?

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Fours sites all come from mountainous terrain, where serpentine soils could be relatively easily accessed.

Geological maps were used to pinpoint possible collection sites.

Permission to collect samples on public land was verbally obtained from Oregon Department of Forestry Service and U.S. Bureau of Land Management.

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For isotopic analysis,108 leaf samples (one from every tree) were dried, ground and weighed into tin capsules at the value of 0.002g.

These samples were combusted in an elemental analyzer coupled to an Isotope Ratio Mass Spectrometer

Elemental analyzer separated the Carbon from the Nitrogen samples by their respective element

Isotope Ratio Mass Spectrometer separated the isotopes by charge, and placed the differing isotopes into Faraday cups. This would gave us the ratio that we were trying to obtain. 15N/14N and 13C/12C

Standards were used to gain the ratio differences from standardized 15N/14N and 13C/12C values

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For endophytic growth analysis, leaves from every tree collected from Sexton Summit and Red Mountain were sterilized, dried, then cut with sterile scissors and allowed to drop onto potato dextrose agar (PDA) plates.

PDA is a universally used generic lab media for growing fungi on. Once endophytes began to grow from the leaf sections on the agar, 8 endophytes were selected. One from each species of tree that was found on both sites, and on both soil types. 2mm2

pieces from the fungi selected were inoculated onto fresh PDA agar.

After a seven day period of growth, the fungi had obtained enough growth to then be inoculated onto 9 different types of media agar in 2mm2 pieces.

Eight tree extract agars were made using crushed leaf tissue, water, agar, and a pinch of sand. Four extracts for non-serpentine fresh and cooked extracts, four extracts for serpentine fresh and cooked extracts. Cooked extracts were made by added the extract to the agar before autoclaving the media. Fresh extracts were made by adding the extract right before plating. All extracts were filtered down to 0.2nm to ensure that all extracts contained the same nutrient consistency. All extracts were sterilized by either autoclaving or by using a sterile 0.2nm filtered syringe.

This resulted in a total of 72 plates being inoculated with fungi. Then the endophytes had their growth measured in millimeters from the centered piece of inoculated ager to the edge of the plate over a seven day period.

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Pic on the right shows how large the pieces of fungi were that were inoculated.Pic on the left shows how varied the fungi were that grew and also shows how the growth was varied for the eight different fungi plated on the nine different types of media.

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Higher amounts of 15N were found on non-serpentine soil systems when compared to serpentine soil systems

Higher amounts of 13C were found on serpentine soil systems when compared to non-serpentine soil systems

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Averaged values for leaf tissue Carbon isotopic ratio of 13C/12C found to be lower on non-serpentine soil than that of serpentine soil

Averaged values for leaf tissue Nitrogen isotopic ratio of 15N/14N found to be lower on serpentine soil than that of non-serpentine soil

T-test statistical analysis affirms that to within a 0.95 certainty, there exists a significant difference between the carbon and nitrogen isotope ratio values found for non-serpentine soil systems when compared to the values found for serpentine soil systems.

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Growth obtained for all eight endophytes isolated were tallied for each media that was used. Some fungi grew very quickly, having growth that exceeded the length of the media plate within a two day period. Some fungi grew at a slower rate. Overall, for every media plate that was used for this experiment, the endophytes from the trees found present on serpentine soil surpassed the growth that occurred for the endophytes originally isolated from a species of tree found on non-serpentine soil.

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d15N values for serpentine soil were lower than that of non-serpentine soil. This indicates that there may not exist a more closed nitrogen cycle occurring on serpentine soil. This could be due to beneficial microbial activity occurring at the soil level.

d13C values for serpentine soil were higher than that of non-serpentine soil. This indicates that Rubisco is not discriminating against 13C as much as it is when the plant is living on non-serpentine soils. This may be due to a lack of soil moisture holding capacity due to a lack of soil development.

Endophytes have only recently become a scientific subject of interest. They have been found to have anti-fungal, anti-bacterial, and anti-microbial properties as well as have been found to be useful antioxidants, nematicides, and have been used in anticancer applications. Selecting endophytes from serpentine soils may be useful in the future due to their indicated heartiness in being able to grow on many different forms of nutrient media.

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Thank you to all helped me with this project! Man did the time fly

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Lots and lots of questions I’m sure

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Just really like this picture

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