Differences in Rhizopogon Colonization on Pines in the EasteTheresa HendrickIntroductionMycorrhizal fungi are extremely important for the growth andPlant material Sugar, Jeffrey, and Lodgepole pine seeds were ordered and shipped to us from the United States Forest Service. Bishop pinecones had previously been collected fromSoil Material The five different soil samples were obtainedExperimental Set-Up and Design Seventy quart-sized jars witDifferences in Rhizopogon Colonization on Pines in the Eastern Sierra Nevada Theresa Hendrick Abstract Rhizopogon is a genus of hypogeous fungi that forms mutualistic associations with many members of the Pinaceae. Rhizopogon maintains spore viability after large-scale disturbances such as stand-replacing fire and is abundant in soil after such disturbances. It provides much of the inoculum necessary for post-fire rehabilitation of pine seedlings and therefore is a key element encompassing fire ecology. Bioassaying pine seeds in soil can successfully capture Rhizopogon. Pinus muricata (Bishop pine) is the preferred pine to bioassay with because it is fast growing, able to capture Rhizopogon in high frequency, and requires little preparation time before planting. Preliminary data from this study shows that Bishop pine is more frequently colonized as compared to the other pines used. In this study Bishop pine was bioassayed to determine whether it could provide a set of similar fungi that colonize the root tips of the native tree populations in the Eastern Sierra Nevada. These native tree populations consist of Pinus jeffreyi (Jeffrey pine), Pinus lambertiana (Sugar pine), and Pinus contorta (Lodgepole pine). Samples of Rhizopogon were obtained directly from the roots of each individual pine in the bioassay containers and screened by ITS-RFLP and unknown Rhizopogon species were further sequenced. I anticipate that similar sets of Rhizopogon will be obtained from Bishop, Jeffrey, and Lodgepole pines while Sugar pine, because of its differences in lineage, may capture a slightly different set of Rhizopogon.Introduction Mycorrhizal fungi are extremely important for the growth and survival of many plant species because they increase the plant’s ability to obtain water and nutrients such as phosphorus and calcium, which can be limiting in soil environments (Kernaghan et. al. 2003). These fungi can also protect roots from adverse conditions such as drought and high metal concentrations within the soil (Brownlee et. al. 1983, Burgess et. al. 1993). Rhizopogon is a genus of ectomycorrhizal fungi with a large number of species specifically associated with members of the Pinaceae (Cairney 1999). Recent work by Kjoller and Bruns (2003) has shown that Rhizopogon species form an abundant sporebank in pine-dominated forests in coastal California, and following severe disturbance, such as stand-replacing fire, this sporebank provides most of the mycorrhizal inoculum for newly establishing pine seedlings. Rhizopogon can be retrieved from soil by bioassaying the soil with pine seedlings. The purpose of this study is to test the hypothesis that different pine species will yield different species of Rhizopogon under identical bioassay conditions. The pine species used in this study are: P. muricata, P. jeffreyi, P. lambertiana, and P. contorta. The different soil types were collected from five different areas in the Eastern Sierra Nevada. P. muricata is the foreign pine of the group because it is native to California coastal regions. It is readily available to us and is easy to germinate and grow. The other pine species are native to Sierra Nevada forests and involve a much longer germinating and growing process. Therefore, if P. muricata (Bishop pine) yields similar Rhizopogon species, researchers would be able to bioassay it with soils from the Sierra Nevada and other mountainous regions. In turn, they would benefit in the faster germinating and growing process as well as have an accurate representation of the different Rhizopogon species within these areas. Little is known about the below ground community in the Eastern Sierras; therefore, gaining an understanding of this otherwise unknown mycorrhizal system benefits future research in this ecologically rich area. According to Kernaghan et. al. (2003) and many others diversity within mycorrhizae in natural ecosystems is yet to be clearly determined.Materials and Methods Plant material Sugar, Jeffrey, and Lodgepole pine seeds were ordered and shipped to us from the United States Forest Service. Bishop pinecones had previously been collected from Point Reyes National Seashore and the seeds were harvested directly from the cones. Each of the Sugar, Jeffrey, and Lodgepole seeds were surface sterilized in 30% hydrogen peroxide with a drop of Tween 20, which acts as a detergent, and placed on a shaker for 20 minutes. The seeds were then rinsed and placed in individual plastic bags with small holes punctured in the bottom and placed under running water for two days followed by a 30-day drip dry in the cold room (33°F). Moist paper towels surrounded the seeds in the plastic bags to retain moisture and they were checked every other day to make sure the moist environment was maintained. After the 30-day drip dry the seeds were once again rinsed, dried, and placed in a new plastic bag and set to dry in the cold room for various periods of time following the suggested protocol obtained from the Forest Service. Jeffrey Pine remained in the cold room for 33 days, Sugar Pine for 61 days, and Lodgepole Pine for 15 days. The seeds require this process to simulate the Sierra Nevada winter conditions. The starting and stopping times for the seed preparation were arranged such that all the seeds would be ready to plant at approximately the same time. This process was started on 7/15/03 and ended on 10/14/03. Bishop Pine seeds were surface sterilized as the others, rinsed, and dried. The Bishop Pine seeds do not require any soaking or drying time and are ready to plant immediately after they are surface sterilized. Soil Material The five different soil samples were obtained from Eastern Sierra pine sites near Mammoth Lakes area (Table 1). Each sample consisted of 10 small scoops of soil, approximately 10 cm into the mineral layer. Each scoop was collected 2 to 3 meters from the previous scoop along a line. The first four samples were under predominately Jeffrey pine and the last
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