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In this experiment, we observed and analyzed the disease called Sudden Oak Death (SOD). Sudden Oak Death is a silent efficient killer of trees in coastal California. It is spreading out fast killing numerous species of Oak and Tanoak in California, especially in Sonoma County. The main species that is most effected by SOD is Umbellularia californica, (Wikipedia.org). According to California Oak Mortality Task force (COMTF), this disease was "first reported killing trees in Santa Cruz and Marin countries in mid-1990s". Now it is spreading out in "14 central and northern coastal counties in California: Humboldt, Mendocino, Lake, Sonoma, Napa, Solano, Marin, Contra Costa, Alameda , San Francisco, San Mateo, Santa Clara, Santa Cruz, and Monterey" (COMTF ). Among these county Sonoma County is facing lots of problem regarding SOD. Many oak trees are dying and lots of trees has already died. According to US forest aerial survey, the map allocation of SOD shows currently "7.5 % of the land in Sonoma Country (75,000 acres) has been affected by SOD mortality in just past three years" (cesonoma.usdavis.edu). Due to this Sonoma Country has been a major area for new SOD infestations and mortality. Sonoma County has "twice as many acres affected by SOD mortality than any other county in California" (cesonoma.ucdavis.edu). Since, Sonoma County has higher rate of SOD mortality, it has been a big threat to Sonoma Country. Its threat can have major impact in environment, ecosystem and economics. The huge number of dead and dying trees can effects the residents of county in various ways. The dead trees can fall in the roadways, house, power line etc. This can cause many hazards including injuries, death or property damages. Death trees can pass disease to other trees and infect them. Oak and tanoak tree are the source of acorns, which are the food for different wild habitats. When large amount of oak and tanoak trees are dead, there will be a loss of food for those habitats. Trees are the source for soil strength and stability, so when there are lots of dead tree soil will lose it strength. As a result, good soil could wipe out from the ground. Fire officials says the dead trees are the main cause for wildfire and increasing dead trees in Sonoma county put the county at a high risk for excessive fire behavior (cesonoma.ucdavis.edu). This entire possible hazard could cause a lot for this county economically and environmentally. To stop the spread of disease many researchers are doing researching regarding the pathogen that cause Sudden Oak Death. Even though researcher are not fully able to understand how the pathogens spread out. Therefore, it is necessary to study on this problem to solve the unanswered questions.
Correspondingly, our main reason of the experiment was also to analyze how those diseases spread in a tree, so we can be able to answer the unanswered question. To begin the experiment we should first know the pathogen that causes SOD. Sudden Oak Death is cause by plant pathogens Phytophthora ramorum (California Oak Mortality Task Force). Phytophthora ramorum is a microscopic organism. This species are water mold so it requires warm and wet weather to produce. California coastal forests are evergreen, fogy and moist, which are the perfect climate for Phytophthora ramorum. Predator P.ramorum is mostly affecting the 'California bay laurel'. In a host tree, it develops hyphae, which grow through bark and leaf tissue. It is produce sexually and asexually. Asexual reproductive structures called sporangia and chlamydospores. Sporangia release zoospores, which have two flagella that propel them through water. Chlamydospores are strong structures that protect the pathogen during hostile conditions, such as heat and drought (whatcom.wsu.edu). For sexual reproduction Phytophthora ramorum needs different mating type (A1 and A2). In California, type A2 mating is found and sexual reproduction has not been studied much outside the laboratory (en.wikipedia.org/sudden oak death). As asexual reproduction is more studied and known, so we should learn it for further study. Sporangia can spread through air and infect plant tissue by direct contact. Zoospores have flagella so it can swim through water. Rainwater or splashing water can spread the Zoospores to other plant. They also stay viable in soil and infect plant tissue. Chlamydospores can live in hostile condition so it can endure for sometime without any host. This spores spread through not only wind, rainwater, plant material, but also human activity. In wet season, human wet shoes can transfer the infected spores to other plant. When spores are spread on other trees leaves and trunk it causes plant to a fatal infection. In this way pathogen spread over a wide area causing a serious infections on trees that leads to a sudden death. With all these knowledge, we are creating our own hypothesis for our experiment. First, we created a reasonable question; Are sun leaves more susceptible to infection than shade leaves? According to the question, our hypothesis was that sun leaves are more susceptible based on previous class experiment. To prove/falsify our hypothesis we create an experiment infecting ten sun leaves and ten shade leaves from each tree. After infecting, we let it to develop the infection and later analyze it comparing frequency and magnitude of infection. This gave us the data and the result for our experiment.
Methods and meterials
To test our hypothesis we set up an experiment in three parts. In first part, we collect the leaves, in second part, we infect the leaves, and in third part, we analyze the infected leaves. We collect our leaves in pepperwood reserve. Pepperwood reserve is located at southland of the northern coastal range. It lied between Sonoma and Napa County. It is stretch toward north of Calistoga to east and Santa Rosa to west. Across the Papperwood reserve there is a panoramic view of Mt.st.Helena. Papperwood reserve covers 3000 acres zone of eye-catching landscape and prosperous biodiversity (papperwoodpreserve.org). It varies in elevation from 240 to 1,560 feet, which create an ecological diversity (papperwoodpreserve.org). Papperwood reserve is an intermediate climate zone where average temperature is around 60. Due to its ecological diversity and intermediate climate there are wide diverse of habitat. Papperwood reserve conserve mixed evergreen woodland, live oak tree, tan oak-redwood forest, grassland, and chaparral (papperwoodpreserve.org). There are various species of birds, reptiles and amphibians, and mammals in pepperwood reserve. Before we collect our leaves, we took a brief tree description such as species name, number of trunks, circumference trunk, and the height. To collect our data and leaves, instructor provided us a required instrument such as measurement tape, cutter, and wrapper bag to keep leave. Our group tree has six trunks with different circumference. Below is the measured circumference.
Number of Trunk
Average height of our tree was twenty feet. To measure the height we just use one of our friend heights and compare it with the tree to approximate height of the tree. After we took a tree description, we cut the 10 sun leaves and 10 shade leaves for the experiment. The process to cut the leaves was quite amusing. The tip leaves of the branch were a new leaves but for the experiment, we need last season leaves. Last season leaves has well develop metabolism so it would be best for pathogen to react. To separate the new leaves from old we feel the bud like surface in the branch. Beyond the bud surface was the leaves we need for the experiment. From sun and shade side of the tree we cut the branches that had good leaves on it, and kept inside the wrapper bag. After cutting leaves, we observed the surrounding and marked the place for further direction. Around our leaves, there were many grasses named Festuca California. In front of our tree, there was also dead fallen tree and oak sprout. All this information will help us to trace our tree for next visit. The collected leaves were brought to the lab. In the lab, we observed the leaves size, roughness and smoothness. Size of the leaves were same for both sun and shade leaves. Sun leaves were little thicker, curved and rough, where shade leaves were thinner, flatter and smother. After observation we choose 12 good sun leaves and 12 good shade leaves. For both sun and shade; 10 leaves were labeled 1 through 10 and other two were label C1 and C2 with tape and marker. Labeled leaves were cleaned with alcohol and cotton balls. After cleaning by alcohols, all the leaves were cleaned with deionized water. Meanwhile when leaves were drying we set up two boxes in the table for leave to lie on. In each box, we labeled our group number, date and either sun or shade. In both box we put a half inch of sterile vermiculite. This helps to absorb the humid environment inside the box. After finishing the box setup, we put dry leaves flat in the box facing lower side up: 1 through 10 leaves lie in a row across the box, and C1 and C2 lie on the side of the other 10 leaves. After putting leaves in the box, we infected our leaves with micropipet. In 1 through 10 leaves we infected it with p.ramorum zoospores and for control we put soil and water. After infecting leaves for both sun and shade, we put box lid on tightly and kept it in appropriate place.
I) Our Group Data for SOD experiment
II) Summary data for class SOD experiment
Table (I) is our group data for SOD experiment. The mean magnitude of sun and shade gives the p-value for our data. Table (II) concludes all class mean data of both sun and shade. Calculating the average of each group mean magnitude gives mean data for whole class. The calculated class mean provides a new p-value. In the summary data for class tree 4 represents our group mean magnitude.
According to our group result, the p-value is high. Our group p-value is 0.4836. There is 48% probability that the difference in magnitude between sun and shade is due to random events. Higher the p-value tells there is no evidence that our hypothesis is true. This means that there is no significance difference between the mean magnitude of infection on sun and shade leaves. Our data concludes that both sun and shade leaves were infects in equal magnitude. Therefore, it falsify our hypothesis that sun leaves infect more than shade leaves. Now comparing to others data, our mean value is lower, because our leaves have less magnitude of infection. This is probably due to an experimental error. While we were infecting the leaves, some drops of zoospores might slip out of the leaves or some drops may slip while moving the box. This could have been due to curve leaves. According to my personal observation, there were some curve leaves and we were having hard time placing the zoospores at the right spot. Due to this, we could probably get less infection in magnitude. Other reason could be due to improper amount of vermiculite, which could effect in the environment of pathogens. If there is more vermiculite than there might not be enough humid that pathogen needs to grow, or if there is less vermiculite than there might be more humid, that pathogen cannot grow enough. Comparing our class group and other class group, our group has less magnitude of infection. This is probably due to the temperature and the environment. The day we went to collect leaves was foggy and the day other class went to collect leaves was sunny. In sunny day, tree leaves will get more energy from sunlight through photosynthesis than in a foggy day. Due to greater energy, their leaves metabolism rate might have been higher than our leaves. Therefore, when leaves were infected, pathogens might have better environment to cultivate in the leaves that were metabolically active. However, looking all classes' data the mean magnitude is still high. Thus, this lab experiment falsify that the sun leaves does not infected more than shade one. According to the result, the magnitude infection was relatively same without any significance.
The experiment conclude that the hypothesis we created were not good enough to know the way pathogen infects in trees. Our whole experiment was not good enough because the reason might be that the sun light does not always come in the same direction. The leaves of the tree are not necessary to face the sun every time and the shade leaves are not always be in shade consistently. In a day period, at a different time, sunlight will evenly distribute. Thus, every side of leaves will have similar amount of light. As a result, they will have similar metabolic rate. Therefore, this might cause that we get the similar magnitude of infection on both sun and shade.
Our group did not collect more good leaves so while preparing leaves we did not have more choice, so we have to use curve leaves. While pipeting it was hard to put drops of zoospores in the leaves due curve, as a result some of the drops were slipping. Especially me, while pipeting I was not holding the pipet straight vertically so some of drops were too small due to air bubble inside the pipet. All this error can improve in next experiment, so we possibly will get good result.
For next experiment a different ideas should be use for a good result. After looking this experiment, the result turns out poor for our group. There was not much infection in leaves. I think this is due to lack of moister environment for pathogens. Based on research, Phytophthora ramorum loves wet and moister environment. Thus, for next experiment we could make moister environment for pathogen, so it can infect leaves out more efficiently. To make a humid condition we can put less vermiculite in our boxes so it cannot absorb humidity. In addition, we can put some water in vermiculite to give a moister environment. As I mentioned above that the shade leaves also gets similar amount of light as sun leaves. Thus, for our next experiment we can go to our tree and cover the shade leaves with black plastic for while so the light cannot pass through it. After collecting the leaves, we can put shade leaves in black plastic and sun leaves in regular plastic black. This creates that shade leaves will not get enough light to make energy and will be metabolically slow. Thus, creating such environment we can predict that sun leaves will be more infected than shade leaves.