Soil Microbial Ecology - Soil Molecular Ecology Laboratory

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Page 10 gently with water, and put spores in a Petri dish in water. Observe spores with the dissecting microscope and identify to the level of genus. Also observe prepared slides of spores under the compound microscopes set up for this purpose. There is also an optional slide set that can be observed in my office if you have special interest in this topic. 2. ROOT COLONIZATION: In the first lab period, you will be given cleared and stained root material to work with. However, at takedown of experiment described below you will prepare your own roots using the following procedure. Put roots from the coarse sieve (see above) into tissue cassette and place in a beaker with 10% KOH. Heat to 90 o C for 10-15 min; do not boil roots. Rinse KOH from roots with 5 changes of water. Acidify with 0.1 N HCl for 5 min., and drain HCl from roots. Cover the samples with Trypan blue-lactic acid (400 ml glycerol, 200 ml lactic acid, 400 ml H 2 O, and 600 mg Trypan blue) and store in a covered container. Heat to 90 o C for 10- 15 min and do not boil roots. Rinse the samples 1-2 times to completely remove all excess stain, or until the water is clear. The samples can now be stored in water in a refrigerator for 2-3 weeks or in double plastic bags. To determine root colonization, prepare a 1/2 inch grid on the outside of the bottom half of a plastic Petri dish. Remove stained roots from capsule and place in grid Petri dish with a small amount of water. Pour in just enough water to cover the bottom of the dish to facilitate uniplanar reading of the roots at 2X magnification of the dissecting microscope. Carefully spread the roots randomly across the plate until you have effectively covered the whole surface area evenly with roots. With the microscope set at 2X magnification and the light source directed from the bottom via the mirror, arrange the Petri dish in such a way that all of the vertical gridlines can be followed one at a time by sliding the dish with your hand in an up and down motion. With the two button counter operated by the other hand, record every root segment that intersects a vertical gridline by pressing one button. If the segment at the gridline is colonized by AM fungi, press both buttons. The final number multiplied by two will give you centimeters of root. The % colonization is determined by dividing the number of root intersections of the grid colonized by AM by the total number of root intersections. Length is based on the following equation: L = (pi * A * n) / 2 H; where A = total area, H = total length of lines, n = # intersections. Note: with 1/2 inch grid, 1 intersection is equal to 1 cm of root length. Determine the % colonization of the root sample provided. From prepared slides learn to recognize arbuscules and vesicles.
Page 11 3. POT CULTURE AM mycorrhizal fungi are obligate symbionts and cannot yet be grown in pure culture. Therefore, these fungi are maintained on plant roots in pot culture. Spores of a Glomus sp. and Gigaspora sp. will be provided. Establish 2 greenhouse cultures with each fungus and 2 controls (noninoculated) on a susceptible host such as maize. After 8 to 12 weeks document the results (% colonization, structures in root, sporulation, growth compared to the control) POT CULTURE METHOD: Add soil to within 10 cm of the top of 6 growth tubes. Place 10 g of the appropriate soil inoculum or sterilized inoculum on the soil surface and cover with additional soil. Seed tubes with either bahiagrass or alfalfa. Water plants as needed and fertilize every other week with a 20-0-20 soluble fertilizer. QUESTIONS: 1. What benefits do AM mycorrhizal fungi provide plants? Are there any disadvantages? 2. How does colonization by Glomus sp. differ from Gigaspora sp.? 3. What are some of the pit falls in the pot-culture method? REFERENCES: Smith, SE. and D.J. Read. 1997. Mycorrhizal symbiosis. 2nd ed. Academic Press Schenck, N.C. (ed.). 1982. Methods and principles of mycorrhizal research. Amer. Phytopath Soc., St. Paul, MN. (Chapters 1, 3, 4, 5 and 6). Sylvia, D.M. 1994. Vesicular-arbuscular mycorrhizal fungi. p. 351-378. In R.W. Weaver and et al. (ed.) Methods of soil analysis, Part 2. Microbiological and biochemical properties. <strong>Soil</strong> Science Society of America, Madison, WI.
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Soil Microbial Ecology - Soil Molecular Ecology Laboratory

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