Isolation of VA mycorrhizal fungi: Numerous techniques are used to recover VAM propagules from soil. The most basic of these is wet sieving and decanting to remove the clay and sand fractions of the soil while retaining spores and other similar-sized soil and organic matter particles on sieves of various sizes.
Wet sieving and decanting (Gerdemann and Nicolson, 1963): Approximately 250 ml of soil were suspended in 1 liter or more of water. Heavier particles were allowed to settle for a few seconds and the liquid was decanted through a sieve fine enough to remove the larger particles of organic matter, but coarse enough to allow the desired spores to pass through. The suspension that passed through this sieve was saved and stirred to resuspend all particles. The heavier particles were allowed to settle for a few seconds and the liquid decanted again through the sieve. The sievings retained on different sieves were washed into separate petri dishes for further observations or purification by sucrose centrifugation (Figure 1).
Sucrose centrifugation (Daniels and Skipper, 19982) Spores and minimal amount of organic particles could be further purified by suspending sievings in the 40% sucrose solution and centrifuging at 2000 rpm (approximate 370 x g) for 1 minute. Pour the supernatant (with spores) through the sieve of 400 mesh and rinse with distilled water to remove sucrose (Figure 1). Root staining (Kormaik and McGraw, 1982)
Root samples could be preserved with Formalin-Aceto-Alcohol (FAA). The standard FAA solution made with 50% alcohol with a v/v/v ratio of 90:5:5 is adequate.
Wash root samples stored in capsules with FAA in tape water.
Place root samples in a glass beaker and cover with a 10% KOH solution.
Heat the specimens in a water bath at 90¢XC for 1 hour or in an autoclave at 15 psi for 10 min.
Pour off the KOH solution and rise the root samples well. * If roots are pigmented, cover the capsules in the beaker with alkaline H2O2 at room temperature for 10 to 20 mins or until roots are bleached. Alkaline H2O2 is made by adding 3 ml of NH4OH to 30 ml of 10% H2O2 and 567 ml of tap water. The alkaline H2O2 solution should be made up as needed; it loses its effectiveness even if stored overnight.
Rinse the capsules in the beaker thoroughly using at least three complete changes of tap water to remove the H2O2.
Cover the capsules in the beaker with 1% HCl and soak for 3-4 mins and then pour off the solution.
Cover the capsules in the beaker with 0.01% acid fuchsin-lactic acid staining solution and heated in water bath at 90¢XC for 30 ~60 mins. The staining solution consists of 875 ml of laboratory grade lactic acid, 63 ml of glycerin, 63 ml of tap water, and 0.1 g of acid fuchsin.
After removing from the capsules, place the root specimens and the capsule top in glass petri dishes for destaining and mycorrhizal assay. The destaining solution is the standard used in step 8, but without the acid fuchsin component.
Making semipermanent slides of VA mycorrhizal fungi: The ability to make a good, semipermanent, diagnostic slide is critical in making a species determination for a specimen of VA mycorrhizal fungi (Schenck and Perez, 1990). Semipermanent mountants, such as PVLG (polyvinyl alcohol-lactic acid-glycerol) (Koske and Tessier, 1983) or Hoyer¡¦s (Cunningham, 1972) allow slides to remain useable for years.
Propagation of VA mycorrhizal fungi: Up to now, three major methods have been commonly used for inoculum production of arbuscular mycorrhizal fungi (AMF)-(i) pot culture, (ii) solution culture, and (iii) root organ culture. Traditionally, AMF are cultivated by sand-based pot cultures (Menge, 1984). However, it is a labor-intensive process and not economical for mass production of inoculum. Solution culture techniques such as nutrient film (Elms and Mosse, 1984; Mosse and Thompson, 1984) and aeroponics (Sylvia and Hubbell, 1986) are mainly adapted for the production of clean, soilless inoculum of AMF. Nevertheless, these methods have proven successful only in cultivating Glomus species (Hung and Sylvia, 1988; Sylvia and Jarstfer, 1994). Recently, several tissue culture techniques for producing contaminant-free AMF inoculum have succeeded. Ri-plasmid transformed-root cultures seem to offer the most efficient method for growing colonized roots (Mugnier and Mosse, 1987). Piche and his colleagues (Becard and Piche, 1989;1990; Chabot, et al.,1992a; 1992b) completed excellent studies on the root organ cultures of Glomus intraradices Schenck & Smith and Gigaspora margarita Becker & Hall.
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