Keywords: allelopathy, arbor species, dish-pack method, sandwich method.Prefectural Green Center, Japan, from April -August in 2002 and 2003. Soon after the fresh leaves were collected, they were used for the experiment.
Sandwich methodIn order to search for leaching substances from leaves, the sandwich method originally developed by Fujii et al. (2003) was revised and employed. For bioassay, a 12-well, multidish plate (3.66 cm 2 area per dish; Techno Plastic Product, Trasadingen, Switzerland) was used instead of a 6-well, multidish plate, as described by Fujii et al. (2003). The revised method is more convenient than the original one in order to screen a large number of samples without decreasing the efficacy. Then, 50 mg of each type of cut leaf were spread in each dish and 1 mL of 0.5% (w/v) agar solution cooled to 50∞C was added to each dish by a callus pipette (Iwaki & Co., Tokyo, Japan). After the leaves were fixed in the agar gel, 3 mL of agar solution were added and the mixture was allowed to harden. Five lettuce seeds (Lactuca sativa cv. Great Lakes 366) per dish were sown on the surface of agar. Each plate was covered with aluminum foil and kept in an incubator at 25∞C in the dark. Three days later, the lengths of the radicles and hypocotyls of the lettuce seedlings were measured. No leaves were put in the control treatment. Each experiment was triplicated.
Dish-pack methodTo find out about the release of allelopathic substances by volatilization, a dish-pack method (Fujii et al. 2000) was employed for this purpose. A 6-well, multidish plate (9.03 cm 2 area of each dish; Techno Plastic Product, Trasadingen, Switzerland) was used for bioassay. Filter papers (34 mm, type 2; Toyo Roshi Kaisha, Tokyo, Japan) were placed in each dish, except one dish in the corner. After the addition of 0.7 mL of distilled water, seven seeds of lettuce (as described earlier) per dish were sown. Also, 2 g of freshly cut leaves were placed in a cor-