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Rhizosphere soil method: a new bioassay to evaluate allelopathy in the field.

Yoshiharu Fujii 1, Akihiro Furubayashi 1 and Syuntaro Hiradate 1

1 Chemical Ecology Unit, Department of Biological Safety, National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba Science City, Ibaraki 305-8604, Japan, Email


We have developed a new bioassay named Rhizosphere Soil Method for the evaluation of allelopathy in the soil. This method is to evaluate the allelopathic activity directly in the soil. Rhizosphere soil was brushed off from the surface of root after removing surface soil by “Air Shaking method”. After sieving the soil by 1.0 mm sieve, 3.0 g (equivalent to 4.0 cm3 volume) of soil was added to the 6-well-multi-dish. 5.0 ml of 0.75% of agar was added to this soil and gelatinized. On the surface of this agar, 3.2 ml of agar was over-layered, and lettuce seeds were put on this surface. After 3 days incubation in phytotoron, germination % and growth were measured. The texture and type of soil did not affect the results. By using this method, allelopathic activity of ground cover plants and weeds were evaluated. Hairy vetch (Vicia villosa), tall goldenrod (Solidago altissima), alflfa (Medicago sativa) showed strong inhibitory activity. These results are in good correlation with Plant Box Method, that we have already established.

Media summary

A new bioassay method to evaluate allelopathy for rhizosphere soil is developed.

Key Words

Rhizosphere soil method, allelochemicals


Root exudates from plants influence the growth of other plants through soils, which is one of the pathways of allelopathy. These exudates tend to be adsorbed on soil components and decomposed into other chemicals by soil microorganisms. To clarify the allelopathic phenomenon, it is important to evaluate plant growth activity of soils. In this study, we have evaluated “soil sandwich method” to explore the allelopathic activity in rhizosphere soils (Furubayashi et al. 2002). We change the name into “Rhizosphere Soil Method” as rhizosphere soil was found to be the most important. In this research, we used rhizosphere soils of 12 plant species under different families as shown in Table 1. We discuss the potentiality of soil sandwich method – a new method for bioassay in allelopathy research.

Materials and Methods


We have selected 12 plant species for our experiment (Table 1). Each plant was grown in plastic pot using mixed soil (Andosol and commercially available soil), and cultivated at the green house at 25 for several weeks.

Soil sampling

The plants were taken out from the plastic pot without disturbance, then plant root were shaken softly to remove the root-zone soils (Figure 1). We collected the soil adhering to the surroundings of a root, called "rhizosphere soil" in general, and the soil shaken off is “root-zone soil”. Collected soils were sieved in a 1mm mesh removing root hair as much as possible. Fresh soil was used for the experiment. Because of the difference in growing of the root hair or soil moisture content, the amount of the rhizosphere soil from one pot differed between plants. In order to obtain the amount of soil required for sandwich method, rhizosphere soils were collected from several pots of same plant species.

Results and discussion

The results are shown in Figure 2. Both Medicago sativa (Alfalfa) and and Vicia villosa Roth (Hairy vetch), belonging to the family Leguminosae, inhibited radicle growth of lettuce strongly in the Rhizosphere Soil Method. Rhizosphere soils of Solidago altissima L. also showed strong inhibitory activity (Figure 2). Solidago altissima is an invader plant from North America. Vicia villosa Roth is a well-known allelopathic cover crop in Japan (Fujii 2001, Fujii et al. 2003), and our group identified cyanamide as the allelochemical from this plant. This finding is the first in the world for the isolation of natural cyanamide (Kamo et al. 2003). The results from Rhizosphere Soil Method showed the allelopathic activity of allelochemicals exudated into soil. The inhibitory activities of rhizosphere soils were stronger than those of root-zone soils in all cases. This suggests that substances released from roots of plants are adsorbed on soil, or decomposed by soil microorganisms.

These results suggest that the Rhizosphere Soil Method is useful as a primary screening method for evaluating the allelopathic potentiality of the soils around the root.


Fujii Y (2001). Screening and future exploitation of allelopathic plants as alternative herbicides with special reference to hairy vetch. In ‘Allelopathy in Agroecosystems’. (Eds RK Kohli, HP Singh, DR Batish), pp.257-275.Food Products Press
Fujii Y, Parvez SS, Parvez MM, Ohmae S, Iida O (2003). Screening of 239 medicinal plant species for allelopathic activity using the sandwich method. Weed Biology and Management 3:233-241.

Fujii Y, Shibuya T, Nakatani K, Itani T, Hiradate S and Parvez M M (2004) Assessment method for allelopathic effect from leaf litter leachates. Weed Biology and Management., 4, 19-23.

Furubayashi A, Hiradate S, Araya H, Horimoto S and Fujii Y (2002). Soil sandwich method: a new method for bioassay to evaluate the allelopathic activity in rhizosphere soils. The Third World Congress on Allelopathy / Abstracts 3 : 236.

Kamo T, Hiradate S and Fujii Y (2003). First isolation of natural cyanamide as a possible allelochemical from hairy vetch Vicia villosa. Journal of Chemical Ecology 29 (2), 275-284.

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