• Analytical Science and Technology
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• v.7 no.3
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• pp.403-411
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• 1994

To isolate, purity and identify of bioactive compounds involved in alfalfa allelopathy and/or autotoxicity, experiment was conducted. Isolation and separation procedures used from an 80% methanol extract of fresh alfalfa leaves(1kg), silica gel thin layer chromatography(TLC), followed by Droplet Counter Current Chromatography(DCCC). Preliminary identification was examined by high preformance lipid chromatography(HPLC). Four phenolic compound, salicylic acid, scopoletin, rutin, and quercetin, were identified and identified all compounds were phytotoxic to alfalfa seed germination and seedling growth. Among these compounds, quercetin treatment($10^{-3}M$) was most inhibitory to alfalfa seed germination and seedling growth. These compounds may be, at least in part, involved autotoxicity and allelopathy.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.42 no.6
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• pp.821-832
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• 1997

The aim of this study was to separate or purify some bioactive compounds from flowers of alfalfa(Medicago sativa L.) and to test of the isolated compounds on alfalfa for their autotoxicity and on Echinochloa crus-galli for their allelopathy for seed germination and seedling weight. Using thin layer chromatography(TLC) of $CHCl_3$ extracts, the most inhibitory band to alfalfa seed germination was determined. Germination inhibition of this extract suggested a complex chemical interaction. Separation and purification of compounds with CHCl$_3$ extract of fresh alfalfa flowers were conducted by a silica gel TLC, and microcrystalline cellulose TLC(MCTLC), followed by droplet countercurrent chromatography(DCCC) bioassay. Preliminary identification was done by high perfomance liquid chromatography(HPLC) on the most inhibitory fractions in DCCC. Ferulic acid, caffeic acid, vanillic acid, rutin, narringin were identified in fraction 5 and ferulic acid, caffeic acid, vanillic acid, rutin, coumarin in fraction 6. The phytotoxicity of their individual compound was tested on alfalfa and Echinochloa crus-galli seed germination and seedling weight. Coumarin and ferulic acid showed the most inhibitory effect on alfalfa seed germination and Echinochloa crus-galli seedling fresh and dry weight. These compounds may be, at least in part, involved in autotoxicity and allelopathy.

• Plant Resources
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• v.3 no.1
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• pp.71-78
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• 2000

Autotoxicity restricts reseeding of new alfalfa(Medicago sativa L.) after alfalfa until autotoxic chemical(s) breaks down or is dispersed into external environments, often requiring up to a year or more. One solution for reducing autotoxicity would be to select germplasms or cultivars with tolerance to the autotoxic chemical(5) and use genetically breeding program. Bioassay of seed germination and early seedling growth was conducted to evaluate autotoxic responses of 3 varieties of alfalfa to the water-soluble extracts(at 4 and 8g/L) from alfalfa ‘Cody’leaf by using agar and filter paper medium in a petri-dish assay. Root length at 5 days after seeding was more sensitive to the extract than was hypocotyl length or seed germination, and was a better parameter of autotoxic effects of alfalfa leaf extracts. Use of an agar medium gave better sensitivity of root length than did use of filter paper. Evaluating tolerance with percent of control was more important indicator than was mean of root length because of significant variation among varieties in root length of control treatment. Bioassay ranked varieties in the following order of tolerance on the basis of relative root length; “Cody” >“ Pioneer 5373” >“ Alfagraze”. Seedling growth from old “Cody” seed was more sensitive to the autotoxic chemical(5) than was that from newly produced seed.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.45 no.2
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• pp.134-138
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• 2000

Most parts of alfalfa plant have been reported to contain autotoxic substances that inhibit seed germination and early seedling growth, however, the chemical(s) is not still studied much. Effect of seed leachates of 'Vernal' alfalfa (Medicago sativa L.) was evaluated for inhibition of alfalfa germination and root growth through bioassay. Alfalfa seeds were extracted in 1 L deionized water for 1 h after soaking and the leachates caused to reduce root length of alfalfa significantly as the soaking time increased. Crude seeds at 4 g L$L^{-1}$ exudated autotoxins that reduce significantly root length by 34 % compared to the control, when the seeds soaked in deionized water for 24 h. However, the extracts did not affect final germination as well as speed of germination. Extracts from ground seeds significantly reduced speed of germination (GT 50) and root length. The results indicate that release of autotoxic substances from seeds during seed imbibition was increased with increase of soaking time and seed amount, and that autotoxicity was more occurred in ground seeds than in crude seeds.

• Proceedings of the Korean Society of Crop Science Conference
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• 1994.06a
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• pp.88-89
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• 1994

• Proceedings of the Korean Society of Crop Science Conference
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• 1995.05a
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• pp.64-65
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• 1995

• KOREAN JOURNAL OF CROP SCIENCE
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• v.42 no.2
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• pp.228-235
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• 1997

This experiments were conducted to determine the effect of allelopathy and autotoxicity in alfalfa. Among several alfalfa cultivars, the substances exudated from seven cultivars including Medicago sativa, c.v. Luna, Sparta, Magnum, Husky, Milkmaker, Challenger and Anchor inhibited the germination and seedling growth of radish, ranging from 60 to 80％. When allelopathic substances were exudated from the soil of the alfalfa-cultivated field, the increased levels of substance retarded the germination rate and seedling growth of radish and alfalfa. The inhibition rate was about 80％, compared with that of control. Thus, this study indicates that the allelopathy and autotoxicity substances of alfalfa influenced the germination and seedling growth in alfalfa itself and radish. When allelopathic substances were analysed by using gas chromatograph, several kinds of phenol compounds were detected as follows; Salicylic acid, hydroxybenzoic acid, vanillic acid, syringic acid, coumaric acid, and ferulic acid.

• Korean Journal of Environmental Agriculture
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• v.18 no.2
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• pp.185-190
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• 1999

Autotoxic substance(s) from alfalfa(Medicago sativa L.) plants reduces germination and growth of adjacent new alfalfa after alfalfa. The autotoxic chemical(s) in alfalfa are clearly unknown. Our objective was to improve the sensitivity of an alfalfa seedling bioassay for evaluating autotoxic leaf extracts. We determined critical extract concentrations that inhibit seed germination and seedling growth, compared two different culture media, and evaluated the effects of extract sterilization on the sensitivity of the assay, by using streptomycin and autoclaving method. An agar medium in petri plate gave better responses of germination and seedling growth to the extracts than using filter paper in the plate. On agar medium, the concentration of extract required to reach 50% inhibition of root length was 2.7 g $kg^{-1}$, and of germination and hypocotyl length were 3.8 and 9.9 g $kg^{-1}$, respectively. Leaf extracts with 100 ppm streptomycin stimulated germination significantly compared to Leaf extract alone but reduced root length of control by 43%. Root length was more sensitive to the autotoxin(s) than was germination or hypocotyl length. These results suggest that agar medium mixed with extract and sterilization by autoclaving could be improved the consistency and precision of bioassay, and that root length was the best parameter of autotoxic effect of alfalfa leaf extract.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.49 no.1
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• pp.7-11
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• 2004

Alfalfa (Medicago sativa L.) plants have been reported to be autotoxic as well as allelopathic. Laboratory and greenhouse experiments through petri-dish and pot test were conducted to determine autotoxic effects of alfalfa leaf and soil extracts on the germination or early seedling growth of alfalfa, and to evaluate allelopathic effects of alfalfa leaf residues on alfalfa, barnyard grass, com, eclipta and soybean. Alfalfa seed germination was delayed depending on aqueous extract concentration, with no difference in final germination after 48 hours. Alfalfa root length was more sensitive to the autotoxic chemicals from leaf extracts than was germination or shoot length. Root growth of alfalfa was significantly inhibited at extract concentration of more than 1 g dry tissue/L (g $\textrm{L}^{-1}$). Hypocotyl growth, however, was not affected by all the concentrations of leaf extracts. Soil extracts from 4-yr-old alfalfa stand significantly reduced alfalfa root length by 66%, while soil extracts from 0,1, and 3yr-old stand stimulated root length up to 14-32% over the control. Residue incorporation with dry matters of alfalfa leaf at 100 g $\textrm{kg}^{-1}$ reduced seedling length of several crop and weed species, ranging from 53 to 87% inhibition. Addition of nutrient solution into alfalfa leaf extracts alleviated alfalfa autotoxic effect. This result indicates alfalfa leaf and soil extracts or residues could exert autotoxic as well as allelopathic substances into soil environments during and after establishment.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.40 no.5
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• pp.619-628
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• 1995

Crude aqueous extracts from dried leaves, stems, roots, and flowers from both field grown and greenhouse grown alfalfa plants inhibited alfalfa seed germination and seedling growth. The degree of inhibition was greater in the field grown plant extracts. Flowers extract of field grown plant most inhibited alfalfa germination and seedling growth. In the concentration study, the highest concentration of extract (9.0%, w/v) significantly inhibited total alfalfa seed germination by 50% as compared to control. In partitioning study using pot hydroponic culture of plant biomass into leaves, stems, root, LAR products of LWR and SLA exhibited significant variation among four species. This result support that the inhibitory effect of autotoxic substances presenting in alfalfa tissue may be possible interference with the patitioning of biomass into leaf component relative to the total biomass produced by the alfalfa plant. Toxicity of extract was not reduced by adding activated charcoal, Dowex-50W, amberlite to the extract. Toxic substances existing in most plant tissues but mainly above ground foliage are water soluble and stable and may persist in old alfalfa fields. Thus, it is recommended to remove as much as possible of the above growth parts, especially vegetative stage, before one tries to re-establish alfalfa in former field of alfalfa.