Natural and environmentally friendly way of managing weeds without herbicides by Allelopathy Yoshiharu FUJII, Tokyo University of Agriculture and Technology, 2.50pm-3.20pm, Monday 24 February 2014 Crown Perth, Burswood 1 Outline of Talk What is Allelopathy • Selection of allelopathic plants by Bioassays • New Concept of Total Activity for the isolation of Allelochemicals. • Cover crop with allelopathic activity provides natural and environmentally friendly way of managing weeds without herbicides. 2 Allelopathy Interaction (inhibitory or stimulatory) between plants or plant to other life by natural chemicals (allelochemicals) Plant to Plants ・・・・・・・・・・weed control Significance of Secondary metabolites* Plant Microorganisms・・・disease (*notto indispensable natural chemicals in plants) control Plant to Insects ・・・・・・・・・・ harmful Insect control Plant to Animals ・・・・・・・・・・nematode, boar, etc. Allelopathy is the same concepts as “Antibiotics” 3 Bioassay for Three Route All these Bioassays are my original Assay 1) Exudation (from root) Plant Box Method 3) 2) Leaching (from leaf or litter) 2) Sandwich Method 2) 3) Volatilization (mainly from leaf as volatile chemicals) Dish Pack Method 1) 4)=1)’ Rhizosphere Soil Method Fig. 1 Route of Allelopathy and Bioassay 4 4 1) Specific Bioassays for Allelopathy For the screening of Allelopathic Plants 5 2.1 Plant Box Method for root exudates (Mixed planting) Root zone separating tube Radicle Length of Acceptor (mm) 80 60 40 20 y = 1.3355x + 14.85 2 R = 0.9066 0 0 10 20 30 40 Sand Culture for１-２ month Distance from the Root of Donor (mm) Plant Box (for tissue culture) Dr. Fujii, Agar Medium (no nutrients) Fujii, Y. et al., Plant-Box Method: A Specific Bioassay to Evaluate Allelopathy through Root Exudates. Allelopathy, New Concepts and Methodology, Science Publisher, 39-56 (2007). 50 1991 1 6 Table 1. Allelopathic activity Leguminosae Family by Plant Box Method Scientific name Pachyrhizus erosus Mucuna pruriens var. utilis Mimosa invisa Crotalaria zanzibarica Medicago scutellata Galega orientalis Coronilla varia Medicago arabica Trifolium album Medicago lupulina Phaseolus vulgaris Vicia villosa var. dasycarpa Abrus praecatorius Canavalia ensiformis Lupinus bicolor Vicia villosa var. villosa Melilotus albus Dolicos lablab Vicia sativa Pueraria lobata Crotalaria juncea Trigonella foenum-graecum Vigna unguiculata Cassia tora Medicago sativa Vicia faba Onobrychis viciifolia Pachyrhizus tuberosus Phaseolus vulgaris Vigna angularis Trifolium incarnatum Cajanus cajan Crotaralia spectabilis Pisum sativum Cicer arietinum Lathyrus odoratus Amorpha fruticosa Latyrus sativus Lupinus albus Arachis hypogaea Calopogonium mucunoides Desmodium paniculatum Vigna unguiculata subsp. sesquipedalis Mimosa pudica Vicia angustifolia var. segetalis Aeschynomeue rudis English name Jicama Velvetbean activity 6 7 9 12 13 14 16 16 16 19 19 19 20 20 20 20 23 25 25 28 30 30 33 34 34 34 35 36 37 38 39 40 40 42 44 44 45 46 46 49 49 49 49 55 55 58 Allelopathic Leguminous Plants Sunn hemp Snail medic Galega Crown vetch Hop clover Kidney bean (Old cv.) Woolly pod vetch Rosary pea Jack bean Hairy vetch White sweet clover Lablab bean Common vetch Kudzu Sunn hemp Fenugreek Cowpea Sickle senna Alfalfa Broad bean Esparcette Ahipa Kidney bean (Modrn cv.) Adzuki bean Crimson clover Pigeon pea Sunn hemp Pea Chickpea Sweet pea False indigo Grass pea White lupine Peanut Calopogonio Tick trefoil Asparagus pea Sensitive plant Karasunoendou Joint vetch Pachyrhizus Mucuna Mimosa Crotalaria Medicago Vicia Plant Box Method Mucuna Mimosa 7 Table Allelopathic activity of Poaceae family evaluated by Plant Box Method (selected results) Scientific name Hordeum vulgare (Barley, cv. HVG-1) Avena strigosa (Wild Oat) Triticum polonicum (Polish Wheat) Avena sativa (Oat, Hokuren) Avena murphyi (Wild Oat) Secale cereale (Rye, Samusa-shirazu) Avena barbata (Wild Oat) Triticum compactum (Club Wheat) Avena longiglumis (Wild Oat) Triticum spelta (Spelta Wheat) Triticum sp. x Secale sp. (Triticale) Avena orientalis (Wild Oat) Chloris gayana (Rhodesgrass) Avena wiestii (Wild Oat) Avena byzantina (Byzantine Oat) Hordeum vulgare (Barley, Sansyu) Phalaris tuberosa (Hardinggrass) Triticum aestivum (Common Wheat, Mulchmugi) Anthoxanthum odoratum (Sweet Vernalgrass) Festuca rubra (Chewing Fescue) Panicum coloratum (Coloured Guinea Grass) Avena fatua (Common wild oat) Triticum aestivum (Common Wheat,cv. Norin 61) Radicle growth 9.7 12.0 13.2 14.8 16.9 17.3 18.3 19.5 21.8 22.4 22.6 22.8 23.5 24.1 24.5 25.5 26.6 28.0 28.1 30.3 32.8 33.0 33.5 Scientific name Radicle growth Scientific name Allelopathic Crops Eleusine coracana (Finger Millet) Triticum monococcum (Einkorn Wheat) Sorghum bicolor (Sorghum, Shikoku-native) Coix lacryma (Coix) Panicum kabulabula (Kabulabula Grass) Avena sativa (Oat, cv. Hayoats) Triticum durum (Durum Wheat) Avena abyssinica (Ethiopian Oat) Avena brevis (Brevis Oat) Panicum maximum (Guinea Grass) Bromus catharticus (Rescuegrass) Triticum dicoccum (Emmer Wheat) Sorghum halepense (Johnson Grass) Panicum antidotale (Blue Panicgrass) Eragrostis tenuifolia (Eragrostis) Festuca arundinacea (Kentucky 31 fescu) Sorghum sudanense (Sudan grass) Agropyron repens (Quackgrass) Lolium subulatum (Wimmera Ryegrass) Sorghum dochna (Common sorghum) Zea mays (Corn, cv. Peter corn) Setaria viridis (Foxtail Grass) Pennisetum glaucum (Pearl Millet) 33.7 34.3 35.5 36.5 36.7 37.0 37.3 38.5 38.8 39.7 40.1 40.5 42.8 43.5 44.6 44.8 45.2 46.0 46.8 47.1 47.7 53.2 54.4 Echinochloa utilis (Green Millet) Euchlaena mexicana (Teosinte) Chloris distichophylla (Weeping Chloris) Zea mays (Corn, cv. Golden dent) Panicum virgatum (Switch Grass) Poa pratensis (Kentucky Bluegrass) Panicum dichotomiflorum (Fall panicgrass) Eragrostis lehmanniana (Lehmann Lovegrass) Dactylis glomerata (Orchard Grass) Eragrostis curvula var. valida (African Lovegrass) Agrostis alba (Red Top) Lolium perenne (Perennial Ryegrass) Zea mays (Corn, cv. Pioneer dent) Cenchrus ciliaris (Buffel Grass) Paspalum dilatatum (Dallis Grass) Paspalum notatum (Bahia grass) Eleusine indica (Goose Grass) Lolium multiflorum (Italian Ryegrass) Poa compressa (Canada Bluegrass) Phalaris arundinacea (Reed Canary grass) Lolium westerwoldicum (Westerwolds Ryegrass) Festuca elatior (Meadow Fescue) Phleum pratense (Timothy) Barley (Hordeum vulgare) Oats (Avena sp.) Rye (Secale cereale) Wheat (Triticum sp.) These upland crops are originally resistant for weed Radicle growth 55.8 55.8 56.9 57.9 58.5 59.8 61.2 61.8 61.9 61.9 62.1 63.6 66.3 70.7 72.4 72.9 73.5 73.9 74.0 76.3 84.8 86.8 96.4 8 Jean-François Millet, Le semeur (1850) bird seed We Japanese love Millet ! Wheat wheat has allelopathic activity amount of seeding rate = 3 to 5 times For bird loss and weed suppression 9 “MACE” is King variety in AU now (by Cristine Zaicou-Kunesch) At today’s morning session Table Allelopathic activity of Triticum family evaluated by Plant Box Method Scientific name Radicle growth (%) Triticum polonicum (Polish Wheat) 13.2 Triticum compactum (Club Wheat) 19.5 Triticum spelta (Spelta Wheat) 22.4 Triticum sp. x Secale sp. (Triticale) 22.6 Triticum aestivum (Common Wheat, cv. Mulchmugi) 28.0 Triticum aestivum (Common Wheat, cv. Norin 61) 33.5 Triticum monococcum (Einkorn Wheat) 34.3 Triticum durum (Durum Wheat) 37.3 Triticum dicoccum (Emmer Wheat) 40.5 Proposal for Project Breeding of Weed Resistant Wheat with high allelopathic activity ! 10 Allelochemicals reported from Oats, Rye, Wheat, Barley O CH3O HO Wheat Rye Oat O O 7-Hydroxy-5-methoxycoumarin (Scopoletin) Many other chemicals are reported, but I guess these traditional compounds are important as allelochemicals based on TA N OH H3CO O N 2,4-dihydroxy1,4-benzoxazine-3-one (DIBOA) HO 2,4-dihydroxy-7-methoxy1,4-benzoxazine-3-one (DIMBOA) H3CO O O N H 2-benzoxazolinone (BOA) CH3 O OH OH CH3 OH N O Barley N,N-dimethyltyramine (Hordenine) O O N H 6-methoxy2-benzoxazolinone (MBOA) As for “TA”, Total Activity, Will explain later. 1. Belz, G.R. and Hurle, K., Dose-Response - A Challenge for Allelopathy? Nonlinearity Biol. Toxicol. Med. 3 (2), 173–211 (2005) 2. Liu, D. L. and Lovett, J. V., Biologically active secondary metabolites of barley. II. Phytotoxicity of barley allelochemicals, J. Chem. Ecol. 19, 2231-2244 (1993) 11 2. Sandwich Method for leaf leachates (Mulching, Litter) 6 well multi-dish make sandwich by agar 10 or 50 mg d.w./10 cm2 For allelopthy by fallen leaves and litters fallen leaves are constant （3 ton / ha / year） = 30 mg d.w./10 cm2 Dr. Fujii, 1991 Fujii, et al., Assessmentmethod method for allelopathic from leafleaf litter leachates. Fujii, Y. Y. et al., Assessment allelopathiceffect effect from litter leachates. Weed Biology and Management, 4(1), 4(1), 19 – 23 (2004). Weed Biology and Management, 1923 (2004) 1 12 Table 3. Allelopathic activity by Sandwich Method Scientific name Cymbopogon citratus Derris scandens Piper betle Tamarindus indica Gliricidia sepium Sesbania grandiflora Acacia farnesiana Duranta repens Diospyros mollis Afgekia sericea Ipomea pes-caprae Jatropha integerrima Melia azedarach Citharexylum spinosum Molineria latifolia Passiflora coccinea Calophyllum inophyllum Amhersita nobilis Cynometra cauliflora Litchi chinensis Radicle 0** 0** 3** 3** 5** 11* 15* 17* 23* 38 39 44 52 70 75 86 94 96 101 113 Hypocotyl 0** 0** 18** 26* 19* 45* 37 51 22 120 90 122 115 60 167 117 146 127 113 115 Radicle and hypocotyl means % growth of lettuce seedling to the control (in agar medium). ** after the data means inhibitory activity stronger than standard deviation value of 60, and * means 55. Sandwich Method Cymbopogon Tamarindus Fujii, Y. et al., Screening of 239 medicinal plant species for allelopathic activity using the sandwich method. Weed Biology and Management, 3(4), 233-241 (2003) 13 4. Dish Pack Method 3 for volatile allelochemicals 6 well multi-dish sealed by tape measure after 3 days plant leaf (2g) CH3 Volatile chemicals were analyzed by GC-MS OH β-pinene H3C CH3 thymol CH3 β-myrcene OH cis-3-hexene-1-ol H3C CH2 limonene Fujii, Y. et al., Dish pack method: a new bioassay for volatile allelopathy. In Proceedings of the 4th World Congress on Allelopathy, August 21 -26, 2005, Wagga 1 Wagga, Australia, 493-497 (2005). 14 Allelopathic plant for biofumigation Cleome Cleome spinosa） Cleome from Takii seed Co. Ｈ Ｎ Ｃ Ｓ Ｃ Ｈ Ｈ Methyl Isothiocyanate Strongest activity by Dish Pack Method Allelochemical : Methyl Isothiocyanate (MITC) EC100 for lettuce is 18 ppm Kill most specise of nematode Concentration of MITC in Cleome 表２ クレオメ各部位における ppm メチルイソチオシアネート濃度 M ethyl isothiocyanate濃度(ppm ) 39 Leaf (fresh) 茎葉 39 Root (fresh) 15 さや 16 Seed pod 16 根 5 Seed 14 種子(＋水) 14 種子( 磨砕＋水) 133 Seed (macerated) 133 15 Table Database for Allelopathic activity evaluated by PB, SW, and DP method 表８今後導入される可能性のある外来植物600種のアレロパシー活性の検定結果（抜粋） 表2.7 今後導入される可能性のある外来植物600種のアレロパシー活性の検定結果（抜粋、データは追加・変更の可能性あり） 学 名 和 名 ｲﾁﾋﾞ ﾔｷﾞﾑｷﾞ 科 名 ｱｵｲ科 ｲﾈ科 ｲﾈ科 ﾋﾒﾇｶﾎﾞ ｲﾈ科 ｲﾄｺﾇｶｸﾞｻ ｲﾈ科 ｲﾈ科 ｲﾈ科 ｲﾈ科 ｲﾈ科 ｲﾈ科 ｲﾈ科 ｲﾈ科 ﾋﾛｰﾄﾞｱｵｲ ｱｵｲ科 ﾋﾕ科 ｲﾇﾋﾞﾕ ﾋﾕ科 ｵｵﾎﾅｶﾞｱｵｹﾞｲﾄｳ ﾋﾕ科 ﾋﾕ科 ﾋﾕ科 ﾋﾕ科 ｾﾘ科 ｲﾈ科 ｲﾈ科 ｱｵｲ科 ｾﾘ科 ｾｲﾖｳﾇｶﾎﾞ ｲﾈ科 ｾﾘ科 ｱｻﾞﾐｹﾞｼ ｹｼ科 ｾﾘ科 ｸﾛｶﾞｸﾓﾒﾝﾂﾞﾙ ﾏﾒ科 ｶﾗｽﾑｷﾞ ｲﾈ科 ｲﾈ科 ｼｸﾞﾅﾙｸﾞﾗｽ ｲﾈ科 ｲﾈ科 ｲﾈ科 ｲﾈ科 ｲﾈ科 ﾁｬﾎﾞﾁｬﾋｷ ｲﾈ科 ｲﾈ科 ｱﾚﾁﾉﾁｬﾋｷ ｲﾈ科 ﾅｶﾞﾐﾉｱﾏﾅｽﾞﾅ ﾂﾊﾞｷ科 ｱﾌﾞﾗﾅ科 ｾﾘ科 ﾔｸﾞﾙﾏｷﾞｸ類 ｾﾘ科 ｾﾘ科 ﾍﾞﾆﾊﾞﾅｾﾝﾌﾞﾘ ﾘﾝﾄﾞｳ科 ﾊﾅﾊﾏｾﾝﾌﾞﾘ ﾘﾝﾄﾞｳ科 ﾑﾗｻｷﾁｮｳﾏﾒﾓﾄﾞｷ ﾏﾒ科 ﾐﾐﾅｸﾞｻ ﾅﾃﾞｼｺ科 ｵﾗﾝﾀﾞﾐﾐﾅｸﾞｻ ﾅﾃﾞｼｺ科 ﾔﾅｷﾞﾗﾝ ｱｶﾊﾞﾅ科 ﾛｰｽﾞｸﾞﾗｽ ｲﾈ科 ｷｸ科 ｾﾘﾆｶﾞﾅ ｾﾘ科 ｻﾝﾄﾘｿｳ ｾﾘ科 ﾄﾓｼﾘｿｳ ｱﾌﾞﾗﾅ科 ﾔｸﾖｳﾄﾓｼﾘｿｳ ｱﾌﾞﾗﾅ科 ﾄﾞｸﾆﾝｼﾞﾝ ｾﾘ科 ﾙﾘﾋｴﾝｿｳ ｷﾝﾎﾟｳｹﾞ科 ｾﾘ科 ｲﾈ科 ﾚﾓﾝｸﾞﾗｽ ｲﾈ科 ﾀﾏｶﾞﾔﾂﾘ ｶﾔﾂﾘｸﾞｻ科 ｶﾔﾂﾘｸﾞｻ科 ｶﾓｶﾞﾔ ｲﾈ科 ﾀﾂﾉﾂﾒｶﾞﾔ ｲﾈ科 ﾖｳｼｭﾁｮｳｾﾝｱｻｶﾞｵ ﾅｽ科 ｺﾒｽｽｷ ｲﾈ科 ﾏﾒ科 ｺﾞﾏﾉﾊｸﾞｻ科 ｲﾈ科 ｲﾈ科 ｱﾌﾞﾗﾅ科 ﾏﾂﾑｼｿｳ科 ﾏﾂﾑｼｿｳ科 ﾏﾂﾑｼｿｳ科 ｲﾈ科 ｲﾈ科 SW法 ＤＰ法 PB法 総合評価 学 名 和 名 31 105 21 1 Echinochloa frumentacea 111 27 1 Echinochloa hispidula 60 119 60 0 Echinochloa oryzzoides ﾉｹﾞﾀｲﾇﾋﾞｴ 44 97 29 0 Echinochloa utilis 36 90 24 1 Echium italicum 42 106 43 0 Echium plantagineum ｼｬｾﾞﾝﾑﾗｻｷ 46 88 43 0 Echium vulgare ｼﾍﾞﾅｶﾞﾑﾗｻｷ 43 100 43 0 Eclipta alba 56 106 37 0 Eclipta erecta 41 134 95 0 Eclipta prostrata 64 91 61 0 Eleusine indica ｼｺｸﾋﾞｴ 31 88 38 0 Emex australis 63 41 109 41 0 Emex spinosa ｲﾇｽｲﾊﾞ 47 106 50 0 Erodium cicutarium 40 107 30 0 Erysimum orientale 25 76 53 1 Eupatorium cannabinum ﾀｲﾜﾝﾋﾖﾄﾞﾘ 31 100 60 0 Euphorbia corollata 42 86 64 0 Euphorbia cyparissias 27 100 31 0 Euphrasia rostkoviana 44 92 15 1 Festuca arundinacea 86 7 2 Festuca ovina 21 96 24 1 Fimbristylis littoralis 50 44 89 64 0 Fimbristylis milliacea 41 59 57 1 Fumaria capreolata ﾆｾｶﾗｸｻｹﾏﾝ 96 34 1 Galium mollugo 24 93 48 0 Geranium molle 56 38 98 24 1 Geranium pratense 34 123 39 0 Geranium pusillum 23 95 35 1 Geranium pyrenaicum 32 101 16 1 Geranium robertianum 95 10 1 Geum rivale 38 88 28 2 24 Geum urbanum 79 29 0 Guizotia abyssinica ｷﾊﾞﾅﾀｶｻﾌﾞﾛｳ 41 115 43 0 Hirschfeldia incana ｱﾚﾁｶﾞﾗｼ 58 85 27 1 Holcus lanatus ｼﾗｹｶﾞﾔ 58 98 18 1 Hyoscyamus niger ﾋﾖｽ 78 105 22 2 20 Hypochaeris glabra 83 63 0 Hypochaeris radicata ﾌﾞﾀﾅ 36 65 20 3 Ipomoea aquatica 23 28 95 22 1 Ipomoea hederacea 26 48 37 2 Ipomoea lacunosa 39 106 57 0 Isatis tinctoria ﾊﾏﾀｲｾｲ 33 83 29 0 Juncus articulatus 40 91 84 0 Juncus bufonius 53 0 60 1 Kichxia commutata 28 11 70 1 Lathyrus latifolius 40 105 46 0 Lathyrus nissolia 20 98 47 1 Lathyrus phaseoloides 55 105 55 0 Legousia speculum veneris ｵｵﾐｿﾞｶｸｼ 17 106 40 1 Leontodon autumnalis 100 47 0 Leontodon hispidus 31 42 95 85 0 Leontodon taraxacoides ｶﾜﾘﾐﾀﾝﾎﾟﾎﾟ 74 91 47 0 Lepidum sativum ｺｼｮｳｿｳ 46 69 48 1 Lespedeza striata 37 20 28 2 Leucanthemum vulgare ﾌﾗﾝｽｷﾞｸ 15 115 28 2 Lolium multiflorum 33 85 25 1 Lolium rigidum 42 61 13 2 Lolium temulentum 39 100 59 0 Lotus corniculatus 39 90 47 0 Lupinus perennis 126 39 0 47 Lychinis viscaria 35 74 75 1 Malva alcea 62 95 77 0 Malva hirsuta 86 32 0 Malva neglecta ｾﾞﾆﾊﾞｱｵｲ 39 107 20 2 Malva parviflora ｳｻｷﾞｱｵｲ 16 33 63 10 2 Marubium vulgare ﾆｶﾞﾊｯｶ 105 82 0 Matricaria discoidea 45 77 94 24 1 Matricaria inodora 44 104 38 0 Matricaria maritima 27 111 58 0 Matricaria perforata 115 82 0 Matricaria recutita 50 22 90 27 2 Matricaria suaveolens 112 83 0 Medicago lupulina 66 54 130 58 0 Medicago sativa 66 116 77 0 Melandrium album ﾏﾂﾖｲｾﾝﾉｳ 99 71 1 26 Melandrium rubrum 90 42 1 Melilotus albus ｼﾛﾊﾞﾅｼﾅｶﾞﾜﾊｷﾞ 23 注１）SW法は葉から溶脱する物質による活性を、DP法は葉から揮発する物質による活性を、PB法は根から滲出する物質による活性を示す。 注２）表注の数字は、検定植物レタスの生育率（％）を表しており、数値が小さいほどアレロパシーによる阻害活性が強いことを示す。 注３）判断基準は、それぞれの活性の全てのデータの平均値から標準偏差を引いた値よりも小さい場合、活性が強いとした。 Abutilon theophrasti Aegilops cylindrica Agropyron repens Agrostis canina Agrostis capillaris Agrostis castellana Agrostis gigantea Agrostis stolonifera Agrostis tenuis Alopecurus geniculatus Alopecurus myosuroides Alopecurus pratensis Althaea officinalis Amaranthus albus Amaranthus lividus Amaranthus palmeri Amaranthus rudis Amaranthus spinosus Amaranthus thumbergii Ammi majus Anisantha madritensis Anisantha rigida Anoda cristata Anthriscus caucalis Apera spica-venti Arctium minus Argemone mexicana Artemesia vulgaris Astragalus danicus Avena fatua Avena sterilis Brachiaria decumbens Bromus arvensis Bromus hordeaceus Bromus lanceolatus Bromus racemosus Bromus rubens Bromus secalinus Bromus sterilis Camelina sativa Cardamine pratensis Cenchrus longispinus Centaurea maculosa Centaurea phrygia Centaurium erythraea Centaurium tenuiflorum Centrosema pubescens Cerastium frontanum Cerastium glomeratum Chamaenerion angustifolium Chloris gayana Chrysanthemum parthenium Cichorium intybus Cnicus benedictus Cochlearia danica Cochlearia officinalis Conium maculatum Consolida orientalis Crepis capillaris Criteson secalinum Cymbopogon citratus Cyperus difformis Cyperus fuscus Dactylis glomerata Dactylocenium aegyptum Datura stramonium Deschampsia flexuosa Desmodium ovalifolium Digitalis purpurea Digitaria adscendens Digitaria ischaemum Diplotaxis muralis Dipsacus fullonum Dipsacus pilosus Dipsacus sylvestris Echinochloa colonum Echinochloa crus galli 科 名 SW法 ＤＰ法 PB法 総合評価 ｲﾈ科 ｲﾈ科 ｲﾈ科 ｲﾈ科 ﾑﾗｻｷ科 ﾑﾗｻｷ科 ﾑﾗｻｷ科 ｾﾘ科 ｾﾘ科 ｾﾘ科 ｲﾈ科 ﾀﾃﾞ科 ﾀﾃﾞ科 ﾌｳﾛｿｳ科 ｱﾌﾞﾗﾅ科 ｾﾘ科 ﾄｳﾀﾞｲｸﾞｻ科 ﾄｳﾀﾞｲｸﾞｻ科 ｺﾞﾏﾉﾊｸﾞｻ科 ｲﾈ科 ｲﾈ科 ｶﾔﾂﾘｸﾞｻ科 ｶﾔﾂﾘｸﾞｻ科 ｹｼ科 ｱｶﾈ科 ﾌｳﾛｿｳ科 ﾌｳﾛｿｳ科 ﾌｳﾛｿｳ科 ﾌｳﾛｿｳ科 ﾌｳﾛｿｳ科 ﾊﾞﾗ科 ﾊﾞﾗ科 ｾﾘ科 ｱﾌﾞﾗﾅ科 ｲﾈ科 ﾅｽ科 ｾﾘ科 ｾﾘ科 ﾋﾙｶﾞｵ科 ﾋﾙｶﾞｵ科 ﾋﾙｶﾞｵ科 ｱﾌﾞﾗﾅ科 ｲｸﾞｻ科 ｲｸﾞｻ科 ｺﾞﾏﾉﾊｸﾞｻ科 ﾏﾒ科 ﾏﾒ科 ﾏﾒ科 ｷｷｮｳ科 ｾﾘ科 ｾﾘ科 ｾﾘ科 ｱﾌﾞﾗﾅ科 ﾌﾄﾓﾓ科 ｾﾘ科 ｲﾈ科 ｲﾈ科 ｲﾈ科 ﾏﾒ科 ﾏﾒ科 ﾅﾃﾞｼｺ科 ｱｵｲ科 ｱｵｲ科 ｱｵｲ科 ｱｵｲ科 ｼｿ科 ｾﾘ科 ｾﾘ科 ｾﾘ科 ｾﾘ科 ｾﾘ科 ｾﾘ科 ﾏﾒ科 ﾏﾒ科 ﾅﾃﾞｼｺ科 ﾅﾃﾞｼｺ科 ﾏﾒ科 38 46 25 22 73 28 39 82 96 77 19 60 57 69 39 37 18 80 63 35 61 47 33 27 39 77 57 72 82 56 73 56 37 17 20 14 81 78 25 45 75 42 54 49 15 23 47 36 42 51 69 50 36 68 35 24 29 50 40 68 48 26 24 21 12 26 63 70 32 65 30 50 45 28 58 37 13 109 109 94 131 89 129 107 124 96 107 135 107 92 131 111 97 64 136 97 110 97 127 100 153 100 77 104 117 89 98 115 102 115 112 100 27 101 88 96 84 109 71 88 111 92 110 77 122 115 96 130 74 94 138 113 84 114 118 18 115 99 108 100 48 88 107 119 110 110 108 98 93 88 91 93 109 61 55 68 30 54 41 43 46 79 62 79 22 54 69 21 16 48 17 39 75 61 29 79 85 30 54 21 38 37 39 33 72 74 65 22 46 47 79 69 57 66 72 25 83 40 59 59 43 40 37 72 61 51 38 27 61 56 81 60 61 36 32 54 81 29 34 83 92 57 47 102 61 94 28 39 28 31 29 0 0 1 1 0 0 0 0 0 0 2 0 0 1 1 0 3 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 2 1 2 0 0 1 0 0 2 0 0 1 1 0 0 0 0 0 1 0 1 0 1 0 0 1 0 0 1 1 2 1 1 0 0 0 0 0 0 1 0 1 0 2 学 名 Melilotus officinalis Melilotus sulcata Mentha arvensis Myosotis arvensis Nicotiana sylvestris Oenanthe crocata Oenothera erythrosepala Onobrychis viciifolia Ononis spinosa Panicum clandestinum Panicum miliaceum Panicum virgatum Papaver dubium Papaver hybridum Paspalum scrobiculatum Pennisetum glaucum Phalaris aquatica Phalaris canariensis Phalaris minor Physalis alkekengi Picris echioides Pimpinella anisum Plantago lanceolata Poa pratensis Poa trivialis Poterium sanguisorba Prunella vulgaris Puccinellia distans Pueraria javanica Ricinus gibsonii Rorippa austriaca Rottboellia exaltata Rubus fruticosus Rumex conglomeratus Rumex crispus Rumex hydrolapathum Rumex patientia Rumex rupestris Rumex sanguineus Rumex stenophyllus Sagina subulata Salvia hormoroides Sanguisorba minor Senecio jacobaea Senecio vulgaris Sesbania exaltata Setaria faberi Setaria macrostachia Setaria verticillata Setaria viridis Sida alba Sida rhombifolia Sida spinosa Sison ammonum Sorghum bicolor Sorghum sudanense Spergularia bocconei Sporobolus cryptandrus Stachys annua Tanacetum vulgare Tephrosia purpurea Trifolium album Trifolium arvense Trifolium campestre Trifolium dubium Trifolium pratense Tripleurospermum maritimum Verbascum nigrum Verbascum thapsus Verbena officinalis Veronica anagalloides Veronica persica Vicia hirsuta Vulpia bromoides Vulpia myuros Xanthium spinosum Xanthium strumarium 和 名 ｾｲﾖｳｴﾋﾞﾗﾊｷﾞ 科 名 ﾏﾒ科 ﾏﾒ科 ｼｿ科 ﾑﾗｻｷ科 ﾅｽ科 ｾﾘ科 ｱｶﾊﾞﾅ科 ﾏﾒ科 ﾊﾘﾓｸｼｭｸ ﾏﾒ科 ｲﾈ科 ｲﾈ科 ｲﾈ科 ﾅｶﾞﾐﾋﾅｹﾞｼ ｹｼ科 ﾄｹﾞﾐｹﾞｼ ｹｼ科 ｲﾈ科 ﾄｳｼﾞﾝﾋﾞｴ ｲﾈ科 ｵﾆｸｻﾖｼ ｲﾈ科 ｶﾅﾘｰｸｻﾖｼ ｲﾈ科 ﾋﾒｶﾅﾘｰｸｻﾖｼ ｲﾈ科 ﾖｵｼｭﾎｳｽﾞｷ ﾅｽ科 ﾊﾘｹﾞｺｳｿﾞﾘﾅ ｾﾘ科 ｱﾆｽ ｾﾘ科 ﾍﾗｵｵﾊﾞｺ ｵｵﾊﾞｺ科 ｲﾈ科 ｲﾈ科 ｵｵｽｽﾞﾒﾉｶﾀﾋﾞﾗ ﾊﾞﾗ科 ｼｿ科 ｱﾚﾁﾀﾁﾄﾞｼﾞｮｳﾂﾅｷﾞ ｲﾈ科 ﾏﾒ科 アカネ科 ﾐﾐｲﾇｶﾞﾗｼ ｱﾌﾞﾗﾅ科 ﾂﾉｱｲｱｼ ｲﾈ科 ﾊﾞﾗ科 ｱﾚﾁｷﾞｼｷﾞｼ ﾀﾃﾞ科 ﾀﾃﾞ科 ﾀﾃﾞ科 ﾀﾃﾞ科 ﾀﾃﾞ科 ﾀﾃﾞ科 ﾀﾃﾞ科 ﾅﾃﾞｼｺ科 ｼｿ科 ｵﾗﾝﾀﾞﾜﾚﾓｺｳ ﾊﾞﾗ科 ｾﾘ科 ﾉﾎﾞﾛｷﾞｸ ｾﾘ科 ﾏﾒ科 ｱｷﾉｴﾉｺﾛｸﾞｻ ｲﾈ科 ｲﾈ科 ｻﾞﾗﾂｷｴﾉｺﾛｸﾞｻ ｲﾈ科 ｴﾉｺﾛｸﾞｻ ｲﾈ科 ｱｵｲ科 ｱｵｲ科 ｱｵｲ科 ｾﾘ科 ｿﾙｶﾞﾑ ｲﾈ科 ｽｰﾀﾞﾝｸﾞﾗｽ ｲﾈ科 ｳｼｵﾊﾅﾂﾒｸｻ ﾅﾃﾞｼｺ科 ｲﾈ科 ｼｿ科 ﾖﾓｷﾞｷﾞｸ ｾﾘ科 ﾏﾒ科 ﾏﾒ科 ﾏﾒ科 ﾏﾒ科 ｺﾒﾂﾌﾞﾂﾒｸｻ ﾏﾒ科 ｱｶﾂﾒｸｻ ﾏﾒ科 ｾﾘ科 ｺﾞﾏﾉﾊｸﾞｻ科 ﾋﾞﾛｰﾄﾞﾓｳｽﾞｲｶ ｺﾞﾏﾉﾊｸﾞｻ科 ｸﾏﾂﾂﾞﾗ科 ｺﾞﾏﾉﾊｸﾞｻ科 ｺﾞﾏﾉﾊｸﾞｻ科 ｽｽﾞﾒﾉｴﾝﾄﾞｳ ﾏﾒ科 ｲﾈ科 ﾅｷﾞﾅﾀｶﾞﾔ ｲﾈ科 ｾﾘ科 ｾﾘ科 SW法 ＤＰ法 PB法 総合評価 2 1 32 64 71 25 65 64 45 39 58 45 44 17 40 36 49 50 26 60 54 25 76 53 32 63 76 15 38 45 16 63 24 73 72 79 84 76 71 55 31 59 70 42 37 16 24 25 28 19 9 35 58 72 21 38 36 51 32 38 22 29 51 59 52 56 59 72 69 77 52 39 29 46 18 21 42 62 37 75 88 100 95 113 105 68 95 81 104 98 104 112 113 42 94 86 88 109 100 101 122 119 89 86 20 122 102 78 108 90 92 147 95 96 103 128 123 81 103 83 141 103 99 124 99 71 86 114 100 121 114 80 30 99 122 106 80 133 110 121 119 82 110 98 126 101 87 148 117 77 81 81 104 120 19 30 97 37 40 93 47 49 32 39 31 62 25 17 65 30 26 34 39 40 46 56 75 55 34 47 91 64 33 75 53 35 67 85 70 75 60 78 82 70 53 23 70 43 77 45 23 37 29 26 51 71 46 56 21 18 44 30 54 93 35 15 48 42 21 65 70 43 24 82 97 46 3 46 30 64 79 3 2 1 0 0 1 0 0 1 0 0 0 1 2 0 0 2 0 1 0 0 1 0 0 0 0 0 2 0 0 1 0 1 0 0 0 0 0 0 0 0 1 0 0 0 1 2 1 1 2 1 0 0 0 2 2 0 0 0 0 1 1 0 0 1 0 0 0 1 0 0 0 1 0 1 1 0 Evaluation of Allelopathy by specific bioassays and evaluated about 4,000 species in 20 years 1) Medicinal Plants 2) Tropical Plants are Allelopathic 3) Endangered Plants 4) Invasive Alien Plants 5) Oats, Barley, Wheat (My dream) Database for Allelopathic activity evaluated by PB, SW, DP method 16 2) Isolation of Allelochemicals based on Total Activity 17 Chromatography (TLC, HPLC …) HO OH H S S H O Zeylanoxide A 試料添加 薄層クロマトグラフィー用 シリカゲルプレート C さらに展開 溶媒（油）で展開 さらに展開 良好な分離！ H H H H HO C OH H S S H O epi-Zeylanoxide A Structure GC-MS, MS, NMR, IR, ESR, X-ray analysis, etc Solvent Extraction Conventional Method for Isolation of natural chemicals 18 New strategies for isolation of allelochemicals in agriculture Comparison of Total Activity and Specific Activity Target Compounds Direct use Allelochemicals of Allelopathy in action in situ Index (g-weight base) Total Activity = 1/EC50×Concentration in Plants 1/（kg/L） x (kg/L in fresh weight) =(L/kg) x (kg/L) = (no unit) high value ～ high activity Use of Natural Bioactive Chemicals Chemicals with high specific activity Specific Activity = EC50 (50% inhibitory Concentration) (kg/L)～ppm low ppm ～ high activity 19 Isolation of L-DOPA from Velvet bean Endemic plant to Himalayas Distributed in Sino-Japan Area Used as food in Japan from more than 1000 years ago Total activity (but now disappearing) = 200 Fujii Y, Shibuya T. and Yasuda T. L-3,4-dihydroxyphenylalanine as an allelochemical candidate from Mucuna pruriens (L.) DC. var. utilis. Agricultural and Biological Chemistry 55, 617-618. (1991) 20 Isolation of cyanamide from hairy vetch Cyanamide was already known as synthetic fertilizer, but never isolated as natural chemical Endemic plant to central Asia, now we are recommending to Japanese farmers for weed control Total activity = 35 - 100 Kamo T, Hiradate S. and Fujii Y. First isolation of natural cyanamide as a possible allelochemical from hairy vetch Vicia villosa.，J. Chem. Ecol.，29 (2), 275-284 (2003) 21 3) Practical use of Allelopathic Cover Crops 22 Mucuna (velvetbean) (Mucuna pruriens) for Green manure, Food, Medicinal plants, and Cover Crop Fujii, Y. Allelopathy of Velvetbean: Determination and Identification of Allelopathic Substances. H. G. Cutler and S. Cutler (Eds), Biologically Active Natural Products: Agrochemicals, CRC Press, pp.33-47 (1999) 23 Ｖｅｌｖｅｔbean : Mucuna pruriens var. utilis High production of forage , Legume, rich in protein 24 Table 1. Assessment of Allelopathic Activity of Leguminous Cover Plants by Plant Box Method Scientific name (English name) Mucuna pruriens var.utilis (Mucuna) Vicia faba (Broad Bean) Vicia villosa (Hairy Vetch) Calopogonium mucunoides Melilotus albus (White Sweet Clover) Vicia sativa (Common Vetch) Medicago rugosa Canavalia ensiformis (Jack Bean) Pueraria phaseoloides (Tropical Kudzu) Vigna angularis (Azuki bean) Medicago sativa (Alfalfa) Trifolium incarnatum (Crimson Clover) Tephrosia candida (White Tephrosia) Cajanus cajan (Pigeon Pea) Latyrus sativus (Grass Pea) Cicer arietinum (Chickpea) Vigna radiata (Mung Bean) Stylosanthes hamata (Stylo) Arachis hypogaea (Peanut) Trifolium pratense (Red Clover) Crotaralia juncea (Sunn Hemp) Astragalus sinicus (Chinese Milk Vetch) Lupinus albus (White Lupine) Trifolium subterraneum (Sub Clover) Trifolium repens (White Clover) Glycine max (Soybean) [%]*1 87 81 80 78 77 75 74 72 71 69 68 64 63 62 59 56 53 52 51 47 43 41 40 30 28 24 Criteria*2 ★★★ ★★ ★★ ★★ ★★ ★★ ★★ ★★ ★★ ★ ★ ★ ★ ★ ★ ★ Plant Box Method (for root exudates) *1 Inhibition (%) means allelopathic activity measured by the Plant Box Test. All data are compared to the control and 100 means complete inhibition. *2 Criteria for the allelopathic activity are ; ★★★ ; >85 %, ★★ ; 70 to 84% , ★ :<69 %, respectively. 25 Table Weed population in continuous cropping fields ────────────────────────────────────── Crop Treatment Weed population Weed species observed 6) (g dry weight per m2) ────────────────────────────────────── Upland Rice 3yr.c 1) 5.11(49.4) 4) 1), 3), 5), 6), 7), 8), 9), 10), 11) 5) Egg plant 3yr.c 16.82(40.1) 1), 2), 3), 5), 6), 7), 8), 9), 10), 11), 12), Tomato 3yr.c 4.92(64.9) 1), 5), 6), 9), 12), 13), 17) Velvetbean 2yr.c 0.00( 0.0) no emergence Fallow 3yr.f 3) 0.97(37.3) 13), 14) 1), 2), 6), 10), 12), 13), 15), 16) ────────────────────────────────────── 1) Continuous cropping for 3 years. After Velvetbean, little weed ! 2) Cultivated for 1 year, followed by fallow next year(test year). 3) Fallow for 3 years, without fertilizer. 4) Numbers in parenthesis are percentages of chickweed, a dominat species. 5) Species appeared in each plot: 1) Sticky chickweed (Cerastium glomeratum), 2) 'Miminagusa' (Cerastium vulgatum var. augustifolium), 3) Annual fleabane (Erigeron annuus), 4) Philadelphia fleabane (Erigeron philadelphicus), 5) Starwort (Stellaria alsine var. undulata), geniculatus), 6) Floating foxtail (Alopecurus 7) Narrowleaf vetch (Vicia angustifolia), (Cardamine flexuosa), 8) Flexuosa bittercress 9) 'Inugarashi' (Rorippa atrovirens), 10) Common dandelion (Taraxacum officinale), 11) Japanese mugwort (Artemisia princeps), 12) Danadian fleabane (Erigeron canadensis), 13) 'Hahakogusa' (Gnaphalium affine), 14) Blady grass (Imperata cylindrica), 15) Meadowgrass (Poa annua), 16) Creeping woodsorrel (Oxalis corniculata), 17) Shepherd's-purse (Capsella bursa-pastoris), 18) Prickly sowthistle (Sonchus asper). Fujii, Y. Shibuya, T. and Usami, Y. Allelopathic effect of Mucuna pruriens on the Source: Fujii, Y. Shibuya, T. and Usami, Y. (1991): Allelopathic effect of Mucuna pruriens appearance ofof weeds. weeds, Weed on the appearance Weed Res. 36, 43-49 Res, Japan 36, 43-49 (1991) 26 Allelochemical in Mucuna (Velvetbean) L-3,4-Dihydroxyphenylalanine (L-DOPA) HO HO COOH NH2 Extremely high concentration in Mucuna pruriens leaves, roots (0.5 to 1.5%) and seeds (5 to 8%) in fresh wt. Relatively strong inhibition to broadleaf weeds (EC50=5-50ppm), but not so active to grass (monocot.) Fujii, Y., T. Shibuya, and T. Yasuda, L-3,4-Dihydroxyphenylalanine as an allelochemical candidate from Mucuna pruriens(L.) DC.var.utilis. Agric.Biol.Chem. 55(2), 617-618 (1991) 27 How to use Mucuna 1) Green Manure 2) Vegetable (Young pod) 3) Seeds as food, but after proper Cooking. 4) Medicine, as a source of L-DOPA 28 Hairy vetch (Vicia villosa) for Green manure and Weed control Fujii, : Screening and Future Exploitation of Allelopathic Plants as Alternative Herbicides with Special Reference to Hairy Vetch. R. K. Kohli, H. P. Singh, and D. R. Batish (Eds), Allelopathy in Agroecosystems, pp.257-275 (2001) 29 First observation of strong weed suppression on the field, August, 1990 30 Table Field Effect of Cover Crops on Weed Control in Abandoned Paddy ; First Trial on the Experimental Station (1992-1993) Cover Crop (English Name) First Sampling (May 7, 1993) Control(No-weeding) Astragalus sinicus（Chinese Milk Vetch) Vicia villosa (Hairy Vetch) A. sativa＋V. villosa（Oat and Hairy Vetch Mix.) Second Sampling (June 10, 1993) Control (No-weeding) Astragalus sinicus（Chinese Milk Vetch) Vicia villosa (Hairy Vetch) A. sativa＋V. villosa（Oat and Hairy Vetch mix.) W (%)*1 0 a *2 82 b 99 b 99 b 0a 59 b 100 c 100 c Crop Yield [g m-2] ---431 a 584 ab 730 b ---135 a 147 b 137 a *1 Percentage of weed control. 100 % means complete control. Dry weight of weeds in the control plot are 281 (First) and 155 (Second) g m-2. Fujii, Y. Screening and future exploitation of allelopathic plants as alternative herbicides with special reference to hairy vetch，J. Crop Prod.，4 (2) , 257-275 (2001) 31 Recommendation of Hairy Vetch In Newspaper, 1994 (Japan Agricultural News) 32 Orange Farm Mr.Ochi （Ehime Pref.) １９９１年 33 34 34 Identification of Cyanamide as Allelochemical from Hairy vetch ★ Cyanamide H N C N H ★ known as active constituents of synthetic fertilizer (Calcium cyanamide) First finding from plants Kamo, T., Hiradate, S. andproducs) Fujii, Y.: First isolation of natural cyanamide as (as natural a possible allelochemical from hairy vetch Vicia villosa . J. Chemical Ecol. 29 (2), 275-283 (2003) 35 36 Hairy Vetch used as cover crop in Japan more than10,000 ha (estimated from 300 tons seed) No.2 as cover crop …after 18 years 37 Other Promising Allelopathic Cover Plants 38 39 Ground Cover Plants with Allelopathic activity Perfume, Medicinal chemicals Resistant for insects, disease & weeds Honey Beautiful flowers O2 ↑ Useful as Forage Prevent Erosion Suppress weeds N2 fixation Food (Fruit, Seed, Tuber...) Clean polluted soil by toxic chemicals Cultivate soil by root system Keep soil moisture Green manure by Nitrogen fixation and organic carbon Resistant for nematoda, and soil disease Phyto-Lark : Ground Cover plants with allelopathy 40 Three key messages • Specific bioassays for Allelopathy, and evaluated about 4,000 plants. • New Allelochemicals Isolated by new methodology, “Total Activity Method” • Ground Cover Plants with allelopathiy (= Phytolark ) provides natural and environmentally friendly way of managing weeds without herbicides. 41 Questions? Contact details [email protected] 42
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