@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Land and Food Systems, Faculty of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Fletcher, Ronald Austin"@en ; dcterms:issued "2011-11-28T18:47:52Z"@en, "1961"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """The knapweeds were introduced into North America in the nineteenth century. They have now established themselves and are spreading rapidly. In British Columbia’s dry interior they grow in dense infestations, tending to exclude as well as out-compete other plants. Because of this characteristic they now present a serious problem. It has been suspected that toxic substances produced by the knapweeds may be partially responsible for enabling them to establish themselves at the expense of other plants. The object of this study, therefore, was to establish the presence and location of inhibitory substances in three species of knapweed: Centaurea repens, Centaurea diffusa, and Centaurea maculosa; and if possible to identify the chemical structure of the compounds responsible. In order to determine whether inhibitors might be present, tomatoes were grown in both infested and non infested soil. Also, dried leaf powder from the knapweed was added to greenhouse soil to observe the effects it might have on the growth of tomatoes. Leaf, stem, root and seed of the knapweed were extracted separately with different solvents: ether, water and ethanol. Germination tests, using lettuce, barley and cress as test crops, were used as methods of bioassay. In order to isolate the inhibitory compound, ascending paper chromatography was employed using the aqueous phase of n-Butanol:acetic acid:water (4:1:5) as the solvent. The inhibitor was located on the chromatographic paper by means of germination tests. Chromogenic sprays and comparisons of Rf values with different solvents were made in order to identify the inhibitor. To further characterize the compound an ultraviolet spectrum was carried out. The findings of the soil studies indicated that knapweed infested soil, and soil containing leaf powder of knapweed, were inhibitory to the growth of tomatoes. The inhibitor was found to be present in all the three species studied, and was located mainly in the leaves. It proved to be both ether and water soluble, and non-specific, being inhibitory to all the crops tested. The inhibitor was found as a pale yellow fluorescent band at an Rf of 0.9. Chromogenic sprays indicated the inhibitor to be an indole derivative. This was confirmed by the ultraviolet absorption spectrum."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/39298?expand=metadata"@en ; skos:note "A GROWTH INHIBITOR FOUND IN CENTAUREA SPP. by Ronald Austin Fletcher B. Sc. (Hons.) Agr., Delhi University, 199+ A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE i n the D i v i s i o n of PLANT SCIENCE We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1961 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ^ > a /LtJt ) The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. Date Mto I,*' ,0tf i i ABSTRACT The knapweeds were introduced into North America i n the nineteenth century. They have now established themselves and are spreading rapi d l y . In B r i t i s h Columbia 1s dry i n t e r i o r they grow i n dense inf e s t a t i o n s , tending to exclude as well as out-compete other plants. Because of t h i s c h a r a c t e r i s t i c they now present a serious problem. It has been suspected that toxic substances produced by the knapweeds may be p a r t i a l l y respon-s i b l e for enabling them to establish themselves at the expense of other plants. The object of t h i s study, therefore, was to e s t a b l i s h the presence.and location of i n h i b i t o r y substances i n three species of knapweed: Centaurea repens, Centaurea d i f f u s a , and Ceritaurea maculosa; and i f possible to Identify the chemical structure of the compounds responsible. In order to determine whether i n h i b i t o r s might be pre-sent, tomatoes were grown i n both infested and non infested s o i l . Also, dried leaf powder from the knapweed was added to greenhouse s o i l to observe the effects i t might have on the growth of tomatoes. Leaf, stem, root and seed of the knapweed were extracted separately with different solvents: ether, water and ethanol. Germination t e s t s , using lettuce, barley and cress as test crops, were used as methods of bioassay. In order to i s o l a t e the i n h i b i t o r y compound', ascending paper chromatography was employed using the aqueous phase of n-Butanol:acetic acid:water (k:1:5) as the solvent. The i n h i -i i i b l t o r was located on the chromatographic paper by means of germination t e s t s . Chromogenic sprays and comparisons of Rf values with d i f f e r e n t solvents were made i n order to i d e n t i f y the i n h i b i t o r . To further characterize the compound an u l t r a -v i o l e t spectrum was carried out. The findings of the s o i l studies indicated that knapweed infested s o i l , and s o i l containing leaf powder of knapweed, were i n h i b i t o r y to the growth of tomatoes. The i n h i b i t o r was found to be present i n a l l the three species studied, and was located mainly i n the leaves. It proved to be both ether and water soluble, and non-specific, being i n h i b i t o r y to a l l the crops tested. The i n h i b i t o r was found as a pale yellow f l u o r -escent band at an Rf of 0.9. Chromogenic sprays indicated the i n h i b i t o r to be an indole derivative. This was confirmed by the u l t r a v i o l e t absorption spectrum. i v TABLE OF CONTENTS Page I. INTRODUCTION 1 I I . LITERATURE REVIEW 2 Nature and structure of i n h i b i t o r s 9 Recent work on i d e n t i f i c a t i o n of in h i b i t o r y compounds 10 Properties of i n h i b i t o r s 12 Methods of extraction and i s o l a t i o n Ik Bioassay methods 15 I d e n t i f i c a t i o n 17 Mode of action and significance 18 I I I . .MATERIALS AND METHODS 23 S o i l studies 2k Proximate analysis 2k Extraction 25 Bioassays 25 I s o l a t i o n 27 Germination tests with eluates 30 C r y s t a l l i z a t i o n 30 I d e n t i f i c a t i o n of the i n h i b i t o r 30 IV. RESULTS 32 V. DISCUSSION 66 VI. CONCLUSION 69 VII. BIBLIOGRAPHY 71 V LIST OF TABLES Table Page 1. Comparative study of growth of tomatoes and barley on infested and non Infested s o i l 33 2. E f f e c t of C. repens leaves on the growth of tomato plants 35 3. Chemical analysis of three Centaurea spp. 37 4-. E f f e c t of C. repens ether extracts on crop seedlings ~~ 39 5. E f f e c t of C. d i f f u s a ether extracts on crop seedlings 4-0 6. E f f e c t of C. maculosa ether extracts on crop seedlings 4-1 7. E f f e c t of C. repens water extracts on crop seedlings ~\" 4-3 8. E f f e c t of C. d i f f u s a water extracts on crop seedlings 44-9. E f f e c t of C. maculosa water extracts on crop seedlings 4-5 10. Fractionation of the ether extract of leaves of C. repens 4-7 11. E f f e c t of ethanol extract of C. repens leaves on growth of two crop seedlings 4-9 12. Comparative study of the toxic effects of^the ether extract of three knapweeds 50 13. Germination percentage of C. repens seeds 51 14-. Location of i n h i b i t o r on the chromatogram by e l u t i o n and lettuce germination: C. repens 54-15. Location of i n h i b i t o r on the chromatogram by e l u t i o n and lettuce germination: C. d i f f u s a 55 16. Location of i n h i b i t o r on the chromatogram by e l u t i o n and lettuce germination: C. maculosa 56 v i Table Page 17. Location of the i n h i b i t o r on the chromatogram by spot germination: C. re-pens 57 18. Location of the i n h i b i t o r on the chromatogram by spot germination: C. d i f f u s a 58 19. Location of the i n h i b i t o r on the chromatogram by spot germination: C. maculosa 59 20. The i n h i b i t o r y band - color reactions with chromogenic sprays 62 21. Ef values of the i n h i b i t o r y compound of Centaurea spp. i n d i f f e r e n t solvents 65 LIST OF ILLUSTRATIONS Figure 1. Morphological i l l u s t r a t i o n s of the three species of knapweed 2. Comparative study of growth of tomato seedling i n knapweed infested and non infested s o i l s 3. E f f e c t of the addition of powdered leaves of C. repens on the growth of tomato plants i n s o i l s k. Response of lettuce seedlings to the ether extracts of the three knapweeds 5. Response of lettuce seedlings to the water extracts/of the three knapweeds 6. Chromatographic resolution of crude ether extract of leaves of the three species of knapweed as observed under u l t r a v i o l e t l i g h t 7. Spot germination tests conducted d i r e c t l y on the successive Rf regions of the developed chromatogram using lettuce seeds 8. U l t r a v i o l e t absorption spectrum of the crude i n h i b i t o r y compound i s o l a t e d from C. repens v i i i ACKNOWLEDGEMENTS The writer i s indebted to a l l the members of his graduate committee fo r t h e i r interest and valuable advice. In p a r t i c u -l a r he wishes to thank Dr. A. J . Renney, of the D i v i s i o n of Plant Science for his deep i n t e r e s t , guidance and constant en-couragement at every step. In addition, the writer wishes to thank Mr. G. J . Brussel, laboratory technician who conducted the chemical analyses of the knapweeds. Thanks are also due to Dr. J . P. Kutney of the Department of Chemistry f o r his help i n interpreting the absorp-t i o n spectrum of the i n h i b i t o r . The writer i s grateful f o r the grants received from the Dominion Department of Agriculture (Extra Mural Research), and the President's Committee on Research, University of B r i t i s h Columbia. These grants made t h i s study possible. A GROWTH INHIBITOR FOUND IN CENTAUREA SPP. I. INTRODUCTION The presence of knapweeds, Centaurea spp., i n North America was f i r s t reported during the nineteenth century. Since then they have established themselves i n many parts of the United States. Three knapweed species now present a serious problem i n the dry i n t e r i o r parts of B r i t i s h Columbia. In t h e i r natural habitats these knapweeds grow i n dense i n f e s t a t i o n s , crowding out other plants, and some workers think that toxic plant products may be partly res-ponsible f o r t h i s condition. While some other weeds are known to produce materials which i n h i b i t the growth of associated species, the production of to x i c materials by knapweeds has not been reported so f a r . The object of t h i s study was therefore to i n v e s t i -gate the p o s s i b i l i t y of i n h i b i t o r y substances being pre-sent i n Centaurea spp., to esta b l i s h i n which part of the plant these substances were located, and i f possible to determine t h e i r nature. 2 II. LITERATURE REVIEW Russian knapweed (Centaurea repens L.) i s a perennial which reproduces by seeds and creeping roots. This weed, a native of southern Russia and central A s i a , has reached serious proportions i n North America, where i t seems to have been introduced i n Turkestan a l f a l f a seed (9k). The deeply penetrating perennial root system forms dense i n -festations that enable Russian knapweed to suppress cereals and many other annual f i e l d crops ( 71 ) . The extensive root system has been described as consisting of the o r i g i n a l root, one to many l a t e r a l roots and t h e i r v e r t i c a l exten-sions ( 2 7 ) . Diffuse knapweed (Centaurea d i f f u s a Lam.) i s a b i e n n i a l or short l i v e d perennial. This weed i s at present found only i n the north western United States and B r i t i s h Columbia. It grows well i n the dry i n t e r i o r of t h i s province, establishing quickly on open disturbed s o i l , and i t i s the knapweed causing the greatest l o c a l concern at the present moment (70 ) . Spotted knapweed (Centaurea maculosa Lam.), l i k e the d i f f u s e , i s a biennial or short l i v e d perennial also found i n the southern i n t e r i o r of B r i t i s h Columbia. Although at present i t i s found only i n waste places, east of the main diffuse area, i t i s feared that i t may become a serious problem i n B r i t i s h Columbia because i t has spread r a p i d l y within the l a s t few years. The knapweeds belong to a group of the Compositae which have d i s t i n c t i v e bracts. The flower heads of Russian knap-weed have entire bracts, while the bracts i n the other species of knapweed have fringed or torn t i p s . In d i f f u s e knapweed the bracts end i n a r i g i d spreading spine, whereas i n spotted knapweed the bracts lack these long spines and always have a terminal blackish fringe (Figure 1). The seeds i n Russian knapweed have a scar at the base and the pappus i s longer than the seed. In the other species the seed hasa'inotch at one side and the pappus i s much shorter than the seed. The blackish scaly roots of Russian knapweed are also d i s t i n c t i v e (9*0. Russian knapweed i s known to be poisonous to sheep and horses. A worker i n South A f r i c a has reported that a f u l l grown sheep died eighteen hours a f t e r administration of just over h a l f a pound of dried Russian knapweed, and a Russian text claims that horses have been poisoned by t h i s weed (9*+). However, animals tend to avoid a l l three knapweeds, where they have any choice of forage. There i s a tendency f o r these three Centaurea spp. to form dense infestations and to suppress the growth of other * species. Based on t h i s observation i n i t i a l work on Centaurea repens i n B r i t i s h Columbia has demonstrated that Russian knapweed infested s o i l has i n h i b i t o r y effects on tomato plants and that water extracts of stem and leaf have a strong i n h i -Figure 1. Morphological i l l u s t r a t i o n s of the three species of knapweed. 5 bitory effect on the germination of turnips ( 6 9 ) . A great deal of confusion exists i n the terminology used i n connection with i n h i b i t o r s . Tukey (9©) defines plant regulators as organic compounds, other than n u t r i -ents, which i n small amounts promote, i n h i b i t or otherwise modify any p h y s i o l o g i c a l process i n plants. This was one of the d e f i n i t i o n s accepted by the Executive Committee of the American Society of Plant Physiologists i n 1953. A minority report by Larsen (51) objected to the word \"regu-l a t o r \" , and made a counter proposal which defined plant growth substances as organic compounds which at low concen-t r a t i o n s promote, i n h i b i t , or q u a l i t a t i v e l y modify growth. Larsen further defined 1 \"growth i n h i b i t o r s \" as substances which retard growth both i n shoot and root c e l l s and have no stimulatory range of concentration. He defines a n t i -auxins as growth substances which competitively i n h i b i t the action of auxins. Evenari ( 2 3 ) defines germination i n h i b i t o r s as substances produced by plants or substances of r e l a t e d structure not found i n plants which i n h i b i t or delay the ger-mination of seeds of the same or other species. The theory that i n h i b i t o r y substances may be deposited i n the s o i l by c e r t a i n plants and may influence the growth of those plants or other species was postulated as early as 1832. DeCandolle (20) showed that s p e c i f i c t o x i c substances produced by Euphorbia sp. and Cnicus sp. were i n h i b i t o r y to f l a x and oats respectively. At that time his theory was not accepted, 6 as the i n h i b i t o r y effects were attributed to competition f o r inorganic nutrients. At the turn of the century, however, interest i n the p o s s i b i l i t y of toxic substances being pro-duced by plants was reawakened. Early investigators, l i k e Schreiner and h i s co-workers (73)» succeeded i n i s o l a t i n g and i d e n t i f y i n g t o x i c substances from c e r t a i n s o i l s . That these substances had t h e i r o r i g i n i n plants was not established. The work of Pickering (63) showed that grass exerts a d i r e c t l y poisonous action on apple trees. I t has also been suggested that the toxin produced by brome grass was to x i c to a l l species, and that other grasses which were sown where brome grass had been sown were pale green and lacking i n vigour ( 6 0 ) . Funke (28) stated that eighteen species of plants which were sown beside a hedge of Artemisia absinthium were severely injured and that one species, Leviticum o f f i -c i n a l e , was even k i l l e d by the chemical excretion. The pre-sence of i n h i b i t o r s i n desert plants has been indicated by Bennet (7). The work of Tourneau (88) showed that extracts from twenty-four species of plants had germination reducing capacity. He also stated that extracts of various weeds and crop plants i n h i b i t germination and growth, and crop extracts appear to be as i n h i b i t o r y as extracts of weed species. It was proved by Patrick (62) that substances capable of markedly i n h i b i t i n g the r e s p i r a t i o n , germination and growth of tobacco seedlings were obtained a f t e r residues from timothy, corn, rye 7 or tobacco had been allowed to decompose in the s o i l ( 6 2 ) . Leopold (52) stated that a great many inhibitors of germin-ation and growth have been found to be present in various plant parts. The presence of inhibitors in different plant parts has also been indicated by other workers. That i n -hibitors are present in leaves has been shown for peas ( 7 9 ) , radish ( 8 0 ) , Encelia farinosa ( 3 3 ) , cocklebur ( 1 2 ) , apple ( 5 6 ) , grapes (5) and Ailanthus ( 2 6 ) . That inhibitory substances exist in seeds has been demonstrated for sugar beets ( 8 6 ) , cabbage ( 1 8 ) , red kidney beans ( 7 6 ) , apple seeds (54-), Kochia indica ( 7 5 ) , wild oats ( 2 1 ) , Betula sp. (11), lettuce (64-), Viburnum trilobum (4-5), maize ( 3 9 ) , and peach seeds (24-). That roots contain inhibitory materials or secrete sub-stances toxic to plants has been claimed for peaches ( 6 7 ) , brome grass ( 6 ) , guayule (14-), red beet ( 6 8 ) , peas (4-3), and Vicia faba (4-9). Inhibitors are present not only in the leaves, seeds and roots but evidence given by other workers indicates the presence of inhibitors in other plant parts as well. Hemberg (36) found a large amount of inhibiting sub-stances in extracts of the peripheral and central buds of Fraxinus. while Hendershot (4-1) extracted an inhibitory sub-stance from peach flower buds. It was also proved by Hemberg (38) that inhibiting substances which are found in potato 8 tubers occurred c h i e f l y i n the peel layer. Evenari (23) established that germination i n h i b i t o r s are found i n many di f f e r e n t species i n most of the d i f f e r e n t f a m i l i e s of the plant kingdom. He also pointed out that i n h i b i t o r s are not confined to seeds or f r u i t but occur i n various parts of plants according to the species: l i k e the f r u i t pulp, f r u i t coats, endosperm, seed coat, embryo, leaves, bulbs and roots. Bentley stated that i t i s now an established fact that n a t u r a l l y occurring i n h i b i t o r s are obviously very widespread ( 9 ) . In a chapter on natural plant growth i n h i -b i t o r s Audus (3) records that there can be l i t t l e doubt from accumulated evidence that roots and underground stems of a very wide range of plant species produce s p e c i f i c chemical compounds that are active i n h i b i t o r s of seed germination and plant root growth. From the array of l i t e r a t u r e c i t e d i t i s obvious that i n h i b i t o r s are very widely occurring, e x i s t i n g as they do i n d i f f e r e n t plant parts of many species. Evenari's review ( 2 3 ) , moreover, states that these i n h i b i t i n g effects are not r e s t r i c t e d to phanerogamous plants. Spores and gemmae of cryptograms show an i n h i b i t e d germination very similar to that of tomato seeds inside the f r u i t , e.g. the gemmae of Marchantia do not sprout inside t h e i r cupules, and spores of Funaria hygrometrlca do not germinate within t h e i r spor-angia. 9 It has been shown that i n h i b i t i n g substances are formed by algae as well (kh). In experiments with Arter-ione11a formosa and Nitzschla palea i t was found that both species formed substances which i n h i b i t e d the growth of other species. Nature and structure of i n h i b i t o r s The pioneer work i n investigation of the chemical structure of t o x i c substances was done by Schreiner et a l . (72). The toxic p r i n c i p l e s found were: picolonic acid, salicylaldehyde, v a n i l l i n and dihydroxystearic acid. Unfor-tunately t h i s work did not prove that these substances had t h e i r o r i g i n i n plants. Schreiner (72) conducted an exten-sive study with a number of compounds which occur n a t u r a l l y i n plants and showed that most of them had i n h i b i t o r y e f f e c t s , e s p e c i a l l y on the roots of seedlings. The toxic effects of sugar beet seed b a l l s has been shown to be due to free ammonia which was hydrolyzed from the organic nitrogen compounds of the seed b a l l during the process of germination (8l). Work done by Evenari (22) established that the i n h i b i -t o r of tomato juice was a non-colloidal substance, and he suggested that i t could be one of three types: e s s e n t i a l o i l s , a l kaloids and glucosides. Of the two substances i s o l a t e d from guayule plants, one was i d e n t i f i e d to be cinnamic acid; the second was an organic a c i d which remained u n i d e n t i f i e d (1*+). It has been stated that many of the i n h i b i t i n g com-10 pounds are normal plant metabolites, e.g. tryptophane and tryptamine produce c e r t a i n physiological actions on plants s i m i l a r to auxins ( 2 ) . The structure of the toxic compound is o l a t e d from leaves of Encelia farinosa was shown to be 3-acetyl-6?methoxybenzaldehyde (34-). One of the blue f l u o r -escing i n h i b i t o r y compounds i n oat roots was reported to be scopoletin ( 3 2 ) . In a review by Evenari (23) the occurrence of a large number of chemicals including: hydrogen cyanide, ammonia, ethylene, mustard o i l s , organic acids, unsaturated lactones, aldehydes, essential o i l s and a l k a l o i d s , which exhibit i n h i b i t o r y properties i s discussed. Recent work on i d e n t i f i c a t i o n of i n h i b i t o r y compounds Bentley (9) thinks that l i t t l e further advance can be made i n the f i e l d of i n h i b i t o r s u n t i l the various n a t u r a l l y occurring compounds have been chemically i d e n t i f i e d and made available f o r physiological studies. Realizing the impor-tance of t h i s several workers i n the past decade have made useful contributions by establishing the single chemicals involved i n plant i n h i b i t i o n . Many investigators have attempted to i d e n t i f y the i n h i b i t o r of the potato tuber. It was suggested that i t .may be trans-cinnamic a c i d (17), or acid i n h i b i t o r B (4-0). Housley (4-2) thought that the i n h i b i t i o n s could not be asso-ciated with any p a r t i c u l a r compound but were due to a 11 complex mixture of a l i p h a t i c acids. He i s o l a t e d a z e l a i c acid and the coumarin, scopoletin, together with an unknown aci d , probably an unsaturated polyhydroxy f a t t y acid. The most active compound present as an i n h i b i t o r i n T r i g o n e l l a arabica was i d e n t i f i e d as coumarin ( 5 3 ) . The compounds i n Thomnosma montana were reported as furocoumarins ( 7 ) , assigning them empirical formulae of C\"i D H15 0£ (OGH3) and C-j^ H^ O3 (OCH^^ , and an i n h i b i t o r y unsaturated keto lactone Xanthinin (C^H^s O3) was i s o l a t e d from the leaves of Xanthium ( 3 0 ) . Koves (hQ) i d e n t i f i e d i n h i b i t o r s which he found present i n twenty-four species of dry f r u i t s . He demonstrated the presence of p-oxybenzoie acid, p-coumaric ac i d and f e r u l i c a c i d, as well as high molecular weight tannic acids, prote-catechuic, c a f f e i c , chlorogenic and s a l i c y l i c acids. Besides the i d e n t i f i e d compounds, the presence of some other i n h i b i t o r s was also noted. These were presumed to be chemically related to the substances demonstrated. A l l the i n h i b i t o r s i d e n t i f i e d from the dry f r u i t s were phenolic acids. The i n h i b i t o r i n Pisum sativum roots was reported as a phenol ( 8 7 ) , while trans cinnamic acid was found to antagon-i z e the growth promoting effect of auxins i n peas ( 9 3 ) . A fa t soluble long chain saturated keto-alcohol with forty-four carbons has been extracted from plants i n the flowering stage ( 8 2 ) . This substance i n h i b i t s the effect of indoleacetic a.-acid (IAA) and has been referred to as an antiauxin. Organic acids were found to be present i n the zone of i n h i b i t i o n 12 around peach seeds (24-), while an unsaturated yellow o i l was responsible f o r i n h i b i t i o n i n beet seeds (4-6), and a saponln-like i n h i b i t o r was associated with germination i n h i b i t i o n i n Kochia indica (75) . Tourneau (89) suggests that the i n h i b i t o r of l e a f y spurge may be a non-alkaloidal nitrogen compound. Luckwill (55) states that l i t t l e i s known about the chemical nature of i n h i b i t o r s , but they probably f a l l into two groups: phenols, and true antiauxins, which block some ess e n t i a l reaction. In an outline of recent work Audus (3) notes that there i s a close r e l a t i o n s h i p between some i n h i -b i t o r s and IAA, and that the i n h i b i t o r s may be either an auxin precursor or an inhibitor-auxin complex. Properties of Inhibitors Inhibitors exhibit a variety of c h a r a c t e r i s t i c s . A review of the germination i n h i b i t o r s q u a l i f y them as being non-specific i n nature ( 2 3 ) , while Audus (3) deals with i n h i b i t o r s which are s p e c i f i c i n nature and which may there-fore be useful i n the f i e l d of a n t i b i o t i c s . The i n h i b i t o r y compounds of Betula sp. was shown to be a non-fluorescent substance ( 11 ) , although many i n h i b i t o r y substances are known to be fluorescent. The fluorescence exhibited by cou-marin and i t s derivatives i s dealt with i n d e t a i l by a number of workers ( 3 1 , 8 3 ) . 13 The i n h i b i t o r y substance of guayule was stated as being organic i n nature (1H-), but at least one other review deals with several i n h i b i t o r y compounds which are inorganic ( 2 3 ) . The i n h i b i t o r i n t h i s plant as well as that formed i n the potato tuber was both ether and water soluble ( 3 5 ) . It has also been shown that there are neutral as well as a c i d i n -h i b i t i n g substances ( 3 7 ) . An i n h i b i t o r y substance i n tomato juice was found to be a non c o l l o i d a l . It was absorbed by animal charcoal and was v o l a t i l e ( 2 2 ) . It i s claimed that some chemical i n h i b i t o r s are photo-dynamic. Galston (29) reported that l i g h t could lead to the formation of an i n h i b i t o r . Using lettuce seed as an i n d i -cator i t has been found that coumarin i s photosensitive aft e r entering the seed and w i l l not exhibit any i n h i b i t i v e e f f e cts on germination i f seeds are soaked i n l i g h t or i f l i g h t s t r i k e s the seed while wet. Thus the i n h i b i t i v e e f f e cts of coumarin are overcome by l i g h t ( 6 ) . Siegel (76) studied the i n h i b i t o r s from red kidney bean seeds and found that i r r a d i a t i o n activated and b o i l i n g i n -activated c e r t a i n extracts, whereas the i n h i b i t o r of grape leaves was found to be stable to heat ( 5 ) . The d i v e r s i t y of i n h i b i t o r s i s apparent, then, as they are: both s p e c i f i c and non-specific i n t h e i r action, both fluorescent and non-fluorescent, organic as well as inorganic, both a c i d i c or neutral i n character, ether and sometimes water soluble, formed by l i g h t as well as destroyed by i t , both heat l a b i l e and heat stable compounds. 1*+ Methods of extraction and Isolation The main object of a satisfactory method is to have an extraction which would be complete. The second aim is to prevent chemical or enzymatic changes in the compounds during and following extraction. In order to reduce the extent of conversion, a number of methods to reduce enzymatic activity can be used, e.g. boiling, addition of specific inhibitors and the use of high temperatures. The experience of a number of workers has indicated however that the most practical methods of reducing conversion is by the use of low tempera-tures and comparatively short periods of extraction. The main features of the extraction procedure outlined by Van Gverbeek (92) are: t o t a l omission of acid, avoidance of crushing the material, one single extraction for a pro-longed time (overnight), and the evaporation of the ether extract to complete dryness. Increased amounts of a growth inhibitor were extracted from the small leaves of Xanthium with continued extraction up to at least twelve hours at 0° C. (13). Black (93) in his work with Betula sp. used methanol^ to avoid proteinaceous material. The different methods of extraction with their advan-tages and disadvantages have been reviewed by Larsen (51) in detail. He deals i n i t i a l l y with the method of trapping and then outlines the extraction with different solvents such as: water, diethyl ether, chloroform and ethanol. \\ 15 Since the crude plant extracts contain various impurities as well as possibly both growth promoting and i n h i b i t i n g sub-stances, a f r a c t i o n a t i o n of the extracts i s necessary f o r i d e n t i f i c a t i o n . Larsen (51) outlines a technique to be used: p a r t i t i o n between solvents, chromatographic p a r t i t i o n on columns, and paper chromatography. Leopold (52) also deals with the chromatographic separ-ation of auxins and discusses b r i e f l y the various types of solvents used by d i f f e r e n t workers. The technique used f o r auxins can also be used f o r i n h i b i t o r s , as Powell (66) has pointed out. A recent review by Thompson (85) deals extensively and intensively with a l l the aspects of p a r t i t i o n chromatography. The thoroughness of t h i s review makes i t unnecessary to deal further with t h i s method. Bioassay methods As most of the growth substances are present i n very low concentrations, b i o l o g i c a l t e s t s , which are more sensi-t i v e than chemical t e s t s , have been adopted by workers i n t h i s f i e l d . The s e n s i t i v i t y of the b i o l o g i c a l assay i s em-phasized by Audus (3) who states that the Avena test i s about 200 times more sensitive , and the root test 50,000 times more sensitive than the most delicate chemical t e s t . Macro methods and f i e l d assesments are s t i l l of value fo r making I n i t i a l evaluations. Benedict (6) added dried 16 roots of brome grass which he suspected contained inhibitory-material to tes t plants and measured the dry weight of the roots and shoots of his test species. Funke (28) made direct f i e l d observations of the growth of eighteen species of plants which were sown beside a hedge of Artemisia absinthium. It was found that the Artemisia absinthium severely injured the growth of these species. Gray (33) conducted both sand and solution culture tests with the leaves of Encelia farinosa. which were found to be i n h i b i t o r y . A very simple and e f f i -cient test i s the germination test which has been elaborated by Evenari ( 2 3 ) . Larsen (51) states that auxin tests based on the use of plant organs r e l a t i v e l y r i c h i n endogenous auxin can be used unchanged for the demonstration and assay of growth-i n h i b i t o r s . He further states that the response of test organs r e l a t i v e l y high i n endogenous auxin ccan be standar-dized against pure preparations of synthetic growth i n h i -b i t o r s . Coumarin seems to be a suitable standard for most purposes. The various tests f o r auxins have been outlined by Leopold ( 5 2 ) which are as follows: avena t e s t , straight growth t e s t , pea root t e s t , root i n h i b i t i o n test and the l a n o l i n paste t e s t . In the avena and l a n o l i n paste t e s t the curvature i n the case of i n h i b i t o r s would be positive whereas i n the case of auxins there would be a negative curvature. Some workers favour other species for these t e s t s , ': e.g. 17 Bentley (8) who recommended that wheat sections are better than oats i n the bioassay of i n h i b i t o r s , while some, l i k e Patrick ( 6 2 ) based t h e i r bioassay tests on manometric methods and depended on the fact that i n h i b i t o r y substances are ca-pable of i n h i b i t i n g r e s p i r a t i o n of the t e s t species. I d e n t i f i c a t i o n As the i n h i b i t o r s which form a part of the growth sub-stances of plants exist i n very low concentrations, e f f o r t s to i d e n t i f y the chemical structure are extremely d i f f i c u l t . Attempts have been made, however, by determining Rf values, colour reactions and b i o l o g i c a l a c t i v i t y of unknown growth substances and comparing these with the c h a r a c t e r i s t i c s of selected synthetic compounds. If the substance can be i s o l a t e d and c r y s t a l l i z e d , then the melting point (M.P.) can be determined. Davig, f o r example* made a comparison of the M.P. of his unknown with that of synthetic juglone before reporting conclusively that the t o x i c p r i n c i p l e of Juglans nigra was juglone.(19). Studies with the i n h i b i t o r y compound found i n Encelia farinosa enabled Gray (3*0 to determine the M.P. He also conducted a sodium fusion test and determined the elements present. By using these methods he i d e n t i f i e d the i n h i b i t o r to be 3-acetyl -6-methoxybenzaldehyde. If the i n h i b i t o r y compounds are due to precursors of 18 auxins or due to higher concentrations of auxins, then the compound may be an indole. Various workers (51,74-,85) deal with the spray and colour reactions used to detect indole compounds. The use of chromogenic sprays and comparison of d i f f e r e n t Rf values by employing d i f f e r e n t solvents i n an e f f o r t to i d e n t i f y coumarin and i t s derivatives as well as organic acids have been l i s t e d by a number of workers ( 3 1 , 8 3 ) . The use of both u l t r a v i o l e t and infrared spectroscopy help i n enabling one to i d e n t i f y the unknown compound. Mode of action and significance Various theories have been postulated by d i f f e r e n t workers regarding the mode of action of i n h i b i t o r s . Skoog (77) reported i n his experiments with bud i n h i b i t i o n that i n h i b i t i n g actions cannot be explained i n terms of concen-t r a t i o n alone. He stated also that IAA can i n h i b i t growth d i r e c t l y i n buds. Later, Skoog (78) confirmed the view that auxin acts as a co-enzyme. He presented a scheme for the action of auxins i n stimulation and i n h i b i t i o n of growth. This scheme accounts f o r the a b i l i t y of auxin to accelerate and i n h i b i t growth, as a quantitative manifestation of a single function, rather than as a result of separate q u a l i -t a t i v e e f f e c t s . Audus (1) i n his work with coumarin indicated that these substances must af f e c t t h e i r i n h i b i t i o n by forming 19 loose combinations, or e a s i l y disassociated compounds with enzymes or metabolites i n the plant c e l l . Mayer (58) r e -ported the a c t i v i t y of coumarin as a germination i n h i b i t o r was due to i t s s p e c i f i c structure, that i s , an unsaturated lactone linked to an unsubstituted benzene nucleus. Any substitution leads to the p a r t i a l destruction of the a c t i -v i t y as a germination i n h i b i t o r . Mayer (59) also found that seed extracts would inactivate or destroy large amounts of coumarin. He suggested that the i n a e t i v a t i o n of coumarin i s effected by a step which involves i t s reaction with some ess e n t i a l metabolite which i s produced enzymatically or with the enzyme producing i t . The exhaustion of t h i s metabolite or the diversion or in a e t i v a t i o n of the enzyme leads to f a i l u r e of germination. The work of Thiamann (8h) established that coumarin and protoanemonin i n h i b i t growth by reacting with a s u l f f hydryl enzyme, t h i s enzyme being normally a l i m i t i n g factor i n growth. This view was also supported by E l l i o t (21). Bentley (9) suggests that the i n h i b i t o r y e f f e c t s of coumarin derivatives are due to the i n a e t i v a t i o n of sulfhydryl-contain-ing enzyme systems, on the basis of the known reaction of t h i o l s with , B-unsaturated lactones. This explanation of i n h i b i t o r action assumes that the primary auxin effect i s exerted v i a a t h i o l compound, e.g. CoA. Such an effect would be a true competitive I n h i b i t i o n . 20 The effects of coumarin and scopoletin upon growth were studied and i t was found that both these substances Inhibit root growth, coumarin being about f i f t y times as e f f e c t i v e as scopoletin (65). Coumarin appears to upset the p o l a r i t y of growth, p a r t i c u l a r l y i n the region of c e l l elongation, but does not eliminate meristematic a c t i v i t y or root hair development. In a study with i n h i b i t o r y J3-eomplex (57) i t was ex-plained that growth i n h i b i t i n g concentrations of JB stimulate oxygen uptake and inte r f e r e with the uptake of inorganic phosphate. It i s suggested that J 3 acts by uncoupling phos-phorylation from the electron t r a n s f e r r i n g systems and by t h i s means deprives the tiss u e of energy (ATP) necessary f o r the performance of the reaction associated with growth. It has been stated that auxin causes an i r r e v e r s i b l e p l a s t i c to an e l a s t i c t e n s i b i l i t y of the c e l l walls during f i r s t phase of elongation (16). It i s assumed auxin medi-ates a r e v e r s i b l e d i s s o l u t i o n of the c e l l wall which explains both i t s growth promoting and growth i n h i b i t i n g action. Cou-marin and daphnetin both destroy the t e n s i b i l i t y of the wall, making i t r i g i d . In his work with apple seeds, Luckwill (5*+) concluded that the i n h i b i t o r y action which resulted i n dormancy of the seed could be broken by the formation of growth promoting substances. It has been stated that the i n h i b i t i o n of growth i n plants at higher auxin concentrations 21 could be due to a natural and predictable consequence of the two point attachment of the auxin molecule ( 2 5 ) . At higher concentrations there i s the formation of an inactive biomolecular complex. Larsen (50) has Indicated that plant growth i s governed by a double system of regulators, growth accelerating and growth retarding substances. That these i n h i b i t o r s have a b i o l o g i c a l function has been discussed by Evenari ( 2 3 ) , under the following headings: prevention of premature germination, extension of the germination period over a long period, and suppression of germination and growth of other species. The signi f i c a n c e of i n h i b i t o r s i n germination has also been dealt with by K o l l e r (4-7) who states that i n h i b i t o r y substances act as a chemical \"rain gauge\" i n dispersal u n i t s . It i s also pointed out that dormancy i s a protection from the vagaries of nature. Regulated germination and prevention of v i v i -p a r i t y i n most fleshy f r u i t s i s due to the presence i n them of substances that s p e c i f i c a l l y Inhibit germination. It has been assumed that growth i n h i b i t i n g substances are s i g n i f i c a n t f or the rest period i n terminal buds of Fraxinus ( 3 6 ) , and the presence of i n h i b i t i n g substance i n dormant flower buds of peach has been reported (4-1). That the dormancy i n potato tubers i s due to i n h i b i t o r s has been suggested by many workers ( 1 0 , 3 7 ) . Patrick (62) points out that toxic substances may per-22 form a s i g n i f i c a n t role as the primary cause of inducing some root rots and i n predisposing plants to attack by organisms not normally regarded as pathogenic. It has been postulated that every growing crop forms a substance which i s t o x i c to the growth of other plants and s t i l l more so to I t s e l f , and that by oxidation t h i s toxin loses i t s toxic properties and enhances the f e r t i l i t y of the s o i l (4-). The work of Proebsting (67) showed that peach roots added to v i r g i n s o i l i n h i b i t e d growth of peach seed-l i n g s . Bonner (15) indicated the importance of toxic sub-stances produced by certa i n species as being responsible for e c ological phenomena, such as competition of plant communities or the sequence of a p a r t i c u l a r species i n a succession. This view was supported by Francois ( 2 6 ) , who suggested that the toxic substance found i n the leaves of Allanthus may be one of the important factors l i m i t i n g . natural succession i n that plant. It i s now evident that although some of the natu r a l l y occurring i n h i b i t o r s exert t o x i c e f f e c t s , other may perform regulatory functions. 23 III. MATERIALS AND METHODS The three species of knapweed used for study were: Centaurea repens L. (Russian knapweed), Centaurea d i f f u s a Lam. (diffuse knapweed) and Centaurea maculosa Lam. (spotted knapweed). In order to c o l l e c t fresh plant material as well as the s o i l In which the three d i f f e r e n t species were growing, a t r i p into the i n t e r i o r of B r i t i s h Columbia was made i n the summer of i 9 6 0 . Seeds and fresh plants of C^ repens were co l l e c t e d from dense infestations found at Cawston. S o i l from infested as well as from neighbouring non infested areas was co l l e c t e d . Fresh specimens of C. d i f f u s a and s o i l from infested and non infested areas were c o l l e c t e d from Okanagan F a l l s . The plants were not i n seed and therefore i t was not possible to get seeds of t h i s species. Fresh speciments of maculosa as well as s o i l from infested and non infested area were co l l e c t e d from the outskirts of an Indian reserve at Canford. Though there were no seeds available at the time of c o l l e c t i o n , seeds of th i s species were obtained through the D i s t r i c t A g r i c u l t u r i s t at Nelson l a t e r i n the season. The leaves, stems and roots of the three species were separated and dried i n an oven at a temperature of 80° C. The oven dried material as well as the seeds of CL repens and C_j_ maculosa were then ground i n a wiley m i l l using a 20 mesh screen. The powdered material was put i n amber co l o r -ed, a i r ti g h t bottles and stored i n the dark at room tempera-ture. 24-S o i l studies Studies with the s o i l consisted of setting up a series of s i x - i n c h pots, three of which contained s o i l from an In-fested area of the di f f e r e n t knapweeds. The other three, as controls, contained s o i l from the neighbouring non infested area i n each ease. In each one of the pots three tomato seedlings (var.Firesteel) which were t h i r t y days old were transplanted. The average height of the three test plants i n each pot was recorded p e r i o d i c a l l y . At the end of ninety days the dry weights of both the tops and roots were recorded. In a second experiment four si x - i n c h pots were f i l l e d with greenhouse s o i l . In three of the pots f i v e , ten and f i f t e e n grams of C. repens a i r - d r i e d l e a f powder were added. A pot to which nothing was added served as a control..Into each pot two t h i r t y day old tomato seedlings ( F i r e s t e e l ) were transplanted. The height of the plants was measured p e r i o d i c a l l y . At the end of sixty days the dry weights of the shoots and roots were recorded and the root-top r a t i o calculated. Proximate analysis A proximate chemical analysis^of the powdered leaves, stems and roots of the three speeies was conducted by the usual methods recommended by the Association of O f f i c i a l A g r i c u l t u r a l Chemists ( i 9 6 0 ) . A l l the analyses was done i n duplicate and the re s u l t s expressed on dry weight basis. 25 Extraction Five grams each of the powdered l e a f , stem, seed and root of the di f f e r e n t v a r i e t i e s was extracted by means of a Soxhlet apparatus using ether as a solvent. The f i n a l volume i n each case was made up to 100 ml. thereby giving a 5% extract. /.The temperature maintained during extraction was approximately 65° C. Five grams each of the powdered l e a f , stem, seed and root was put into 200 ml. Ehrlenmeyer f l a s k s . To each was added 100 ml. of d i s t i l l e d water and the f l a s k then placed on a shaker f o r twelve hours. The extract was f i l t e r e d through a Buchner funnel and the f i n a l volume brought up to 100 ml. thereby giving a 5% extract. In order to determine the s o l u b i l i t y of the i n h i b i t o r , the ether extract was pa r t i t i o n e d with an equal volume of water. Each of the two fra c t i o n s thus obtained was used f o r germination studies. Another attempt was made to extract the i n h i b i t o r with ethanol. The procedure was sim i l a r to that of Soxhlet ether extraction except that a higher temperature around 95° C. had to be employed. Bioassays Germination tests were used as a method of bioassay to determine the presence of i n h i b i t o r s . The indicator plants used i n t h i s study were: lettuce (var. New York), barley 26 (var. Vantage) and cress (var. Pepper). These crops were chosen because they represented d i f f e r e n t families and In-cluded both large and small seeds. Lettuce and cress give a more sensi t i v e response than does barley. P e t r i dishes, 9 cm. i n diameter, were thoroughly cleansed and repeatedly rinsed with hot water. The washed dishes were then s t e r i l i z e d i n an autoclave at a temperature of 125° C. and a pressure of 15 pounds for 15 minutes. In each p e t r i dish an extra t h i c k Whatman f i l t e r paper was placed. In the case of the ether extract, 5 ml. of the ether extract were added to each dish. Then 10 minutes were allow-ed to elapse so that the ether could evaporate, to avoid Inh i b i t i n g effects because of ether contamination. Then 5 ml. of d i s t i l l e d water were added. In these tests there were two controls. One with d i s t i l l e d water alone, and one with d i s t i l l e d water added to a dish where ether had been allowed to evaporate. With the water extract 5 ml. of the di f f e r e n t extracts were added to each dish and 25 seeds each of the^kinds men-tioned above were equally spaced on the f i l t e r paper. One dish with only d i s t i l l e d water,served as a control. In the germination studies with the ethanol extracts, 5 ml. of the di f f e r e n t extracts were added to the f i l t e r paper. The p e t r i dishes were then gently heated on a hot 27 plate u n t i l the f i l t e r papers were dry and the ethanol had evaporated. Then 5 ml. of d i s t i l l e d water was added and 25 seeds of the d i f f e r e n t v a r i e t i e s mentioned were placed i n p o s i t ion. Again these two controls; one with d i s t i l l e d water and one with d i s t i l l e d water added to the ethanol dried f i l t e r paper, were used. In a l l the germination studies the p e t r i dishes were covered and placed i n a Mangelsdorf germinator. The tem-perature of the germinator ranged between 25° C. to 27° C. After a period ranging from 5 to 8 days the number of seeds germinated was determined and the average height of both the shoots and roots of 10 seedlings picked at random was measured. Those seeds where the r a d i c l e had emerged to more than 1 mm. were considered as having germinated. The germination per c e n t , p a r t i c u l a r l y i n C. repens, was determined. A few pre-treatments of the seeds were employed pr i o r to germination, which consisted of either soaking the seeds f o r 6 hours In water at room temperature, or c h i l l i n g the dry seeds f o r 6\\hours at 5° C. i n a r e f r i -gerator, or planting the seeds d i r e c t l y (without pre-treat-ment) i n the p e t r i dishes containing either the f i l t e r paper or greenhouse s o i l . I s o l a t i o n The common procedure of fr a c t i o n a t i n g the crude extract into a c i d i c and basic parts was not attempted, because i t 28 might have led to the addition of other substances, or created conditions which might have interfered with the germination t e s t s . In order to i s o l a t e the i n h i b i t o r , paper chromatography was used. Since i t was i n i t i a l l y determined that the i n h i -b i t o r was mainly concentrated i n the leaves of the three spe-cies of knapweeds a l l further work of i s o l a t i n g the i n h i b i t o r was done only with the crude ether extract of the leaf powder. The crude leaf extract of the three d i f f e r e n t species consisted of 5 grams of powdered leaves In 100 ml. of ether extracted by the Soxhlet procedure. The 100 ml. volume was i n i t i a l l y reduced at 4-0° C. to 15 ml. i n a f l a s h evaporator. The concentrated extract was then centrifuged at a high speed of 10,000 r.p.m., t h i s done i n order to get r i d of im-p u r i t i e s which might usually cause overloading of chromato-graphic paper. Whatman No. 3 f i l t e r paper was used and 5 ml. of the concentrated extract was applied as a band about 5 n i m . wide and two inches away from the edge of the paper. The band was applied with the a i d of a c a p i l l a r y tube, one end of which had a s l i g h t curvature to f a c i l i t a t e a p p l i c a t i o n . The following solvents were used: 1. Aqueous phase 4-. Isopropanol:ammonia:water Butanol:acetic:water 10 : 1 : 1 1 5 70% ethanol 2 . Organic phase Butanol:acet ic:wat er 6. Butanol:ammonia:water h : 1 : 5 100 : 3 : 18 3. Water 7. Butanol:ethanol:wat er 4- : 1 : 1 29 Descending chromatography did not give s a t i s f a c t o r y re-solution, so a l l chromatographic work was done by employing the ascending method. After application of the band the paper was made into a cylinder and both the edges were stapled. The solvent was poured into a t a l l c y l i n d r i c a l glass jar and the jar was covered with a wax-coated l i d . The solvent was allow-ed to d i f f u s e into the atmosphere of the glass jar overnight before the Whatman paper cylinder was placed i n i t . Of the various solvents mentioned the one which was most sa t i s f a c t o r y was the lower aqueous phase of n-butanol:glacial acetic aeidtwater i n the r a t i o of *+:l:5. The solvent was obtained by mixing the three ingredients and allowing the mixed solvent to stand i n a separatory funnel overnight. The lower phase was drained off and used. Any organic phase pre-sent i n t e r f e r e d with the resolution. The solvent was allowed to r i s e u n t i l the solvent front reached a distance of 20 cm. which occurred i n approximately sixteen hours. A f t e r t h i s the paper was taken out, the staples removed and the;; paper was a i r dried. The a i r dried o paper was viewed under u l t r a v i o l e t l i g h t (long wave 3 6 6 0 A) and the d i f f e r e n t bands as well as the solvent front were marked and the colour noted. Each band was cut out as a long s t r i p , folded at one end and placed i n a trough containing water as the eluting solvent. The other end of each band was allowed to just dip into a beaker. After a period of eighteen hours, about 30 15 ml. of the eluate was obtained from each s t r i p . Germination t e s t s with eluates To the d i f f e r e n t p e t r i dishes, which contained extra-th i c k f i l t e r paperV 5 ml. of the eluate from each band was added. For the germination t e s t s , 25 seeds of lettuce were used. The area on the Whatman paper immediately preceding the band or spot of ap p l i c a t i o n of the extract served as a control f o r any i n h i b i t i o n due to the solvent. To confirm the Rf values of the i n h i b i t o r y substance, the chromatographic paper which had been banded and developed was cut out into equal parts representing successive Rf values. On each of the s t r i p s so obtained, usual germination t r i a l s were conducted using lettuce seeds. C r y s t a l l i z a t i o n The volume of eluates from the i n h i b i t o r y bands was reduced at i t s b o i l i n g point to an optimal l e v e l f o r crys-t a l l i z a t i o n . The Ehrlenmeyer f l a s k containing t h i s eluate was then placed i n the r e f r i g e r a t o r . As the results were unsatisfactory, another method was also used. In t h i s case, methanol was added to hot and concentrated eluate and then ra p i d l y cooled. Identification! of the i n h i b i t o r The following methods were attempted i n an e f f o r t to i d e n t i f y the i n h i b i t o r : 31 1. the Rf values of the i n h i b i t o r were recorded i n dif f e r e n t solvent systems 2. the colour under v i s i b l e l i g h t as well as under u l t r a v i o l e t l i g h t was recorded 3. the i n h i b i t o r y band was exposed to ammonia and i t s reaction noted, both under v i s i b l e and u l t r a v i o l e t l i g h t *+. spray tests - the i n h i b i t o r y band was psprayed with the following reagents and the reaction recorded: (a) 2 N-NaOH (f) 1% aqueous FeCl^ (b) 2'H-Hcl (g) FeCl. - HClG k (SalKowski) * (c) 1% ethanolic A l C l o - (h) 1% P-dimethylamino-benzaldehyde (d) 1% aqueous KMnO^ (Ehrlich) (e) 1% ethanolic FeGl^ ( i) KN02 - HHC3 (j) fe innamaldehyde 5. one milligram of the crude c r y s t a l l i z e d product was dissolved i n 2 ml. of water and 3 ml. of ethanol to deter-mine the u l t r a v i o l e t absorption spectra 32 IV. RESULTS A comparative study of the knapweed infested and non infested s o i l gave res u l t s as shown i n Table 1. The infested s o i l of the Russian and spotted knapweed produced marked i n h i -b i t i o n both to tomatoes and barley. The height of the shoot was reduced and there was a reduction of the dry weight of both shoots and roots. In the case of the d i f f u s e knapweed the r e -sults were the opposite, possibly due to the fact that the non infested s o i l was co l l e c t e d from, an old railway siding, of poor growing c h a r a c t e r i s t i c s . The i n h i b i t i o n of growth with two of the species i s shown i n Figure 2. Addition of 5, 10 and 15 grams of a i r dried leaf powder to green house s o i l produced i n h i b i t i o n to the growth of t o -matoes. There was a consistant decrease of height as well as a decrease i n the dry weight of shoots and roots (Table 2). The difference i s graphically represented i n Figure 3. Results of the chemical analyses of the root, stem and leaf of the three v a r i e t i e s are represented i n Table 3. The nitrogen content and consequently the amount of protein i n the leaves of C. repens was considerably higher than i n the other two species. Preliminary e f f o r t s to detect the presence of i n h i b i -tors were accomplished through growth and germination studies. The re s u l t s obtained with the ether extract of the d i f f e r e n t 33 SOIL'. STUDIES Table 1. Comparative study of growth of tomatoes and barley on infested and non infested s o i l . (a) Averages of 3 tomato plants after 90 days. Type of Height Dry Weight Dry Weight S o i l of Shoot of Shoot of Root 1 (cms.) (gms.) (gms.) Russian 19 .5 1.10 O.38 Non Russian 35 .2 1.79 0.61 Diffuse 33.6 1.59 0.4-9 Non Diffuse 2 9 . 5 0 . 8 5 0.19 Spotted 18.2 0.68 0 . 2 0 Non Spotted 4-9.1 2.4-'+ 0.73 (b) Averages of 25 barley seedlings a f t e r 25 days. Type of Height Dry Weight Dry Weight S o i l of Shoot of Shoot of Root (cms.) (gms.) (gms.) Russian 24-.0 1.14- I . 76 Non Russian 26 . 5 1.56 2.4-0 Diffuse 24-. 0 1.20 1.94-Non Diffuse 2 2 . 5 1.10 1.58 Spotted 18.2 0 .68 0.20 Non Spotted 21 .0 1.10 1.52 -r Figure 2. Comparative study of growth of tomato seedlings in knapweed infested and non infested soils. Table 2. Effect of Cj. repens leaves on the growth of tomato plants. Averages of two tomato plants. Dry Dry C. repens Height of Wt. of Wt. of Root leaves . Shoot Shoot Root Top (gms.) (cms, .) (gms. ) (gms.) 1st 10th 20th 30th *+0th 50th 60th day day day day day day day Control 8.5 10.9 1U..1 17.3 21.*+ 25.2 35.1 2.11 0.19 5 8.7. 10.8 13.5 16.5 20.5 2V..1 33.2 2.02 0.32 0.16 10 8.6 10.6 12.8 15.1 18.5 22.9 30.1 1.63 0.25 0.15 15 8.0 10.3 11.9 1>+.1 17.3 21.8 28.5 1.52 0.22 0. Ih 36 10 20 30 ko 50 60 DAYS Control weight of shoots (gms.) 15 gms... C. repens weight of roots (gms.) Figure 3. E f f e c t of the addition of powdered leaves of C. repens on the growth of tomato plants i n s o i l . 37 Table 3. Chemical analysis of 3 Centaurea spp.* (a) C. repens Root Stem ~ Leaf Moisture 7.22 4-.60 4-. 12 Ash 8.06 7.30 11.10 Fat 0.80 1.1+9 2.33 Crude f i b r e 50.60 4-2.80 15.65 Nitrogen O.83 1.46 3.39 Protein 5.20 9.14- 21.20 Nitrogen '\"' free extract 35.34- 39.27 4-9.72 (b) C. d i f f u s a Root ' ' Stem Leaf Moisture 3.51 4-. 52 2.4-3 Ash 13.73 4-.07 15.62 Fat 1.19 0 .80 2.34 Crude f i b r e 29.40 4-1.80 21.75 Nitrogen 0 .59 0.4-3 1.33 Protein 3.71 2.74- 8.31 Nitrogen free extract 51.97 50.59 51.98 (c) C. maculosa Root Stem Leaf Moisture 1.37 2.86 3.73 Ash 4-3.60 6.53 lS.18 Fat O.78 1.23 2.87 Crude f i b r e 3 L 1 0 4-3.55 16.85 Nitrogen 0.4-2 O.87 0.92 Protein 2.62 5.4-0 5.68 Nitrogen free extract 4-3.29 46.4-2 * The values obtained above are expressed i n per cent on dry weight'basis. 38 parts of the three knapweeds are presented i n Tables 5 and 6, and Figure k. It was found that an i n h i b i t o r was present i n a l l three species. The i n h i b i t i n g substance was found mostly i n the leaves and to a lesser extent i n the seeds. The i n h i b i t o r was non-specific, being i n h i b i -tory to a l l three test crops (barley, lettu c e , cress). There was both germination and seedling growth i n h i b i t i o n ; and the growth of roots was more i n h i b i t e d than that of the shoots. In order to determine whether the i n h i b i t o r was water soluble, both growth and germination studies were again conducted with the water extract. The r e s u l t s are repre-sented i n Tables 7, 8 and 9, and Figure 5. The i n h i b i t o r was found to be water soluble, and was again present to the greatest extent i n the l e a f extract., It was i n h i b i t o r y to both the test plants used (barley and l e t t u c e ) , exhibiting germination as well as growth i n h i b i t i o n . An experiment to establish whether the i n h i b i t o r was more water or ether soluble proved the l a t t e r to be the case. It was also found, however, that the i n h i b i t o r was not com-p l e t e l y taken up by the ether f r a c t i o n . Inhibitory a c t i v i t y of a lesser degree was also found i n the water f r a c t i o n , (Table 10). An ethanol extraction was attempted but i t did. not prove s a t i s f a c t o r y . It was found very d i f f i c u l t to remove 39 Table l+. E f f e c t of C. repens ether extracts on crop seedlings.* Test Crop Knapweed Test Germination Shoots Roots Extract. % (cms.) (cms.) Control water 96 lh.2 13.5 Control ether 96 13.5 12 .5 Leaf 1+0 2.8 l.i+ BARLEY Stem 80 1 5 A 12.8 Root 80 12.1+ i+.l Seed 96 13.3 10 .9 Control water 100 3 . 5 3 . 2 Control ether 96 3-5 3 .3 Leaf 72 0.6 LETTUCE St em 92 3 . 0 2 .8 Root 96 2.3 3.1 Seed 81+ 2.6 2.2 . . . . . . Control water 96 3.1 12.2 Control ether 96 2.9 10.3 Leaf 8 1.3 1.0 CRESS Stem mm — — — • •'• Root 81+ 2.3 7.i+ Seed * Measured 8 days a f t e r sowing. 1+0 f Table 5. E f f e c t of G. d i f f u s a ether extracts on crop seedlings.* BARLEY Test Crop Knapweed Test Germination Shoots Roots Extract % (cms.) (cms.) Control water 92 9 . 5 11.1 Control ether 76 10.1 11.2 Leaf 68 3.1 3 . 2 Stem 88 9 .2 11.M-Root 88 7.3 7 .2 Seed 96 96 80 96 92 3.6 3.2 1.8 3.0 2 .5 3 .6 3 . 5 0 . 8 3 .1 3 .0 96 96 16 3.1 2 .9 1.6 12.2 10.3 1.*+ 96 2.1+ 8.2 LETTUCE Control water Control ether Leaf Stem Root Seed CRESS Control water Control ether Leaf St em Root ' Seed * Measured 7 days a f t e r sowing. 1+1 Table 6 . E f f e c t of C. maculosa ether extracts on crop seedlings.* Test Crop Knapweed Test Extracts Germination % Shoots Roots (cms.) (cms.) BARLEY. Control water Control ether Leaf St em Root Seed 92 92 80 81+ 81+ 80 11.5 10.6 l+.O 10 .5 1 0 . 5 10.2 10 .0 9.1 5.3 9 A 7.5 7.1 LETTUCE Control water Control ether Leaf Stem Root Seed 100 100 81+ 96 88 88 3.7 3 A 1.8 3 .1 2.9 2.8 3 . 5 3 . 3 0 . 9 3 . 2 3 . 0 2.1+ Control water 96 3.1 12.2 Control ether 96 2.9 10.3 Leaf 16 1.6 1.5 CRESS Stem Root 96 2.7 9 . 5 Seed * Measured 7 days a f t e r sowing Control Control water ether 1+2 Leaf Stem Root Seed Centaurea repens Germination % 10G 96 72 92 96 84-Centaurea diffusa Germination 96 96 80 96 92 Centaurea maculosa Germination 100 100 84- 96 88 88 Figure 4-. Response of lettuce seedlings to the ether extract of the three knapweeds. The length of histogram above the line indicates the length of shoot, and below the line, the length of root. **3 Table 7. E f f e c t of repens water extracts on crop seedlings.* Test Crop Knapweed Test Extracts Germination Shoots Roots (cms.) (cms.) LETTUCE Control water 96 3 A 3.5 Leaf 6*+ 2.5 0.9 Seed 92 2.6 2.0 Root -96 3.6 3.2 BARLEY Control water 8*+ 12.6 10.2 Leaf 76 7.5 5.9 Seed 76 3.5 5.0 Root 80 11.1 9 A •Measured 7 days a f t e r sowing. 4-4 Table 8. E f f e c t of C^ . d i f f u s a water extract on crop seedlings.* Test Crop Knapweed Test Germination Extract % Shoots Roots (cms.) (cms.) LETTUCE BARLEY Control water Leaf Seed Root 96 84-84-Control water Leaf Seed Root 84-72 80 3.4-2.6 3 .5 1.0 3.1 3 . 5 12.6 7.7 10.2 6.4-11.4- 9 .1 * Measured 7 days a f t e r sowing i+5 Table 9. E f f e c t of C. maculosa water extracts on crop seedlings.* Test Crop Knapweed Test Germination Shoots Roots Extract % (cms.) (cms.) Control water 96 3 A 3 . 5 Leaf 80 2.6 1.5 LETTUCE Seed 80 2.0 1.9 Root 92 3 . 2 3 .2 Control water 6% 12.6 10.2 Leaf 72 7.8 6 . 5 BARLEY 3.6 Seed 8i+ 3 .5 Root 81+ 12.1 9.8 * Measured 7 days after sowing 4-6 Control water Leaf Root Seed Centaurea repens Germination % 96 64- 96 92 Centaurea diffusa Germination % \"84- 72 80 Centaurea maculosa Germination % 96 80 92 80 Figure 5. Response of lettuce seedlings to the water extract of the three knapweeds.. The length of histogram above the line ^indicates the length: of shoot and below the line, the length of root. Table 10. Fractionation of the ether extract of leaves of C. repens. Lettuce measured after 5 days. C. repens leaf fraction Germination % Shoots (cms.) Roots (cms.) Control water 96 2.k 2.5 Control ether fraction 92 2.2 2.1+ Control water fraction 96 2A 2.2 Leaf ether fraction 60 0.6 0.3 Leaf water fraction 82 1.2 0.6 1+8 the i n h i b i t i o n caused by the solvent i t s e l f . The re s u l t s are presented i n Table 11. In a comparative study of the le a f extracts of the three d i f f e r e n t knapweeds i t was found that the i n h i b i t o r i n the leaves of C. repens was more toxic than that from. C. d i f f u s a which was again more tox i c than that from C. maculosa (Table 1 2 ) . As the i n h i b i t i o n was also found to be present i n small amounts i n the seeds of C. repens and C. maculosa a study was made to obtain the germination percentage of C. repens seeds which were one year old. Results are given i n TaBle 13. The germination percentages aft e r 15 days i n the ger-minator were very low and i t was only after a period of a month that the seeds showed maximum germination. Because of the slowness of germination and the low germination percentage i t was decided to forgo any further experiments of t h i s type. Of the various solvents t r i e d the one which gave the best resolution was the lower aqueous phase of n-butano}.: acetic acidtwater i n the r a t i o of *+:l:5. The ascending method of chromatography proved more suitable than the descending one. In a l l other solvents, and with the des-cending method, there was poor resolution due mainly to t r a i l i n g of associated plant materials. A diagrammatic representation of the various compounds v i s i b l e under u l t r a v i o l e t l i g h t (long wave) i s presented i n Figure 6. A pale yellow fluorescent compound at an Rf of 4-9 Table 11. E f f e c t of ethanol extract of C^ repens leaves on growth of 2 crop seedlings.* Test Crop Knapweed Test Germination Shoots Roots Extract. % (cms.) (cms.) Control water 84- 8.8 8.4 BARLEY Control ethanol 50 3.8 k.2 Leaf 4-8 2.8 2.7 3 .0 3.1 1.8 1.7 1.3 0.9 Control water 92 LETTUCE Control ethanol 60 Leaf 56 •Measured 6 days afte r sowing. 50 Table 12. Comparative study of the toxic effect of the ether extract of 3 knapweeds.* Test Crop Leaf Extracts Germination Shoots Roots * (cms.) (cms.) Control water 100 7.8 8 .1 Control ether 96 7.7 8.1 BARLEY ho G. repens 3.0 1.5 C. d i f f u s a 68 3.1 2.2 C. maculosa 68 i+.O 2.9 Control water 92 3 . 0 3.1 Control ether 8*+ 3 . 0 o 3 . 2 LETTUCE C. repens 10 0.2 0 .2 C. d i f f u s a ho 0 . 5 0 .3 C. maculosa 52 2.0 1.0 * Measured 5 days after sowing. 51 Table 13. Germination percentage of repens seeds. Treatment Germination % A f t e r 15 days After 30 days Untreated Soaked C h i l l e d S o i l 35 ho 30 30 85 85 56 60 52 C. repens C. d i f f u s a C. maculosa '/////////, Rf values solvent T r o n t _ 0-9 _ 0.8 . 0.7 _ 0.6 0.2 0.1 s t a r t i n g point i I I I I | Yellow . M i l l fluorescence Faint ////A absorption 1 Strong *• absorption i-, - . . - . . i Blue : I fluorescence Figure 6. Chromatographic resolution of crude ether extract of leaves of the three species of knapweed as observed under u l t r a -v i o l e t l i g h t . 53 0 . 9 was common to a l l three species. A dark absorbing com-pound was found at an Rf of 0 . 5 i n C. d i f f u s a and C. maculosa. Another l i g h t absorbing compound was present at an Rf of 0 .6 i n C. maculosa. Two blue fluorescent compounds were present only i n C. repens. In locating the i n h i b i t o r y compound on the chromato-graphic paper, germination studies with the eluates of the di f f e r e n t bands yielded p o s i t i v e r e s u l t s . These findings are presented i n Tables 1M-, 15, and 16. In a l l three v a r i -eties studied the eluate from the pale yellow fluorescent band at an Rf of 0 . 9 produced severe i n h i b i t i o n to the growth of shoots and roots of lettuce seedlings. There was also a depression of the germination percentage. Growth stimulation, e s p e c i a l l y of the shoots was observed i n the eluates obtained from the solvent front. A confirmatory method of locating the i n h i b i t o r proved to be simpler and more e f f i c i e n t than the e l u t i o n studies. This was done by means of the spot germination t e s t s , r e s u l t s of which are presented i n Tables 17, 18 and 19. Again, severe germination and growth i n h i b i t i o n were encountered at the pale yellow fluorescent band (Rf 0 . 9 ) . A l i t t l e i n h i b i t i o n was also found at an Rf of 0 . 8 , the band immediately preceding the i n h i b i t o r y band. This was considered to be due to t r a i l -ing. In these studies again a stimulation of the shoots was found at the solvent front. In a l l three of the knapweeds there was a greater i n h i b i t i o n of the lettuce roots as com-pared with the shoots. Figure 7 represents the length of 5H-Table lh. Location of the i n h i b i t o r on the chromatogram. Method I. By elu t i o n and lettuce germination. C. repens Rf Nature of Band Germination Shoots Roots % (cms.) (cms.) Control water -- 96 3.6 3 A Control solvent Band before spot 92 3.1 3 .3 0 . 0 Spot 92 3.0 3.1 0.1 — 96 3 . 5 3 .7 0.2 Blue 96 3.5 3 A 0 . 3 — 92 3 . ^ 2.3 O A — 96 3.6 3 . 2 0 . 5 Light, absorbing 92 3.2 3 .3 0.6 — 96 3.3 3 .2 0 .7 — 96 3.1 3 . 0 0 . 8 Blue 80 2.4 2.1 0 . 9 Yellow 61+ 1.1 0 .6 1.0 Solvent front 96 5.8 3 .9 55 Table 15. Location of the i n h i b i t o r oh the chromatogram. Method I. By el u t i o n and lettuce germination. C. d i f f u s a Rf Nature of Band Germination Shoots Roots \\% (cms.) (cms.) Control water Control solvent Band before spot 0 . 0 Spot 0.1 0 . 2 0 . 3 Dark absorption 0.1+ Dark absorption 0 . 5 Dark absorption 0 . 6 0 . 7 0 . 8 0 . 9 Yellow 1.0 Solvent front 92 3.8 3 . 2 92 3.5 3 .3 88 3.3 3.2 81+ 3.7 3 . 0 88 3.7 3 .2 92 3 . 5 3.1 92 3.1+ 3.3 88 3 . 5 3 .1 8*+ 3.1+ 3 .3 88 3 .2 3 . 0 80 2.0 1.6 68 1.2 0 .8 96 1+.5 3 . 2 56 Table 16. Location of the i n h i b i t o r on the chromatogram. Method I. By elu t i o n and lettuce germination. C. maculosa Rf Nature of Band Germination Shoots Roots % (cms.) (cms.) Control water — 92 3 .3 3 . 8 Control solvent Band before spot 88 3.3 3.6 0 . 0 Spot 85 3.2 3 .6 0 . 1 — 92 3.3 3 . 5 0 .2 — 88 3.2 3.^ 0 . 3 — 80 3.1 3 A O A Dark absorption 81+ 3 A 3 . 5 0 . 5 Dark absorption 88 3.2 3 .3 0 .6 Light;? absorption 92 3 . 0 3 . 2 0 . 7 — 81+ 3.1 3 A 0 . 8 — 80 3 A 3 . 0 0 . 9 Yellow 76 1.1+ 1.0 1.0 Solvent front 88 i+.O l+.O 57 Table 17. Location of the i n h i b i t o r on the chromatogram. Method I I . By Spot germination C. repens Rf Nature of Band Germination Shoots Roots % (cms.) (cms.) Control water — 100 3.6 3 .5 Control solvent Band before spot 95 2.9 2 .8 0 . 0 Spot 85 2.8 2 .8 0.1 — 80 3.0 2 .8 0 . 2 Blue 85 2.8 2.9 0 . 3 — 90 2.9 2.8 O.k — 85 2.7 2.8 o .5 Light absorption 90 2.9 2 .8 0.6 — 100 2.6 2 .7 0 . 7 — 95 2.7 2.6 0 .8 Blue 80 1.5 0 . 9 0 . 9 Yellow 25 0 . 5 0 . 1 1.0 Solvent front 95 3.9 2.8 58 Table 18. Location of the i n h i b i t o r on the chromatogram. Method I I . By Spot germination C. d i f f u s a Rf Nature of Band Germination Shoots (cms.) Roots (cms.) Control water — 95 3.6 3 . 5 Control solvent Band before spot 95 3.0 2.7 0 . 0 Spot 90 2.9 2.7 0 .1 — 100 2.5 2 . 5 0 .2 — 85 2.7 2 A 0 . 3 Dark absorption 90 2.8 2 . 0 o.k Dark absorption 85 2.9 2.6 0 . 5 Dark absorption 80 2.9 2.6 0.6 — 90 2.6 2 A 0 . 7 — 85 2.0 2.1 0 . 8 — 80 1.0 O A 0 . 9 Yellow 35 0.6 0 . 2 1.0 Solvent front 95 k.O 2.9 59 Table 19. Location of the i n h i b i t o r on the chromatogram. Method I I . By Spot germination G. maculosa Rf Nature of Band Germination % Shoots (cms.) Roots (cms.) Control water ~ 100 3.6 3 . 5 Control solvent Band before spot 95 3.2 3 . 0 0 . 0 Spot 90 3 . 0 3 . 0 0 . 1 — 95 2.9 3 .1 0 . 2 — 100 3.2 3 . 5 0 . 3 — 90 3.1 3.4-0.1+ Dark absorption 85 3 A 3.*+ 0 . 5 Dark absorption 90 3.5 2.9 0.6 Light absorption 90 3 A 3 . 2 0 .7 — 80 3.2 3 . 0 0 . 8 — 65 2.0 1.9 0 . 9 Yellow 1+0 0.8 0 . 2 1.0 Solvent front 95 H-.9 2.9 C. repens C. d i f f u s a C. maculosa Rf values solvent front — 0 . 8 0 .6 — 0.4-0 . 2 s t a r t i n g l i n e — control solvent control water Length of root i n cm. u> TO OJ ro u> ro Figure 7. Spot germination tests conducted d i r e c t l y on the successive Rf . regions of the developed chromatogram using lettuce seeds. 61 the roots i n the different Rf *s. The greatest i n h i b i t i o n was again at an Rf of 0 . 9 . The eluates.of the i n h i b i t o r y bands were concentrated by reduction under vacuo and stored at 5° c « i n an attempt to c r y s t a l l i z e the compound. After a period of one month the results were negative. As there was no \" c r y s t a l l i z a t i o n , methanol was added and the solution then heated. This method produced an immediate p r e c i p i t a t i o n which was consi-dered to be due to the i n h i b i t o r y compound. The i n h i b i t o r y compound on the chromatogram was tested with various sprays outlined f o r coumarins and t h e i r deriva-t i v e s . In addition, sprays f o r aromatic acids were used. The r e s u l t s of a l l these t r i a l s proved negative; the com-pound remaining yellow (Table 2 0 ) . The spray tests outlined f o r indole and i t s derivatives yielded p o s i t i v e r e s u l t s . The compound was reactive to both the Salkowski and the E h r l i c h reagents, turning pink and pinkish brown respectively. The results obtained with the chromogenic sprays f o r indoles are presented i n Table 20. In order to confirm the presence of an indole nucleus, an u l t r a v i o l e t absorption spectrum was run (Figure 8). The absorption spectrum i s similar to that expected of compounds 0 with an indole nucleus, having a maximum at 2250 A and secon-dary maxima at 2700 A and 2790 A (91) . In order to characterize the unknown i n h i b i t o r y com-pound further, studies of the Rf's obtained with di f f e r e n t 62 Table 20. The i n h i b i t o r y band -color reactions with chromogenic sprays. Treatment C. repens CL, d i f f u s a C. maculosa V i s i b l e l i g h t U l t r a - v i o l e t (short wave) fa i n t yellow f a i n t yellow f a i n t yellow greenish yellow U l t r a - v i o l e t ( (long wave) NH3 vapour SPRAYS FOR COUMARIN 2 N-NaOE\" 2 B-Hcl 1% ethanolic A l CI3 - NH3 yellow* aqueous KMnOl^ . ethanolic FeCl 3 -1% aqueous FeCl^ SPRAYS FOR INDOLE COMPOUNDS FeCl3 \" HClOlf pink 1% P-dimethylamino-benzaldehyde pinkish mauve KN02-- HNO3 -C innamaldehyde yellow brown yellow brown greenish yellow yellow* pink pinkish mauve-greenish yellow yellow* pink pinkish mauve yellow brown yellow brown yellow brown yellow brown •fluorescence 6h solvents were made (Table 21). It was observed that the com-pound moved r a p i d l y with aqueous solvents and very slowly, being almost immobile, i n organic solvents. The Rf's obtained with synthetic indole acetamide and tryptophane are also pre-sented, as the i n h i b i t o r y compound was suspected of being one of these. The Rf• s for both compounds were run. Those f o r indole acetamide were not available i n the l i t e r a t u r e . The comparative study Indicated that the i n h i b i t o r was neither tryptophane nor indole acetamide and Rf's did not coincide with those of the known indoles. 65 Table 21. Rf values of the inhibitory compounds of Centaurea spp. in different solvents, compared with the Rf values of indole acetamide and tryptophane. Rf values Solvent Centaurea spp. inhibitor Indole acetamide Trypto-phane* Aqueous phase Butanol:acetic:water 4- : 1 : 5 0.90 0.7k 0.76 Organic phase Butanol:acetic:water 4- : 1 : 5 0.24-Water 0 . 8 2 0.64- 0.63 Is opropanol:ammonia:wat er 10 : 1 : 1 0.16 0.78 0.19 70% ethanol 0.60 0.76 0.4O Butanol:ammonia:wat er 100 : 3 : 18 0.10 m»mm Butanol:ethanol:water 4- : 1 : 1 0.12 0 . 8 0 0.26 * Some of the Rf values for tryptophane are recorded from literature, (740. 66 V. DISCUSSION The presence of n a t u r a l l y occurring Inhibitors i n plants i s now an established f a c t . These i n h i b i t o r s occur not only i n a wide v a r i e t y of plants but also i n d i f f e r e n t parts of the plant. It was also observed that the knapweeds grew i n dense infestations and outcompeted other species. The r a p i d i t y with which they have established themselves, and t h e i r tendency to exclude other species leads to the b e l i e f that the knapweeds could be making the s o i l t o x i c to other species and may con-t a i n some i n h i b i t o r y substances. The present work with the leaves, stems, seeds and roots of the three d i f f e r e n t knapweeds: Russian, spotted and d i f f u s e , has confirmed the presence of an i n h i b i t o r y substance i n a l l three of the species studied. The i n h i b i t o r y substance was found to be located mainly i n the leaves and i n smaller amounts i n the seed. No i n h i b i t o r y substance was found either i n the stem or the roots. It was also found that the knapweed infested s o i l was i n h i b i t o r y to the growth of tomatoes and barley. This leads one to believe that the t o x i c i t y found i n the knapweed',:soil i s due to the i n h i b i t o r of the leaves becoming incorporated into the s o i l , either by r a i n washing i t out of the leaves, or because the leaves are broken down i n the s o i l when they f a l l . Though the i n h i b i t o r was not found i n the roots i t would be wrong to assume that i t may not be excreted as a 67 root exudate. At the time of c o l l e c t i o n which was i n summer the i n h i b i t o r was found to be located mainly i n the leaves. In another season i t i s quite possible that the i n h i b i t o r could also be translocated to the roots. Though the presence of i n h i b i t o r s i n a number of spe-cies of plants has been established, yet the exact nature of the i n h i b i t o r y substance has been shown only i n a very few cases. In the present study, the i n h i b i t o r was found to be both ether and water soluble, being more soluble i n ether. By chromogenic sprays as well as by means of an u l t r a -v i o l e t absorption spectrum i t was found that the i n h i b i t o r y compound was an indole. S p e c i f i c colour reactions with the i n h i b i t o r y compound indicated that i t might be indole aceta-mide or tryptophane. However, comparison of Rf's with the synthetic compound's did not confirm either of these. It could be concluded,therefore, only that the i n h i b i t o r found i n the leaves of the knapweeds studied was an indole d e r i -vative, the exact nature of which was not known since the color reactions and Rf's did not coincide with any other known compound. The i n h i b i t o r y compound having been i d e n t i f i e d as an indole, provides further evidence that precursors of auxin, or auxin i t s e l f i n higher concentrations, could be an i n -h i b i t o r . The function of these i n h i b i t o r s can only be speculated upon. The i n h i b i t o r s may perform a part i n the dual regu-68 l a t i o n of growth. In the form of a precursor they might be i n h i b i t o r s , thereby causing such phenomena as dormancy. When converted to IAA they then could act as an auxin, promoting growth. They could also be responsible for the production of t o x i c materials which are sometimes poisonous to the parent plant. Usually, however, the i n h i b i t o r i s toxic to enzymes systems of other species, enabling the species pro-ducing i t to outcompete and exclude other forms. This may p a r t i a l l y explain why the knapweeds are r a p i d l y establishing themselves at the expense of other plants. 6.9 VI. CONCLUSION This study conducted on the presence, l o c a t i o n , nature and role of growth i n h i b i t i n g substances i n Centaurea spp. gave evidence of the following: 1. a growth i n h i b i t i n g f r a c t i o n i s present i n a l l three species of Centaureas studied 2. s o i l on which d i f f e r e n t knapweeds were growing was found to be i n h i b i t o r y to the growth of tomatoes 3. addition of a i r dried leaf powder of C. repens to greenhouse s o i l also produced i n h i b i t o r y effects on the growth of tomatoes the i n h i b i t o r i s mostly located i n the leaves of the three species tested, but i s present i n smaller amounts i n the seed of C. repens and C. maculosa 5. studies with the stems and roots did not i n d i -cate the presence of any i n h i b i t i n g substance 6. the i n h i b i t o r was found to be both ether and water soluble, though the i n h i b i t o r i n the leaves was more ether soluble, and that of the seeds was more water soluble 7. the i n h i b i t o r was located as a pale yellow f l u o r -escent band at an Rf of 0.9 8. as the i n h i b i t o r was water soluble the i n h i b i t o r y band could be eluted using water as the eluting solvent 9. attempts to c r y s t a l l i z e the water eluate of the i n h i b i t o r yielded negative r e s u l t s , however, addition of 70 methanol and evaporation resulted In p r e c i p i t a t i o n 10. chromogenic sprays and comparison of Rf's i n d i -cated that the unknown i n h i b i t o r was not coumarin or one of i t s derivatives 11. the sprays f o r indole compounds gave posi t i v e tests thereby i n d i c a t i n g that the i n h i b i t o r y compound i s an indole derivative 12. the u l t r a v i o l e t spectrum was also t y p i c a l of com-pounds having an indole nucleus 13. a comparison of the various spray tests showed s i m i l a r i t i e s between the unknown and both indole acetamide and tryptophane but Rf values i n various solvents would i n -dicate that i t i s neither. 71 VII BIBLIOGRAPHY 1. 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