ALLELOPATHY IN CUCUMBER (CUCUMIS SATIVUS L) Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY 7 RONALD HOLLIS LOCKERMAN 1977 ‘ \: rad-{lia- I} 3" J [PIE/iflY IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII7IIIIII .1: n 1:11C1: "33m SLate 1293 103963 UflVCLahy-JJ This is to certify that the thesis entitled ALLELOPATHY IN CUCUMBER (CUCUMIS SATIVUS L.) presented by Ronald Hollis Lockerman has been accepted towards fulfillment of the requirements for Ph. D. degree in HOPt‘ICUItUY‘e gig/5’ I. Pram. Major professor .WWW ‘ ABSTRACT ALLELOPATHY IN CUCUMBER (CUCUMIS SATIVUS L.) BY Ronald Hollis Lockerman Selected cucumber accessions were compared with the cv 'Pioneer' for growth interference of several weed indicator species. Plant introduction (PI) 16939l suppressed proso millet (Panicum miliaceum L.) populations l0-57% in three of five field evaluations, whereas 'Pioneer' had no suppressive effect on germination. Reduced weed suppression by cucumber coincided with increases in rainfall and soil organic matter content. Inhibition of weed germination decreased as the distance from cucumber to weed seed increased. Inhibition of germination was attributed to allelOpathy rather than competitive interactions. Growth analyses during the time period associated with biochemical interactions indicated that the allelopathic PI 169391 is not quantitatively superior to 'Pioneer' in net assimilation or relative growth rate. Leaf area ratios indicated that PI 169391 may have a greater competitive shading advantage than 'Pioneer'. Allelopathy appears to be the more important component of interference during the early growth stages of PI l69391. Ronald Hollis Lockerman Seed tissue and extract bioassays demonstrated that PI 169391 seed testa contain a water-soluble germination inhibitor which is both intra- and interspecific. Fermenta- tion, leaching, or the addition of the adsorbant activated charcoal eliminated the growth inhibition. Similar toxicity was obtained with PI 169391 fruit juice which may indicate the source of the seed coat toxin. Cucumber leaf and root extracts inhibited proso millet, but not cucumber growth. Application of leaf, root and juice extracts to soils of increasing adSorptive capacity decreased their toxicity. Applications of PI 169391 leaf, root and fruit tissue as a soil amendment could reduce weed populations under certain edaphic and environmental conditions. ALLELOPATHY IN CUCUMBER(CUCUMIS SATIVUS L.) By Ronald Hollis Lockerman A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1977 ACKNOWLEDGMENTS The author wishes to express his appreciation to Dr. Alan R. Putnam for both his friendship and guidance during the course of graduate study and in the prepara- tion of this thesis. Appreciation is also given to Drs. s. K. Ries, D. R. Dilley, o. Penner and M. J. Zabik for their editorial assistance. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . . v1 LIST OF FIGURES . . . . . . . . . . . . . . . . . . . viii INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 CHAPTER 1: LITERATURE REVIEW . . . . . . . . . . . . 3 Kolines in Higher Plants . . . . . . . . . . .V. . . 3 Classes of Kolines . . . . . . . . . . . . . . . . . 12 Mechanisms of Action of Kolines . . . . . . . . . . l6 Factors Influencing Koline Production . . . . . . . 17 CHAPTER 2: FIELD EVALUATION OF ALLELOPATHIC CUCUMBERS 19 Abstract . . . . . . . . . . . . . . . . . . . . . . l9 Introduction . . . . . . . . . . . . . . . . . . . . 20 Materials and Methods . . . . . . . . . . . . . . . 24 Selection for Field Evaluation . . . . . . . . . . 24 Primary Field Evaluations . . . . . . . . . . . . 25 Secondary Field Evaluations . . . . . . . . . . . 26 Results and Discussion . . . . . . . . . . . . . . . 28 Selection for Field Evaluation . . . . . . . . . . 28 Primary Field Evaluations . . . . . . . . . . . . 3] Secondary Field Evaluations . . . . . . . . . . . 36 Literature Cited . . . . . . . . . . . . . . . . . . 47 CHAPTER 3: MECHANISMS FOR DIFFERENTIAL GROWTH INTERFERENCE BY CUCUMBER . . . Abstract . Introduction . Materials and Methods Cucumber Exudate Bioassay Growth Analyses Results and Discussion . Cucumber Exudate Bioassay Growth Analyses Literature Cited . CHAPTER 4: ASSAYS FOR INHIBITORS ASSOCIATED WITH CUCUMBER SEED GERMINATION . . . . Abstract . . . . . . . .-. . . . . Introduction . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . Fermentation . . . . . . . . . . . . . . . . Leaching . Fruit Maturation . Seed Source . . . . . . . . . . . . . . . Activated Charcoal Bioassays Cucumber Interactions . . . . . Results and Discussion . Fermentation . . . . . . . . . . Leaching . . . . . . . . J . . . . . . . Fruit Maturation . 3 Seed Source iv 9 Page 49 , 49 SO 53 . 53 54 56 56 59 64 65 65 66 69 69 69 7O 7O 7T 7T 73 73 73 76 76 Page Activated Charcoal Bioassays . . . . . . . . . . . 79 Cucumber Interactions . . . . . . . . . . . . . . 81 Literature Cited . . . . . . . . . . . . . . . . . . 83 CHAPTER 5: ERRHTH INHIBITORS IN ALLELOPATHIC CUCUMBER L NTS . . . . . . . . . . . . . . . . . . 85 Abstract . . . . . . . . . . . . . . . . . . . . . . 85 Introduction . . . . . . . . . . . . . . . . . . . . 86 Materials and Methods . . . . . . . . . . . . . . . 88 Seed Tissue Bioassay . . . . . . . . . . . . . . . 88 Seed Extract Bioassay . . . . . . . . . . . . . . 88 Cucumber Plant Bioassay . . . . . . . . . . . . . 89 Cucumber Plant Bioassay in Different Media . . . . 90 Results and Discussion . . . . . . . . . . . . . . . 9l Seed Tissue Bioassay . . . . . . . . . . . . . . . 9l Seed Extract Bioassay . . . . . . . . . . . . . . 93 Cucumber Plant Bioassay . . . . . . . . . . . . . 93 Cucumber Plant Bioassay in Different Media . . . . 97 Literature Cited . . . . . . . . . . . . . . . . . . 99 SUMMARY AND CONCLUSION LIST OF REFERENCES . LIST OF TABLES CHAPTER 2 1. Growth interference of proso millet in association with selected cucumbers Suppression of proso millet by leachates from selected cucumbers Needs per plot and percent inhibition 10 days after planting in the presence and absence of selected cucumbers (l975) . . . . . . Needs per plot and percent inhibition lO days after planting in the presence and absence of selected cucumbers (l976) . Needs per plot and percent inhibition at harvest in the presence and absence of selected cucumbers (l975) . . . Average fresh weight per plot and percent inhibition at harvest in the presence and absence of selected cucumbers (l975) . Total weed growth per plot in the presence and absence of selected cucumbers (l975) . Average fresh weight per plant of cucumber vines grown in the presence and absence of weeds (l975) . . . . CHAPTER 4 l. The effect of fermented cucumber seed on germination of cucumber and proso millet . . The effect of leaching cucumber seed on germination of cucumber and proso millet . The effect of cucumber fruit maturity on cucumber and proso millet germination vi Page . 29 . 3O . 37 . 39 . 4T 43 . 44 . 46 . 75 . 77 The effect of seed source on cucumber and proso millet germination . The effect of cucumber leachate on growth of cucumber . . . . . . The effect of cucumber leachate on growth of proso millet . . CHAPTER 5 l. The effect of cucumber seed tissue on germina- tion and hypocotyl length of cucumber and proso millet . . . . . . The effect of partitioned cucumber seed extracts 94 on percent germination of proso millet . The effect of cucumber plant tissue extracts on germination and hypocotyl length of cucumber . The effect of cucumber plant tissue extracts on germination and hypocotyl length of proso millet . . . . . . . . . . . . . . . . The effect of PI l6939l plant tissue extracts on percent germination of proso millet in selected growing media vii Page . 78 . 8O , 82 . 92 , 95 , 96 , 98 CHAPTER 2 1. LIST OF FIGURES Page The fresh weight of indicator plants grown in the presence and absence of an allelopathic and non-allelopathic cucumber plant. The allelopathic accession inhibited proso millet and redroot pigweed at all spacings at the l% probability level and the F value for the interaction of spacing times accession was not significant . . . . . . . . . . . . . . . . . . 32 The emergence of indicator plants grown in the presence and absence of an allelopathic and non-allelopathic cucumber plant. The allelo- pathic accession inhibited emergence of proso millet at all spacings and redroot pigweed at O, l. 27, and 2.54 cm at the 5% probability level . . . . . . . . . 34 CHAPTER 3 l. The effect of cucumber leachate at 10 day growth intervals on percent germination and dry weight of proso millet. Each observation is the mean of 20 plants. * indicates that means are significantly different at the 1% probability level with Duncan S Multiple Range test . . . . . . . . . . . . . . . . . . 57 Net assimilation rate of cucumbers. Each observation is the mean of six plants . . . . . 60 Relative growth rate of cucumbers. Each observation is the mean of six plants . . . . . 6l Leaf area ratio of cucumbers. Each observation is the mean of six plants . . . . . . . . . . . 62 INTRODUCTION Plants growing in both natural and agronomic ecosystems continuously interact to influence their growth and subsequent survival. Some plants are segregated into exclusive associa- tions due to similar adaptability and response to specific environments. Many species are predominant due to a morpho- logical advantage, whereas, others may gain advantage by tolerance to unfavorable growth conditions. Undoubtedly, the composition of many plant communities is influenced by compe- titive parameters. However, all growth interference cannot be attributed to plant competition. It is well established that many lower organisms influ- ence each other biochemically. Penicillium notatum suppres- ses the growth of many bacteria. The importance of the utility of antibiotics has resulted in a rapid accumulation of Specific information on microbial biochemical interactions. Although postulation that certain plants release phytotoxins is not new, Specific knowledge involving allelopathic inter- actions among higher plants is limited. Toxic plant mechanisms are often obscure due to the similar effect of many competitive and biochemical growth parameters. Detailed investigations are necessary to deter- mine the components of plant interactions. The objective of this study was to identify and measure the components of plant growth interference in cucumber. The utility of allelo- pathic traits in crop plants for suppression of nondesirable plant species indicates potential as a supplemental means of weed control. Manipulation of plant interference may provide several effective and inexpensive techniques for integrated pest management. CHAPTER I LITERATURE REVIEW Kolines in higher plants. Kolines is the term used to des- cribe phytotoxins produced by higher plants. Knowledge of the utilization of natural plant products in pest management has existed for centuries. Democritus (5th century B. C.) advised soaking seeds in the juice of leek flowers to con- trol blight and Theophrastus advocated the use of olive juice to suppress tree root growth (86). DeCandolle (24) in l832 was one of the first scientists to suggest that one plant can produce and release a substance toxic to another plant. Liebig (56) originally supported DeCandolle's theory but later as a result of his depletion of soil minerals theory, discounted toxic plant substances as being important in regulating plant growth. Until recent- ly, plant growth interference has been almost unanimously interpreted as resulting from competition. According to Loehwing (58), the l9th century literature did not provide evidence for an important role of any toxic substance in normally aerated soils. The classical report of Stickney and Hay (88) in 188] on the injurious effect of black walnut (Juglans nigra L.) to surrounding vegetation was not accepted as a biochemical interaction until the middle of the century. Pickering (7l) in 1903 suggested that an inhibitory effect of grass on the growth of apple trees was due to the release of toxic 3 4 substances. Nutrient competition and oxygen exclusion was ruled out by experimentation and soil poisoning was conclud- ed to be the causal factor. The toxic theory was further emphasized by Schreiner et al. (83) when water cultures from monoculture soils were found to be toxic to wheat seedlings. Toxic chemicals were isolated and identified as picolinic acid, salicylaldehyde, vanillin and dihydroxystearic acid. Davis (23) provided evidence for the existence of toxic interactions among plants with the identification of juglone as the substance associated with the injurious effect of black walnut. In 1905, Livingston (57) proposed that plants fail to grow in peat bogs because of toxic chemical substances. Cowles (l8) suggested in l9ll that plant toxins were important as causative agents in plant succession. Annual plants rarely occur in the Rosmarino-Ericion in France (25). Field soil from this shrub association con- tained toxic compounds that inhibited the growth of numerous annuals. Parks of black locust (Robinia pseudoacacia L.) are nearly devoid of all other vegetation, and bark and wood of black locust contain compounds toxic to barley (Hordeum vulgare L.) (94). Went (96) reported that certain organic compounds endogenously produced by plants stimulate or retard the growth of other plants. In some cases, the liberation of organic substances from higher plants has been associated with the 'soil sick- ness' phenomenon. The difficulty of re-establishing peach (Prunus Persica (L.) Batsch.) trees in old peach orchards 5 has been recognized for years. Proebsting and Gilmore (72) showed that soil incorporated peach roots are toxic to peach seedlings. The effect was duplicated by alcohol extracts of peach roots with the alcohol-insoluble residue being non- toxic to peach trees. It was further demonstrated that the peach roots contain a growth inhibitor but it was not estab- lished that the substance leaches into the soil or is active under field conditions. Benedict (6) reported a condition known as 'sodbinding' in which old bromegrass (Bromus inermis L.) stands thin out. Leachates from old plants growing in sand culture were toxic to seedlings. Toxicity was further demonstrated by incorporating dried bromegrass roots into the media where seedlings were grown. However, it was not shown that toxic substances from bromegrass roots accumulate in the soil of old stands. Here again, the presence of a toxic substance in plant parts was established but inhibi- tion of plant growth under field conditions could not be directly attributed to the substance. Recent discoveries of natural occurring plant growth regulators have renewed interest in the biochemical influ- ence of higher plants on each other. Molisch (63) coined the term allelopathy in 1937 to refer to any biochemical interaction among plants and microorganisms. The suggestion that allelopathy should cover both detrimental and benefi- cial biochemical interactions is erroneous since the term is derived from two Greek words meaning mutual harm. Grummer (4l) in l955 suggested a system of naming natural occurring inhibitory plant compounds on the basis of the type of plant that produced the toxin and the type of plant affected. Antibiotic was suggested to describe a chemical inhibitor produced by a microorganism that is ef- fective against another microorganism. Phytoncide was pro- posed as a term for an inhibitor produced by a higher plant that is effective against another microorganism. Further- more, the term marasmims was suggested for compounds pro- duced by microorganisms that are harmful to higher plants and koline was defined as a chemical produced by higher plants which is toxic to other plants. The mutual interactions of microorganisms and higher plants are now grouped into individual areas, with allelo- pathy being used to refer to the effect of a higher plant on another. The currently accepted definition of allelo- pathy as pr0posed by Rice (76) in 1974 refers to any direct or indirect harmful effect by one plant on another through the production of chemical compounds released into the environment. The occurrence of allelopathy is dependent upon a chemical compound being added to the environment. Therefore, allelopathy can be differentiated from competi- tion which involves the reduction or removal of some growth factor from the environment that is required by other organisms sharing the same habitat. Muller (65) has suggest- ed the term plant interference for situations where both allelopathy and competitive parameters are evident and inseparable. Many plant growth responses have been directly associ- ated with allelopathy. Bonner and Galston (10) observed that the edge rows in guayule (Parthenium argentatum L.) plantings had much larger plants than the center rows and that the differences could not be eliminated by heavy water- ing and/or mineral application. Additionally, roots of adjacent plants did not intermingle but grew in separate areas and the guayule seedlings never grew under large guayule plants. Conversely, the seedlings were commonly found to grow under other shrubs. Experiments with nutrient solutions and distilled water leachates from guayule roots enabled Bonner (9) to identify the principle compound as trans-cinnamic acid, a substance highly toxic to guayule seedlings with growth reduction resulting from as little as 1.0 mg/l of culture solution. The toxic substances produced and released by guayule are rapidly destroyed under cultiva- tion as evidenced by toxicity being associated only with the outside rows in cultivated guayule plantings. Curtis and Cottam (21) reported that the fairy-ring pattern of the prairie sunflower (Helianthus rigidus L.) is due to an autotoxic inhibitor. A large reduction in plant numbers and flowers was evident in the center of the clone and additional water and nutrients did not alleviate the toxicity. However, when prairie sunflower plants were grown in soil from which all its roots and rhizomes were removed, the plants grew normally and flowered. It was concluded that inhibition resulted fromthe degradation of plant parts. 8 Substances capable of inhibiting the germination of tobacco seeds and the respiration and growth of seedlings have been isolated from residues of timothy (Phleum pratense L.) and rye (Secale cereale L.) (70). Mergen (62) in 1959 reported that natural succession appears to be slow in areas containing tree-of—heaven (Ailanthus altissima L.). Alcohol extracts of the rachis, leaflets, and stems of this plant caused rapid wilting of similar plants when applied to the cut surface of a stem. Toxicity also occurred when these extracts were applied to 35 gymnosperm and 11 angiosperm species. Approach grafting of Ailanthus with several species gave results similar to those with extracts. Flax (Linum usitatissimum L.) stands are reduced when a small percentage of flaxweed (Camelina alyssum L.) is grow- ing among them (43). Plants in close proximity to flaxweed plants produced 40% less dry matter than control plants in which the same amount of water was applied directly to the soil instead of allowing it to fall on the leaves and drip to the soil. Twenty species of native trees, shrubs, and grasses in northern Arizona inhibit the growth of wheat (Triticum aestivum L.) radicles (49). Schlatterer et al.(80) suggests that arid regions are abundant with allelopathic compounds due to the lower leaching effects caused by sparse rainfall. There are numerous examples of weeds suppressing the growth of other weeds or crop plants, but there is limited information on the economic potential of crop plants being biochemically injurious to weeds. Putnam and Duke (73) in 1974 evaluated the world collection of cucumber (Cucumis sativus L.) and identified several aCcessions toxic to proso millet (Panicum miliaceum L.) and white mustard (Brassica DIXIE Moench.) in sand culture under controlled environmental conditions. One accession inhibited indicator plant growth by 87% and 25 inhibited growth by 50% or more. The transfer of toxic leachates from pots containing inhibitory cucumbers to indicator plants germinated in separate containers con- firmed allelopathy. Several plant organs as well as different plant species have been identified as being allelopathic. Leaf secretions appear to play a major role in nature as plants interfere for space. According to Rice (76), leaves are the most consistent source of growth inhibitors. Volatile secretions from the burning bush of Moses (Dictamnus alba L.) are so strong that the air surrounding the shrub can be burned during calm weather (1). Seedling growth and seed germination of fennel (Foeniculum vulgare Mill.) is inhibited by incorporating the leaves of wormwood (Artemisia absinthium L.) into the soil (1). Encelia farinosa L. produces 3-acetyl-6-methoxybenzal- dehyde which is toxic to the growth of many plants, but not Encelia (38). The inhibitor produced and released by the leaves is non-volatile, heat stable, and appears to be a neutral compound. The active material was isolated from the leaves by successive fractionations with benzene, water, lO benzene, and petroleum ether. Recrystallization from ether yielded 0.5 mg of inhibitor per original gram of leaf tissue. Ten grams of Encelia leaves per tomato plant caused severe growth suppression and a 2X rate was lethal under field conditions. Chrysanthemum (Chrysanthemum morifolium L.) produces a phytotoxin that leaches from the leaves and is inhibitory to the growth and development of other Chrysanthe- mums (54). The question of active or passive release of growth inhibitors from plants is still obscure. Oxalic acid is reported to be released by living root tissue of wood sorrel (Oxalis stricta L.) (34) and viable legume roots are thought to release large quantities of toxic amino acids (92,93). Recently, it has been demonstrated that numerous kinds of organic compounds can be exuded from the roots of donor plants and absorbed by adjacent plants (32,39,48,79). Accord- ing to Rice (76), it is very difficult to determine if com- pounds on the outside of the roots are exuded or result from the sloughing off of dead outer cells. Eberhardt and Martin (27) with the aid of the florescence microscope observed the secretion of scopoletin (7-hydroxy-6-methoxy-coumarin) from oat (Avena sativa L.) root cells growing in distilled water. This seems to be the only case in which secretions from liv- ing cells have been experimentally demonstrated. Further- more, they found that secretion was greater under unfavorable growth conditions. There are very few cases where inhibitory root exudates ll of crop plants have been implicated. Schreiner and Reed (8l) and Schreiner and Sullivan (84) have reported toxic root exudates for wheat, oats, and cowpeas (Vigna unguiculata (L.) Nalp.). Conversely, there are numerous suggestions of toxic root exudations for noncrop species (2,5,8,12,68,69,74,75, 78,99). Fruits and seed possess and secrete growth suppressing substances. Many allelopathic effects may be a secondary consequence of self-imposed dormancy, resistance to seed decay and inherent anti-infection mechanisms. The release of endogenous protective toxins into the environment may well be a common strategy for survival. The occurrence of substances inhibiting germination is a common phenomenon and has been found in more than a hundred species (1). Cox et al. (19) reported the presence of water-soluble seed germination inhibitors in the testa of cabbage (Brassica oleracea L. var. capitata). Germinating beet (Beta vulgaris L.) seeds liber- ate ammonia which prevents the germination of corn cockle (Agrostemma githago L.) (29). These effects are similar to the natural function of antibiotics excreted by soil fungi and bacteria which inhibit or destroy other organisms. Many germination inhibitors appear to be nonspecific (29). Seed leachates of red clover (Trifolium pratense L.) and the fruits of table beets inhibited the germination of seeds in 28 species belonging to 14 families (35,36). Kuhn et al. (55) reported that mountain ash (Sorbus aucuparia L.) produces parasorbic acid which inhibits germination of field 12 cress (Lepedium campestre L.) seeds in a dilution of 1:1000 and allows 10-80% germination at l:l0,000. Ahshapanek (3) found that seed extracts of buffalobur (Solanum rostratum Dunal) are inhibitory to buffalobur seedlings. Many fruit juices also contain high concentrations of growth inhibitors. Tomato (Lycopersicon esculentum Mill.) juice inhibits the germination of wheat and oats (53). The fruit juice of Solanum coagulans inhibits the germination of wheat until diluted to 1:64 (29). Classes of kolines. Growth inhibitors have been found in most families of the plant kingdom. Their presence seems to be wide spread and not restricted to phanerogamous plants. Many of the chemical inhibitors have been termed secondary compounds because they occur sporadically and do not appear to play a role in basic metabolism (33,97). According to Rice (76), there are many thousand secondary compounds, but only a limited number have been implicated in allelopathy. Many of the growth inhibitors that have been identified are phenolics or derivatives of phenolic compounds (15). Aamisepp et al. (1) states that flavones, unsaturated lac- tones, and phenols constitute the majority of known inhibitors. Rice (76) proposes that most growth inhibitors arise through the acetate or the shikimic acid pathway. Amino acids, nucleosidic and proteinaceous compounds originate via the acetate pathway and inhibitors originating from amino acids are formed through the shikimic acid cycle. Whittaker I3 and Feeny (97) indicate that most toxic compounds are classi- fied as phenylpropanes, acetagenins, terpenoids, steroids, and alkaloids. They further pointed out that phenylpropanes and alkaloids originate from a small number of amino acids and the rest originate from acetate. The flavonoid compounds are considered hybrids because one ring arises from phenyla- lanime and the other forms from acetate. Simple water-soluble organic acids, straight-chain alcohols, aliphatic aldehydes and ketones have been identi- fied as natural plant growth inhibitors. Acetaldehydes, propionic aldehyde, acetone, methanol, and ethanol are released as volatile growth inhibitors by beet, tomato and common morninglory (Ipomoea purpurea (L.) Roth.) leaves and by carrot (Daucus carota L.) roots in a closed environment (22). Patrick et al. (70) reported that acetic and butyric acids are among the toxins produced by decomposing rye. The natural occurring unsaturated lactone, patulin, is toxic to many higher plants (67). Penicillum expansum produces patulin during decomposition of apple root and leaf residues (11). It is further stated that this may be implicated in the apple replant problem.‘. Juglone (5-hydroxy-a-naphthaquinone) is the only quinone that has been identified as a natural growth inhibitor (76). Robinson (77) reports that higher plants produce a variety of terpenoids but only a small number have been associated with plant toxicity. The monoterpenoids are the major components of the essential oils of plants and also represent 14 the largest group of terpenoid inhibitors that have been identified as toxic to higher plants. Muller et al. (66) identified camphene, camphor, cineole, dipentene, c-pinene, and 3-pinene as the volatile inhibitors produced by three species of dessert sage. Wormwood produces three sesquiter- pene inhibitors, B-carophyllene, bisabolene, and chamazulene (42). The cinnamic acid derivatives have been commonly identi— fied as higher plant toxins. Vanillic and p-hydroxybenzoic acid are the most common benzoic acid derivatives that have been identified as allelopathic agents (76). Guenzi and McCalla (44) reported occurrence of toxic cinnamic acid derivatives in wheat, sorghum (Sorgum vulgare Pers.), and oats, whereas, Schreiner and Reed (82) demonstrated that cin- namic acid, g-coumaric and g-hydrocoumaric acid are produced by higher plants and are toxic to seedling growth of many plants. Caffeic, ferulic and chlorgenic acids have also been implicated as allelopathic agents (29). Coumarins are widely distributed in the plant kingdom and several are involved in allelopathic interactions. Scopoletin and scopolin have been reported as inhibitors in oat roots (30,37,60). Van Sumere and Massart (91) list several coumarins as inhibitors of seed germination in many species from several families of plants. Flavonoid compounds are widespread in seed plants (45). Aglycones and glycosides are extremely potent toxins to seed germination (76). Kohlmuenzer (52) identified the flavonoid, 15 diosmetin trioside as one of the growth inhibitors in bed- straw (Galium mollugg_L.). Quercitrin has been identified as an additional inhibitor in the leaves of wormwood (42). Tannins are widespread in dicotyledonous plants but only a few have been reported as possible allelopathic com- pounds. Corcoran et al. (16) reported that B-l-O-galloyl- D-glucose inhibits hypocotyl growth induced by gibberellic acid in cucumber seedlings. Plant residues that contain hydrolyzable tannins often contain gallic or ellagic acid which are potent inhibitors of lower plant life (76). Tan- nins in the grains of certain sorghum hybrids inhibit pre- harvest seed germination (46). Although Evenari (29) emphasized the importance of alkaloids as seed germination inhibitors, Rice (76) indicated that there is no recent evidence of alkaloids playing a role in toxic plant interactions. No sulfur compounds have been identified as allelopathic. Robinson (77) indicated that there is no conclusive evidence for the production of di- or polysulfides in plants and that they probably arise through secondary transformations initi- ated by plant enzymes such as the production of allicum when garlic (Allium sativum L.) is crushed. The only purine nucleoside that has been identified in allelopathic reactions is caffeine (76). Evenari (29) includes caffeine as one of the most potent inhibitors of seed germination. Miscellaneous compounds such as ethylene (63), abscisic acid (4) and agropyrene (29) which is produced by quackgrass I6 (Agropyron repens (L.) Beauv.) have been implicated as possible allelopathic agents. Abscisic acid and 6-methoxy- 2-benzoxazolinone have been identified as growth inhibiting compounds in the primary roots of corn (98). Gross (40) suggests that auxins, cytokinins and gibberellins may be involved in allelopathic plant interactions. Mechanisms of action of kolines. There is a limited amount of information on the mode of action of natural occurring plant growth inhibitors, however, they apparently affect a variety of plant processes. A saturated aqueous solution of coumarin blocked mitosis in onion (Allium cepa L.) roots within 2 to 3 hours (17). Jensen and Melbourne (50) found a decrease in pea (Pisum sativum L.) root cell numbers 4 to 8 hours after treatment with an aqueous extract of black walnut hulls and trans-cinnamic acid. Volatile terpenes from sage leaf tissue inhibited mitosis in roots of cucumber seedling (64). Toxins from perennial sowthistle (Sonchus arvensis L.), common lambsquarters (Chenopodium album L.), and Canada thistle (Cirsium arvense L.) reduced cell division in wheat and rye (l3). Croak (20) reported that ferulic acid regulates the uptake of four macronutrients and three micronutrients. Scepoletin inhibits photosynthesis of tobacco and redroot pigweed (Amaranthus retroflexus L.) (28). The flavonoids, naringenin and 2',4,4'-trihydroxychalcone inhibits oxidative- phosphorylation in higher plants (87). Turner (90) reported 17 that victorin reduces the transpiration rate of oats. Some growth inhibitor effects are highly specific. Hydrolyzable tannins inactivate peroxidase and catalase, while a condensed waffle tannin inhibits the activity of polygalacturonase (7). It is evident that some allelopathic agents inhibit pectolytic enzyme processes. Schwimmer (85) found that chlorogenic acid, caffeic acid and catechol inhi- bit phosphorylase activity in potato. Cysteine and gluta- thoine have a pronounced effect in deactivating parasorbic acid in higher plants (47). Factors influencing koline production. Many factors affect the quantity of growth inhibitors produced by higher plants. Ionizing radiation increases phenolic inhibitors in tobacco and sunflower (31,51). Lott (59) increased the chlorogenic content of tobacco (Nicotiana tabacum L. var. Mont Calme brun) 550% in the greenhouse by supplemental uv radiation. Long days increase phenolic acids and terpenes in plants regardless of the photo-inductive cycle needed for flowering (14,89,100). Natanabe et al. (95) discovered a 20-fold increase in scopoletin content of tobacco plants growing in a boron-free solution for 38 days. Subjection of sunflow- er plants to water stress with a NaCl culture solution resulted in a 16-fold increase in isochlorogenic acids (26). Martin et al. (61) found that 7 times as much scopoletin exuded from roots of oat plants in 72 hours at 30 C than at 135 hours at 19 C. Exudation is no indication of the amount 18 that is actually produced by a plant, but may represent the potential intensity of an allelopathic mechanism. Koeppe et al. (51) reported that scopolin and chlorogenic acid content decreased with age of tobacco leaves. The occurrence of biochemical interactions among plants is no longer obscure. Natural plant toxins may be directly or indirectly responsible for the regulation of species distribution and density in natural plant communi- ties, and furthermore, may influence weed and crop growth in agroecosystems. CHAPTER 2 FIELD EVALUATION OF ALLELOPATHIC CUCUMBERS ABSTRACT Cucumber (Cucumis sativus L.) accessions which had demonstrated allelopathy under controlled environmental conditions were evaluated against indicator weeds in several field tests.over three seasons. Plant introduction (PI) l6939l gave the greatest reduction in populations of proso millet (Panicum miliaceum L.) with a range of 43 to 90% of control in three of five evaluations. The reduction in interference by cucumbers coincided with increases in rainfall and soil organic matter content. Plant introduc- tion 285605 was approximately half as effective as P1169391 in suppressing weed growth. Toxicity of PI 169391 to proso millet and redroot pigweed (Amaranthus retroflexus L.) decreased as the distance from cucumber to weed seed was increased. Early reduction of weed numbers was attributed to allelopathy rather than competition. Although weed control with allelopathic cucumbers was not consistent in the field, these tests demonstrated that excellent activity could be obtained under certain edaphic and environmental conditions. 19 20 INTRODUCTION Higher plant species influence one another in both natural and agronomic plant communities. This can be par- tially attributed to their differential ability to compete for abiotic growth factors, however, suppression of growth cannot always be explained by competition. Although know- ledge of toxic biochemical interactions among higher plants is still limited, allelopathy has been observed in many ecosystems (16). The term allelopathy was coined by Molisch in 1937 to refer to any biochemical interaction among plants,including microorganisms (10). Although Molisch's definition includes both detrimental and beneficial interactions, Rice (16) recently proposed that allelopathy should be defined as any direct or indirect harmful effect by one plant on another through the production of chemical compounds released into the environment. By accepting Rice's definition, competi- tion can definitively be separated from allelopathy. Plant competition occurs through a reduction or removal of a growth factor needed by both plants, whereas, allelopathy occurs by the addition of a toxic factor to the environment. Until recently, allelopathy was commonly considered a part of competition or completely ignored by scientists. 2] Only in the last few years has there been an increasing awareness of the importance of separating these two concepts. Even so, there are inherent limitations in identifying an allelopathic response since many competitive and allelopath— ic effects are difficult to separate experimentally. Cultivars within species with superior competitiveness have been reported and may often be characterized as vigor- ous plants with large root systems or leaf canopies. In addition, there are numerous examples of growth suppression that can be directly associated with allelopathy. Inhibi- tion of germination and acute root inhibition among seeds and seedlings during the early stage of growth and develop- ment are indicative of allelopathy. Growth inhibition by leachates introduced from plants grown in separate contain- ers is excellent evidence for allelopathy (15). Competition may become a crucial factor in growth sup- pression during late season in the field, especially if one plant gains advantage by biochemically suppressing another during the initial stages of growth. Total growth suppres- sion of a plant at maturity is the result of the combined effect of allelopathy and competition. In view of this, plant interference better describes overall deleterious effects of one plant on another, encompassing both allelo- pathy and competition (11). Interference should probably be substituted for the term competition in many previously published papers on weed-crop interactions. De Candolle in 1835 was one of the first scientists to 22 postulate that plants may excrete compounds injurious to other plants (3). Since that time there have been numerous classical examples of interference among higher plants in which allelopathy is implicated (2,4,6,7,9,17,l8). Patrick et al. (14) demonstrated that toxic products from decaying plant residues are produced under field con- ditions. Hater extracts of large crabgrass (Digitaria sanguinalis (L.) Scop.) have also been shown to inhibit the germination of coronilla (Coronilla varia L.) (l). Neustruyeva and Dobersova reported in 1974 that wheat (Triticum aestivum L.), oats (Avena fatua L.), peas (Pisum, sativum L.), and buckwheat (Fagopyrum esculentum Moench.) suppress common lambsquarters (Chenopodium album L.) through biochemical activity (12). Recently, Rice (16) has compiled a thorough review of the literature on allelopathy which indicates common occurrence of this phenomenon among higher plants. In agronomic cases, most of the allelopathic evidence has been associated with the effect of weeds on crops and crops on crops, however, an important economical potential of allelopathy may be the ability of crops to suppress weeds. Disease and insect resistance as well as Stress adaptations have been genetically incorporated into commercial cultivars from wild types. Therefore, the possibility exists that weed suppressing traits may be derived from prototypes possessing an allelopathic mechanism which favors their establishment in natural plant communities. Incorporation 23 of an allelopathic trait into commercial cultivars may enable cr0p plants to gain an advantage over other species through biochemical activity and subsequent competitiveness. In 1974, Putnam and Duke screened the world collection of cucumber (Cucumis sativus L.) against proso millet (Panicum miliaceum L.) and white mustard (Brassica hirta Moench.) (15). 0f the plant introductions (PI) tested in a controlled environment, one accession inhibited indicator plant growth 87% and 25 inhibited growth by 50% or more. Leachates transferred from several toxic PIs to proso millet grown in separate containers inhibited emergence and plant growth. It was demonstrated that within the world collec- tion of cucumber there are accessions capable of biochemi- cally inhibiting the growth of certain species in relatively sterile media. To demonstrate the economic potential for weed suppres- sion by a crop it was necessary to determine if the allelo— pathic cucumber accessions were active in the field. The objective was to determine if selected cucumber accessions could inhibit the growth of several economically important weeds. It is hypothesized that with cucumber, inhibition of germination and early stage weed suppression would be sufficient to allow a vigorous vining crap to gain an advantage over weeds in a field Situation. Additionally, high density cucumber plant populations would favor weed suppression in the field. 24 MATERIALS AND METHODS Selection for field evaluation. Fifty-one accessions of cucumber and the commercial hybrid cv 'Pioneer' were evaluated against proso millet in 10.2 Styrofoam pots con- taining sterilized spinks loamy sand (1.47% organic matter). Twenty indicator seed were planted in a 5 cm diameter circle around each cucumber seed with controls maintained in the absence of cucumber. Plants were grown in a randomized complete block design with ten blocks. A 16 hr day-length was maintained using supplemental cool white fluorescent light (160 u E m'zsec'l), and the approximate day and night temperature was 32 and 210, respectively. Treatments received 50 ml of half-strength Hoagland's solution at 24 hr intervals after initial satura- tion. Suppressive growth effects were recorded as inhibition of germination and suppression of shoot fresh weight 28 days after planting. Allelopathy was verified as being partially responsible forgrowth interference in the initial selection screen by applying cucumber leachate to indicator plants grown in the absence of cucumber plants. Three accessions (PI 285605, 169391, and 175694) that had previously been shown to inter— fere with the growth of proso millet, and the non-allelopathic 25 cv 'Pioneer' were grown in 30.5 cm Styrofoam pots containing sterilized quartz sand. Four cucumber seeds were placed in each pot and leached every 12 hr for15 days with 300 ml of distilled water. A 16 hr day-length was maintained in the greenhouse using supplemental cool white fluorescent light and the approximate day and night temperature was 29 and 18(3, respectively. Cucumber leachate was collected in aluminum pans and applied daily to 10 replications of 20 proso millet seed in 10.2 cm diameter Styrofoam pots. Growth reduction was re- corded as inhibition of germination and shoot fresh weight 15 days after planting. Primaryifield evaluation. Plant introduction 169391 and the commercial cv 'Pioneer' were field tested during August 1974 against proso millet and redroot pigweed (Amaranthus retroflexus L.) at the Horticultural Research Farm in East Lansing. Twenty indicator weed seed were planted in a circle at diameter spacings of 0, 1.27, 2.54, and 5.08 cm around each cucumber seed with controls maintained in the absence of cucumber. Plants were grown in a randomized complete block design with five blocks. Spinks loamy sand containing 1.47% organ- ic matter was fumigated with methyl bromide in early June to eliminate the existing weed population. Two months prior to planting, 66 kg/ha N, K20 and P205 was broadcast over the experimental area. Rainfall was supplemented with sprinkler 26 irrigation to maintain a minimum of 1.27 ha cm per 72 hr. Inhibition of germination and suppression of shoot.fresh weight were recorded 28 days after planting. Secondary field evaluatiOns. Cucumber accessions PI 169391, 285605, and the commercial cv 'Pioneer' were evaluated at high density populations against a broad spectrum weed popu- lation in 1975 and 1976. The field evaluations included tests on the same soil previously used and an additional test the second year on a Miami silt loam with 3.0% organic matter. Four weeks prior to planting, 55 kg/ha of N, K20 and P205 fertilizer was broadcast over the experimental area supplemented with 60 kg/ha of N 4 weeks after cucumber emer- gence. Four grasses, large crabgrass, proso millet, barn- yardgrass (Echinochloa crus-galli (L.) Beauv.), yellow foxtail (Setaria glauca (L.) Beauv.) and four broadleaves, prostrate pigweed (Amaranthus graecizans L.), redroot pigweed, common ragweed (Ambrosia artemisiifolia L.), and common lambsquarters were overseeded prior to planting. Twenty-five cucumber seeds were planted on the square, 23 cm apart in 1.3 m2 plots, and weeded controls were main- tained in the absence of cucumbers. Within blocks a comparison of hand weeding and no weeding effects on the growth and development of cucumber was made in 1975. Needed plots were rogued twice a week to eliminate any effects from other plant species on growth of cucumber. The condi- tions of the 1976 tests were similar to the previous year 27 with the following exceptions: a) hand-weeded plots were eliminated, and b) PI 285605 was not included. Plants were grown in a randomized complete block design with four blocks. Rainfall was supplemented with sprinkler irrigation to maintain a minimum of 1.27 ha cm per 72 hr. The weed population in all tests was recorded by indi- vidual species 2 weeks after planting and at cucumber maturity. Average fresh weight for individual weed species, cucumber vine fresh weight and total plant population were recorded at harvest in 1975. Harvest date was determined by fruit maturity associated with once-over mechanical harvesting of commercial pickling cucumbers. 28 RESULTS AND DISCUSSION Selection for field evaluation. From the fifty-one acces- sions evaluated for growth interference in sand culture, PI'S 285605, 169391, and 175694 suppressed fresh weight and germination of proso millet 78, 70, and 70%, respectively (Table 1). The preliminary investigation by Putnam and Duke showed that fifteen accessions of the world collection of cucumbers inhibited growth of indicator species by more than 75%. The results of the field experiments compared favorably to the earlier experiments in sand culture. Fresh weight inhibition by the cucumber accessions after 28 days must be attributed to the combined influences of competition and allelopathy. Inhibition by 'Pioneer' was attributed primarily to competition since leachates trans- ferred to indicator plants showed no suppression of fresh weight or germination. Inhibition of germination is logi- cally attributed to allelopathy. Leachates from the cucumber accessions applied to proso millet in separate containers inhibited emergence and plant growth while leachates from the control and 'Pioneer' had no suppressive effects (Table 2). These data support the previous report that 'Pioneer' is non—allelopathic (15). PI 169391 was the most effective, suppressing fresh weight 29 Table 1. Growth interference of proso millet in association with selected cucumbers. Inhibitiona Accession or Fresh weight Germination cultivar (%) (%) PI 285605 78 b 66 b PI 169391 70 b 68 b PI 175694 70 b 66 b 'Pioneer' 27 a O a aMeans within a column followed by the same letter are not significantly different at the 5% probability level with Duncan's Multiple Range test. 30 Table 2. Suppression of proso millet by leachates from selected cucumbers. Inhibitionb Accession or Fresh weighta Growth Germination cultivar (mg) (%) (%) No cucumber 42 c - - 'Pioneer' 44 c - - PI 285605 27 b 39 a 21 a PI 169391 20 a 55 b 42 b PI 175694 29 b 34 a 26 a aMeans within a column followed by the same letter are not significantly different at the 5% probability level with Duncan's Multiple Range test. bInhibition was calculated by using 'Pioneer‘ as a control. 31 and germination 55 and 42%, respectively. Selection of accessions for field evaluation was based on inhibition of germination, suppression of fresh weight and the consistency within a given accession. The initial investigations with allelopathic cucumbers demonstrated that accessions and cultivars differed greatly in their ability to alter plant growth in sand culture (15). These tests in soil also indiCated a wide range of variation with- in a given accession as well as among accessions. PI 169391 was selected for the primary field evaluation since it pro- vided the most consistent growth inhibition and germination and growth was more uniform than either 175694 or 285605. Primary field evaluations. There were no significant differ- ences between the fresh weight of proso millet and redroot pigweed grown in the presence of 'Pioneer' or in the absence of cucumber (Figure 1). However, PI l6939l significantly inhibited the fresh weight of both indicator Species at all spacings. There was no difference in cucumber plant weights among spacings indicating the absence of a dilution effect. Maximum growth suppression of redroot pigweed and proso millet was respectively 73 and 90% at the closest spacing to the cucumber plants. There was no significant difference in indicator emer- gence between the control and 'Pioneer' treatments (Figure 2). The allelopathic plant introduction suppressed indicator emergence at all spacings except 5.08 cm for redroot pigweed. Figure 1. 32 The fresh weight of indicator plants grown in the presence and absence of an allelo- pathic and non-allelopathic cucumber plant. The allelopathic accession inhibited proso millet and redroot pigweed at all spacings at the 1% probability level and the F value for the interaction of spacing times accession was not significant. FRESH WT (g) 33 P3080 IILLIT —CON'I'ROI. <- . PIONEER 'I’llpl 160391 RIDEOOT PIGWIID O 1.27 2.54 5.08 SPACING FROM CUCUMBER (cm) Figure 2. 34 The emergence of indicator plants grown in the presence and absence of an allelo— pathic and non-allelopathic cucumber plant. The allelopathic accession inhibited emer— gence of proso millet at all spacings and redroot pigweed at 0, 1.27, and 2.54 cm at the 5% probability level. 35 PROSO MILLIT 20 15 10 lo- lani. ner- and O REDROOT PIGWEED a N PLANT NUMBER d O _ CONTROL -- PIONEER 4 s\\\‘ Pl 169391 0 1.27 2.54 5.08 SPACING FROM CUCUMBER (cm) 36 Toxicity of PI 169391 to emergence of proso millet and redroot pigweed decreased as the distance from cucumber seed to weed seed increased. The difference between cucum- ber lines for the spacing extremes was greater for redroot pigweed than proso millet. This may be explained on the basis of differential susceptibility among proso millet and redroot pigweed or the presence of a toxic gradient. Addi- tionally, the root system of proso millet may have had a greater lateral distribution than redroot pigweed resulting in more contact near the cucumber seed. The growth and development of a plant is often modified by the proximity of other plants. In a field situation it is often very difficult to separate the influence of compe- tition and allelopathy on suppression of plant weight. However, suppression of germination during the early stage of growth and development can best be attributed to allelopathy. Secondary field evaluations. Emergence of proso millet and barnyardgrass was suppressed 10 days after planting by PI 169391 and 285605 in 1975 (Table 3). This suppression demonstrates that allelopathy occurs early in the growth and development of field grown cucumbers. The similarity in the control and 'Pioneer' weed population supports pre- vious evidence indicating that the commercial cultivar is non-allelopathic. Plant introduction 285605 was approxi— mately half as effective in suppressing proso millet and 37 Table 3. Weeds per plot and percent inhibition 10 days after planting in the presence and absence of selected cucumbers (1975). Speciesa Accession or Proso millet Barnyardgrass Redroot pigweed Cultivar (No.) (%)b (No.) (%) (No.) (%) No cucumber 25 c - 31 c - 58 c - 'Pioneer' 21 c - 26 c - 39 ab - PI 169391 4 a 81 7 a 73 23 a 41 PI 285605 11 b 48 17 b 35 41 ab 0 aMeans within a column followed by the same letter are not significantly different at the 5% probability level with Duncan's Multiple Range test. bPercent inhibition calculated by using 'Pioneer' as a control. 38 barnyardgrass emergence as PI 169391 and non-effective on redroot pigweed. This indicated differential quantitative and/or qualitative toxicity between cucumber accessions and resulted in the elimination of PI 285605 for future field evaluation. Plant introduction 169391 had the greatest suppressive effect on prOSo millet and barnyardgrass 10 days after planting with inhibition of 81 and 73% respectively in 1975 (Table 3) and 43 and 33% respectively in 1976 (Table 4). During the three year evaluation involving five experiments, PI l6939l suppressed weed growth in three of the five tests. Suppression of redroot pigweed emergence by PI l6939l which occurred in 1975 was not evident in 1976. P0pulation densi- ty and fresh weight was not suppressed by any treatment 10 days after planting or at cucumber maturity on the Miami silt loam. Rainfall occurred 12 days after planting the first year as compared to 2.29 ha cm 3 days after planting the following year. The decreased suppression of shoot fresh weight and weed emergence the second year on Spinks loamy sand may have resulted from leaching. Evidence which indi- cates a high degree of water solubility and early release of the toxic constituents suggests that leaching may remove the chemical from the germination zone. The lack of inhibi— tion on Miami silt loam may have resulted from a combined effect of leaching and/or adsorption by soil. Population densities of barnyardgrass and redroot pig- weed indicated no difference between the control and 'Pioneer' 39 Table 4. Weeds per plot and percent inhibition 10 days after planting in the presence and absence of selected cucumbers (1976). Speciesa Accession or Proso millet Barnyardgrass Redroot pigweed cultivar (No.)' (%)b (No.) (%) (~0.) (%) No cucumber 15 b - 18 b - - 32 a - 'Pioneer' 14 b - 16 b - 28 a - PI 169391 8 a 43. 12 a 33 30 a 0 aMeans within a column followed by the same letter are not significantly different at the 5% probability level with Duncan's Multiple Range test. bPercent inhibition calculated by using 'Pioneer' as the control. 4O 10 days after planting (Table 3), with a subsequent signifi- cant difference at harvest (Table 5). Three factors that may interact to regulate population density are: a) germi- nation, which initially determines the number of plants per unit area; b) plasticity, which affects the Size and weight of survivors; and c) mortality which determines the number of survivors (5,8,13). Monitoring plant populations during the later stages of crop growth and deve10pment is important but may be misleading if mortality is not separated from initial germination data. Both germination and mortality must be considered when reporting effects on plant density since the physiological superiority of an older plant on a germinating seedling could result in mortality. It is also conceivable that inhibition of germination may result from reduction of light intensity by a plant that is morphologi- cally superior. In view of this, stand densities should be monitored during the early and late stages of plant growth and development. Early inhibition of weed germination by crops can be directly associated with a biochemical effect whereas later reductions in populations may also be related to competitive parameters.‘ Suppression of weed populations by PI'S 169391 and 285605 remained relatively constant between 10 days after planting and harvest in 1975. The reduction in proso millet and barnyardgrass populations by PI l6939l evident 10 days after planting in 1976 was not present at cucumber harvest. The possibility of early leaching in conjunction with a 41 Table 5. Weeds per plot and percent inhibition at harvest In th? presence and absence of selected cucumbers 1975 . .Speciesa Accession or Proso millet Barnyardgrass Redroot pigweed cultivar (No.) (%)b (No.) (%) (No.) (%) No cucumber 28 b - 38 c . - 54 c - 'Pioneer' 20 b - 21 b - 40 b - PI 169391 3 a 85 7 a 67 21 a 47 PI 285605 8 a 60 11 a 48 39 b 0 aMeans within a column followed by the same letter are not significantly different at the 5% probability level with Duncan's Multiple Range test. bPercent inhibition calculated by using 'Pioneer'as a control. 42 dilution effect apparently allowed the weed seeds to germi- nate later and regain normal population density. This suggests that timing and persistence is just as critical for an allelopathic effect as it is in regulating efficacy of synthetic herbicides. Need fresh weight at harvest indicated no difference between the control and 'Pioneer' except for a reduction in fresh weight of barnyardgrass (Table 6). PI l6939l reduced the fresh weight of proso millet, barnyardgrass and redroot pigweed 54, 39 and 37%, respectively. PI 285605 inhibited all three indicator species at approximately half the efficiency of PI 169391. Total weed fresh weight at harvest was reduced by all cucumbers (Table 7). PI 169391 inhibited weed biomass 84%. 'Pioneer' suppressed fresh weight 53% indicating its competi- tive ability. Strong competitive ability was expected for all lines as the cucumber plants vined and formed a canopy over the soil. There was no difference in the total weed numbers be- tween the control, 'Pioneer' and PI 285605. Although PI 285605 reduced the number of a few overseeded species, the total weed number was not reduced. Total plant popula- tion was reduced approximately 50% when grown in association with PI 169391. It is postulated that the combined effect of allelopathy and competition resulted in the long-term interference of PI 169391 on both weed number and biomass. PI l6939l produced larger plants than either 'Pioneer' 43 Table 6. Average fresh weight per plant and percent inhi- bition at harvest in the presence and absence of selected cucumbers (1975). . a . Spec1es Accession: compmcpELBw acmEummcp comm ”capo zmmm< pmmmmp Ha .mecowm. mmuczom ummm . m gumcmp Paaouoa»; use cowpmcweme co mamm“upuwwMagmmmumzwcwocmmwmwwumflm .P mpnmh 93 Seed extract bioassay. Partitioning of cucumber extracts between water and chloroform demonstrated that the toxic compound is polar and localized only in the seed testa (Table 2). Localization of a leachable toxin in seed testa leads to speculation that the inhibitor may also be a con- stituent of cucumber fruit juice. According to Evenari (8), many fruits contain non-specific chemicals inhibitory to seed germination such as cyanide, ammonia, ethylene, alka- loids, organic acids, aldehydes, essential oils, phenols, phenolic acids, and unsaturated lactones. Cucumber plant bioassay, PI l6939l juice extract from green and yellow fruit suppressed cucumber germination 82 and 22%, respectively (Table 3). Hypocotyl length was also suppressed by fruit juice and decreased with maturity. All 'Pioneer' and stem, leaf and root extracts of PI 169391 had no auto- inhibitory effect on germination or hypocotyl length. Con- versely, PI l6939l cucumber fruit juice, leaf, and root extracts suppressed germination and hypocotyl length of proso millet (Table 4). These bioassays indicate interspecific toxicity of PI l6939l leaf and root extracts accompanied by both intra- and interspecific activity of juice. It is postu- lated that the plant and seed toxins and/or mechanisms are different, however, this has not been qualitatively demonstrat- ed. Quantitative differences in susceptibility of cucumber and proso millet should not be discounted since growth responses may be a direct effect of concentration. 94 .ummp magma opavppzz m.:mu::a guvz pm>mp Rm we» no ucmgmmtvn appeau*wwcmrm pa: men smupmp «Sam on» »a umzoppoe caspou a cpgpvz mcmmzo a mm a mm a m a mm mummh m em a on u aw m om oaLnEm e mm a mm o em a mo coumpxuou m cm a am a mm a mm ummm a ma Elam o am a as _oeoeoo Fammop Ha . .mecowa. memop Ha .Lomcova. mama?» uczoew mpaapomisgoeogopgo. mpnspomuemuoz acoruomgm compuocuxm .umppws omega to cowpmcpsgmm pcmugma co muuoguxm ummm Lanszoau umcowpwgcma mo powwow one .N mpnoh unequuaoo gowuuaaunm .pmmp magma mpapppaz m.=mu:=o cup: Fm>w_ gm mg» pm pcmcmmwwn apucmchwcmFm yo: man mepmp mEom mgp an vmzoFFoe cszpou a argue: mcmmzo a ~.F 3 mm a m.m m mm marsh uwsge 3oPFo> a n.o m NP a o.m m cm marsh upset :moew u o.~ a No a m.~ a om uoom o m.P n no a o.m m cm wow; u N.~ a on . a F.m m mm Emum o m._ a he a ~.~ a mm . Poeoeou m” Azov ARV “soy ARV pzuoquA: cowumcFEme quouoa»: cowumcwseou “cospowcp ,mmmop Ha .eooeoea. umucaom uumguxm .Lmn23usu co gumcmp quouoax; uco cowpmcwscmm co muoocuxo mammmu peopa amassuau mo uummwm one .m mpamp .ummp mmcmm mpawppzz m.:muc:o sup: Pm>mp Nm asp an pcmemmmvu appcoowewcmwm uo: wee cmppwp msom on» »n vmzoP—oc cszpou m argupz mammzo u o.p a we a m.o a ma mowsn “was; zap—m> a m.o a m_ m ~.P m mm mowaw awaem cmmgc n o.o 3 mm a o._ m cm poem a m.o a mm m m.p a co wow; u _.— o co m ~.P m cm scum u P.p u mm a p.~ m om Fogpcou ,o 9 :3 :3 :3 :3 quouoax: corumcwscmw pxuouoa»: :ovumcrscmw acmEpmmgp Pmmmm~ Ha .cmmcowa. mmugzom pumgpxm .umppws omega to gumcmp quouoa»; vcm cowpmcweemm co mpuoeuxm mammww “capo emasaoau co pumwtm one .e mpnmh 97 The inhibitory effect of fruit juice further indicates the testa as a possible source of the seed toxin. It is also possible that inhibitors in the juice could be absorbed by the seed testa. Qualitative analysis of juice and testa extracts must be utilized for verification. Cucumber plant bioassay in different media. Leaf, root and juice from green and yellow fruit suppressed germination of proso millet in media with 0.0 and 1.5% organic matter (Table 5). Juice from green fruit was the most toxic and all inhibitory effects decreased as organic matter increased. The adsorptive capacity of increased amounts of organic mat- ter and clay is suggested as the probable cause of decreased toxicity. However, persistence may have been affected by soil permeability, chemical reactions, pH, and/or microbial degradation. These data further indicate the possible use of PI 169391 leaf, root, and green fruit tissue for incorpo- ration or soil amendment weed control mulches in low adsorp- tive capacity soils. 98 .ummu mmcmm mpawupaz m.cmu::o saw: Pm>mp gm mzu pm “cosmeyvu >~ucoowmwcmvm 9o: mom emuHmF mEom on» xa nmzoppom caspou a cmcpwz meame u em 6 we on mm movzm upagm zoPPm> 6 mm a c4 m a mowan peace :mwsw u mm o mm. o co poem u cm 6 mm u no mom; n ma u mm , v mm posucou stop “pew esowz. . neon AEooF mx=_qm ccom Npeozc “coauomch wwwbmz .mwuwe mcwzogm umpum_mm cw pmppws omega to cowpocfisemm pcmuema co mpooepxm mammru “capo memmp He mo pumewm och .m mpaoh 10. 11. 99 LITERATURE CITED Baskin, J. M., G. T. Lublow, T. M. Harris and F. T. Wolf. 1967. Psoralen, an inhibitor in the seeds of Psoralea subacaulis. Phytochem. 6:1209-1213. Bell, 0. T. and C. H. Muller. 1973. Dominance of California annual grasslands by Brassica nigra. Amer. Mid. Natur. 90:277-299. Bennett, E. L. and J. Bonner. 1953. 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New physiological and biological aspects in the interrelationships between higher plants. Symp. Soc. Exp. Biol. 15:229-244. SUMMARY AND CONCLUSION Plant introduction l6939l suppresses the growth of several plants by the combined effect of competition and allelopathy. Early inhibition of weed germination by a plant can be directly attributed to a biochemical effect, whereas, later population reductions may also result from competition. It is postulated that the combined effect of allelopathy and competition resulted in the long term inter- ference effect of PI 169391 on both weed number and biomass. However, the fact that it was not a larger or more vigorous plant under weed-free conditions indicated that it gains advantage more by biochemical rather than competitive factors. Although growth interference was not consistent in the field, growth suppression could be obtained under certain edaphic and environmental conditions. Growth interference on low organic and clay content soils and during periods of limited rainfall indicated potentially high adsorptive and water-soluble prOperties. Undoubtedly, the influence of soil type and moisture level on the action of allelopathic compounds will follow the trends already demonstrated for synthetic compounds. The potential use of crop plants with strong 101 102 interspecific growth interference as a supplemental means of weed control has increased the importance of identifying and separating allelopathic and competitive factors. The simi- larity of many allelopathic and competitive growth effects and their interaction demonstrates the need for a system of assessing both biochemical and physical suppressive mecha- nisms. One major danger of basing conclusions on the obser- vation of one growth parameter is exemplified by the fact that two different plant responses may be inversely related at various stages of growth and development. Total growth analysis was demonstrated to be a definitive means of moni— toring growth interference. Total growth analysis during the early growth stage of PI l6939l indicated that allelo- pathy is the main component of plant interference. Fermentation, leaching and activated charcoal bioassays indicated that PI l6939l seed is both autoallelopathic and allelopathic during the early stages of seedling growth and development. It is hypothesized that growth suppression of plants in the presence of PI 169391 may have resulted from the release of endogenous germination inhibitors associated with after-ripening and seed dormancy. Growth differences for seed with and without testa compared to seeds without testa germinated in the presence of the removed seed coat indicated biochemical activity. Cucumber seed extract par- titioning demonstrated that the toxin is polar and associated only with the testa. The inhibitory effect of fruit juice further indicated the testa as a possible source of the seed 103 toxin. Additionally, it is possible that the inhibitors in the juice could be adsorbed and/or absorbed by the seed coat. Plant bioassays demonstrated interspecific toxic activi- ty for leaf and root extracts accompanied by both intra- and interspecific activity of juice. PI l6939l may possess several allelopathic systems. It is possible that compart- mentalized leaf and root toxins may be released during certain natural conditions. There are at least two important implications for alle- lopathic plants. Isolation and identification of the toxic products could result in synthesis of them or their analogs for use as herbicides- Additionally, incorporation of the toxic mechanism into cultivars by genetic manipulation may provide at least partial weed resistance. The utility of allelopathy may provide another strategy for integrated pest management in agricultural ecosystems. 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