”WWWIW’I‘ilvlfiit‘ilfifl‘flfil'l‘lflifliflll 3 1293 10702 7702 MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from ._;_. your record. FINES win be charged if book is returned after the date stamped below. ‘ the-n , A «cote-W .. 0/) 1"}. 79% g. Mo 1:172; ALLELOPATl-IIC ACTIVITY OF RYE (Secale cereale L.) By Jane Patricia Barnes A DISSERTATION Submitted to Michigan State University in partial ful filluent of the require-ents for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1985 ABSTRACT ALLELOPAIHIC ACTIVITY OF RYE (Secale cereale L.) By Jane P. Barnes In previous field studies, residues of both fall and Spring- planted rye (Secale cereale’Lm) reduced weed germination (69%) and growth (32%) when compared to control mulch plots in no-till systems. In simulated no-till greenhouse experiments, residues of 35 day-old 'Hheeler‘ rye shoots reduced emergence of both lettuce (Lactuca sativa L.) and proso millet (Panicum milaceum L.) by 58% and 35%, respectively, over that obtained with a control mulch. In petri dish assays of rye residues in soil, the degree of phytotoxicity to several species was a function of the proximity of seed relative to the residue. Radical elongation was a more sensitive measure of phytotoxicity than germination, 23; fig. Shoot tissue was twice as phytotoxic as root tissue and remained toxic in the absence of soil uficroorganisms. The phytotoxic agent was water extractable and could be partitioned into organic solvemts. A cress (Lepidium sativum L. 'Curly') seed germination and seedling growth bioassay was used to monitor separations for compounds with the greatest inhibitory activity. Further chromatographic Separation of the diethyl ether extract resulted in identification of two compounds, 2,4-dihydroxy- l,4(2fl)-benzoxazin-3-one (DIBOA) and 2(3fl)-benzoxazolinone (BOA), the structures of which were confirmed by mass and NMR spectrometry. DIBOA and BOA differentially inhibited germination and seedling growth of six monocots and dicots tested. In addition, concentrations of 7:5 x l0’5M DIBOA and 1.0 x io-3M BOA provided 50% inhibition of Chlamydomonas rheinhardtii over a 28 h period. Both compounds inhibited emergence of barnyardgrass (Echinochloa crusgalli L. Beauvg), lettuce, and cress when applied to the soil prior to emergence. Based on a colorimetric determination, greenhouse grown root and shoot tissue contained ca 171 and 2865 pg hydroxamic acids/g dry tissue, respectively. The benzoxazinones were the most phytotoxic compounds in rye residues and were present in relatively high concentrations. Since residues, extracts, and pure compounds all provided similar plant injury, it was concluded that allelopathy is an important component of the interference produced by rye residues. DEDICATION This dissertation is dedicated to all my family and friends, who” for the past ten years, have wondered if I would ever finish school and get a 'real' job. I realize that 'it's about time', but the good things in life take time. ii ACKNOHLEDGEMENTS In looking back over the ups and downs of the last three years, I am grateful for the many friends, family, and colleagues who have helped sustain my P.M.A. during completion of the research necessary for this dissertation. I am especially'indebted to Dr. Alan R. Putnam, who believed in me enough to take me back. His advice, expertise, and enthusiasm have helped make the research what it is, while his friendship will always be valued. I would like to thank Dr. S. Tanis, Dr. R. Perry, Dr. A. Cameron, and Dr. D. Penner for their time and effort in reviewing and critiqueing this dissertation. I have enjoyed the opportunity to work with an interesting and lively group of pe0ple. I appreciate and will remember Rod Heisey for his ability to listen as his unique perSpective and point of view initiated many lengthy discussions. As "bioassay“ is the bottom line in allelopathy research, I owe many thanks to Jean, Jeff, Mike, Karl, Karen, Jim, and Charlotte, for their time and energy spent on tedious, but important measurements. Lastly, I would like to thank Jackie Schartzer for her friendship and expertise. TABLE OF CONTENTS PAGE LIST OF TABLES .......................... vi LIST OF FIGURES ......................... vii CHAPTER 1: LITERATURE REVIEN INTRODUCTION . . . . .................... l ALLELOPATHY ......................... 2 Proof of AlleTOpathy ....... . . . ......... 3 Sources of Allelochemicals ............... 5 Classes of Allelochemics and Factors Affecting Activity . . . .......... . . . . . ...... 5 Role of Allelopathy in Natural and Agroecosystems .................... lO Controversy Associated with Allelopathy ......... 12 RYE (Secale cereale L.) ....... . ........... l5 Growth and Development. . . . . . . ........... 15 Prior Evidence for Allelopathy in Rye .......... 16 LITERATURE CITED . . . . . . . ............. . . 23 CHAPTER 2: EVIDENCE FOR ALLELOPATHY BY RESIDUES AND AQUEOUS EXTRACTS OF RYE (Secale cereale L.) ABSTRACT ......................... . 36 INTRODUCTION ....................... . 37 MATERIALS AND METHODS. . . . . ............... 39 General......... ................ 39 Inhibition by Rye Residues .............. . . 4O Inhibition by Aqueous Extracts .............. 42 RESULTS AND DISCUSSION . . . . . . ......... . . . . 42 Inhibition by Rye Residues ................ 42 Inhibition by Aqueous Extracts .............. 53 LITERATURE CITED ...................... 57 iv PAGE CHAPTER 3: ISOLATION AND CHARACTERIZATION OF ALLELOCHEMICALS IN RYE (Secale cereale L.) SHOOT TISSUE ABSTRACT .......................... 59 INTRODUCTION ........................ 60 MATERIALS AND METHODS .................... 61 Genera] O O O O O O O O O O O O O O O O O O 0 O O O O O I 6" Bioassay. . . . . . . .................. 61 Extraction ..... . . . . ............... 62 Isolation of Unknown Compounds. . . . .......... 65 Identification of Pure Compounds ............. 68 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . 71 Extraction Efficiency and Relative Activity . . . . . . . 71 Preliminary TLC Bioassay of the Crude Ether Extract . . . 73 Isolation, Characterization and Activity of Compound l. . 73 Isolation, Characterization and Activity of Compound 2. . 78 LITERATURE CITED . . . . . . . . . . . . ...... . . . . 89 CHAPTER 4: ROLE OF BENZOXAZINONES IN ALLELOPATHY BY RYE (Secale cereale L.) ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . 95 INTRODUCTION . . . . . . . . . ............... 96 MATERIALS AND METHODS. . . . . . . . . . . . . ...... . 99 Comparison of Organic Extracts from Field and Greenhouse Grown Rye ... . . .. . .. ....... . 99 Preparation of Extracts for Quantification ....... .100 Quantification of Hydroxamic Acids. . . . . . . . . . . .100 Purification of DIBOA for Bioassays . . . . . . . . . . .100 Seed Germination and Seedling Growth Bioassays ..... .101 Algae Growth Bioassay . . . . . . . . . . . . . . . . . .102 Soil Activity of Hydroxamic Acids . . . . . . . . . . . .103 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . .104 Comparison of Organic Extracts from Field and Greenhouse Grown Rye . . . . . . . . . . . . . . . . . .104 Quantification of Hydroxamic Acids. . . . . . . . . . . .107 Activity on Seed Germination and Seedling Growth. . . . .108 Activity on Growth of Algae . . . . . . . . . . . . . . .116 Activity of DIBOA and BOA in Soil . . . . . . . . . . . .119 LITERATURE CITED . . . . . . . ..... . . . . . . . . . .125 i LIST OF TABLES TABLE PAGE CHAPTER 2: 1. Percent emergence of four indicator species in residues of greenhouse grown 'Nheeler' rye . . ......... . . . . . 44 CHAPTER 3: 1. Activity of rye shoot tissue on cress . . . . 72 2. Cress bioassay of preliminary TLC separation ........ . ......... 74 CHAPTER 4: 1. Determination of DIBOA and BOA by TLC in organic extracts from aqueous extractions of rye root and shoot tissue. . ....... 105 2. Recovery of organic fractions obtained from sequential partitioning of root and shoot extracts. ........... . . . . 106 3. Concentration of hydroxamic acids in greenhouse grown rye roots and shoots and field rye shoots. . . . ......... 107 4. Emergence of barnyardgrass (BYGR), cress, and lettuce (expressed as a percent of control) 4 days after Spray application of DIBOA and BOA to soil ..... . . . . . . 120 5. Emergence of barnyardgrass (BYGR), cress, and lettuce (expressed as a percent of control) 14 days after spray application of DIBOA and BOA to soil. ..... . . . . . 120 vi LIST OF FIGURES FIGURE PAGE CHAPTER 2: 1. Effect of rye residue (1.0 g per 150 9 soil) placement relative to seed in soil on germination and root growth of four indicator Species Figure 3; cress, b; lettuce, g; proso millet, g; barnyardgrass. . 45 2. Effect of rye root and shoot residues in soil on root growth of four indicator Species. Figure a._cress, b. lettuce, (g; proso millet, g; barnyardgrass . . . . . . 48 3. Root growth of cress and barnyardgrass (as a % of control) in response to varying rates of rye shoot residue in sterile and non-sterile soil ....... . . . . . . . . 51 4. Effect of aqueous extracts of rye shoots on root growth (as a % of control) of cress and barnyardgrass in sterile and non-sterile soil. ......... . . . . . ..... . 54 CHAPTER 3: 1. Flow diagram for initial extractions of rye shoot tissue ....... . . . . . . . . . . 63 2. Isolation scheme for compound 1 ....... 66 3. Isolation scheme for compound 2 . . . . . . . 69 4. Structures for (a) Glycoside, previously identified in non-injured rye (b) DIBOA and (c) BOA isolated from rye residues. . . . 76 5. Response of cress to various concentrations of DIBOA after 72 h incubation. LSD for root length (5%-20), shoot length (5%-12), and germination (NS) ..... . . . . . . . . . . 79 6. Response of cress to various concentrations of BOA after 72 h incubation. LSD for root length (5%-16), shoot length (5%-11), and germination (NS). . . . . ...... . . . . 82 7. Response of barnyardgrass to various concen- trations of DIBOA and BOA after 72 h incubation. . . . . . . . . . . . . . . . . . 84 vii CHAPTER 4: FIGURE 1. PAGE Comparison of activity of BOA from Aldrich Co. and BOA extracted from rye on cress seedling grouth O O I O O O O O O O O O O O O O O O I O O] 09 Response of (a) large crabgrass, (b) barnyard- grass, and (c) proso millet to various concen- trations of DIBOA, BOA, PLA, and HBA in petri dishes ............ . . . . . . . . .112 Response of (a) redroot pigweed, (b) tomato, and (c) lettuce to various concentrations of DIBOA, BOA, PLA, and HBA in petri dishes. . . .114 Response of Clamydomonas rheinhardtii Dangeard to various concentrations of DIBOA and BOA based on absorbance at 652 nm . . . . . . . . .117 viii CHAPTER 1 LITERATURE REVIEH INTRODUCTION Man's intervention in the natural ecology of an area is radically disruptive of the balanced and mutually dependent system of interrelated plant and animal life. The original plants disappear and are replaced by other, better adapted, species (Hhittaker, 1975). The vast monocul tures of crops in agriculture create new ecosystems for plants to interact with other plant species. On agricultural lands, weeds are often the better adapted species. They reduce crap yields and quality, interfere with harvesting, serve as hosts for other pests, and increase the time and costs involved in crop production. The detrimental influence of one plant on another through release of chemicals into the environment has been termed allelopathy (Molisch, 1937) and has been identified as an important component of plant interference in both natural and agroecosystems (Borner, 1950; Tukey, 1969; Holm, 1974; Putnam and Duke, 1978; Rice, 1979; Lovett and Levitt, 1981; Lovett, 1982; Putnam, 1985). Detrimental weed/crop interference found in agroecosystems has traditionally been attributed to differential utilization of space, light, water, and nutrients (competition). The potential of'weeds.to chemically'interfere with crop growth may also prove to be an important factor responsible for their detrimental influence and aggressive behavior. 1 As plants (other than seeds) are essentially immobile, chemicals offer a rational approach to self-defense against many organisms. Assuming a plant has evolved the chemical defense capabilities in response to a host of environmental factors, it could have also evolved control for chemical production and release to gain advantage throughout critical stages in the life of the plant. Elucidation of the compounds responsible and factors involved in expression of allelOpathic activity in plants may clarify the nature of the interference noted between weeds and cr0ps in agricultural ecosystems. ALLELOPATHY The phenomenon of a1 lelopathy, where one plant influences the growth of other plants through release of chemicals into the environment, has been observed for decades. As early as 1832, de Candolle suggested that the 'soil sickness' problem in agriculture may be due to exudates of crap plants, and that crop rotation may help alleviate the problem. From Molish's noteworthy paper in 1937, to the more recent reviews by Tukey (1969); Putnam and Duke (1978); Rice (1979, 1984); and Putnam (1985), allelopathy is gaining acceptance in the scientific world. Recently, there have been several international symposia resulting in entire books on the subject (North American Symposimn of Allelopathy, 1983; The Chemistry of Allelopathy, 1984; International Chemical Congress of Pacific Basin Societies, 1984). Since several recent treatises on this topic already exist, this review will provide limited information on known allelochemicals and proof of 3 a1 lelopathy but will focus on studies related to the rye. Molisch (1937) coined the term allelopathy and defined it as the ”biochemical interactions between all types of plants including microorganisms)‘ His broad definition was meant to cover both detrimental and beneficial reciprocal biochemical interactions. Rice (1979) modified Molisch's definition by stating that “the effect depends on a chemical being added to the environment.“ Rice further clarified the difference between allelopathy and competition by stating that, "competition involves the removal or reduction of some factor from environment that is required by some other plant sharing the same habitatf A major obstacle in proving allelopathic effects has been to eliminate aspects of competition in the plant/plant interactions. Perhaps a more appropriate term for the overall detrimental influence of one plant on another is "interference" as suggested by Muller (1969L. Components of plant interference include competition or al leloSpoly (depletion of physical factors necessary for growth such as light, water or nutrients), a1 lelopathy, and other indirect effects which modify the physical and biological environment. The number of variables and their potential interactive effects have created a situation difficult to completely define and conclusively prove (Harper, 1977). Proof of Allelopathy. Most research in the area of allelopathy has been hindered by the lack of any protocol for its proof. In attempts to alleviate variable and inconclusive results, Fuerst and Putnam (1983) have proposed a set of criteria to be fulfilled for proof of allelopathy. Initially, the symptoms of interference must be identified in the natural state with experiments designed to eliminate competitive and indirect forms of interference. Supportive assays should examine factors which may influence activity, ie: (1) the primary source of toxicity; (2) the method of release; and (3) the role of microorganisms in the expression of toxicity. Where ever possible, several relevant indicator species should be tested and symptoms of injury should be carefully examined. After identification of the symptoms of the interference, the second step in proof oflallelopathy relies on chemical isolation, identification, and assay. Phytotoxicity should be monitored throughout the separations and care must be taken to exclude identification of artifacts during the separations. Lastly, the release, movement, uptake, and quantity of the toxin should be monitored, since final proof relies on simulating the interference by supplying the toxin as it is supplied in nature. In other words, one must determine if a plant releases chemicals which accumulate in sufficient quantity to affect plant growth. The chemicals must either persist for some period of time or be continually released in order to have lasting effects. For example, juglone (5- hydroxyba-naphthaquinone), which has been implicated in allelopathy by black walnut (Juglans nigra LJ, is relatively persistant in wet soil (Fisher, 1978) lending greater significance to its impact in certain ecosystems. This is in contrast to compounds of a more ephemeral nature where concentrations and resultant physiological activities may rapidly change over time and, therefore, remain undetected (Patrick, 1971). Rapid and sensitive assay methods to detect phytotoxic compounds during their short interval of production and disappearance are necessary. It is only through careful extraction, separation, identification, and quantitation of the chemicals responsible for the 5 inhibitory activity that al lelopathy can be proven. Sources of A1 lelochenicals. Potentially a1 lelopathic compounds may leave a donor plant through four pathways: 1) volatilization (Muller, 1964, 1965; Muller et a1, 1968; Heisey and Delwiche, 1983), 2) leaching from above ground parts (Bode, 1940; Grummer and Beyer, 1960; Tukey, 1966), 3) root exudation of water soluble toxins (Bell and Koeppe, 1972; Fay and Duke, 1977; Kossanel et al, 1977; Kozel and Tukey, 1968), and 4) litter decomposition (Guenzi and McCalla, 1962; Stotzky, 1974; Cochran et a1, 1977; Patrick, 1971; Chou and Patrick, 1976). Unfortunately most of these pathways do not lend themselves to easy _i_n m set-ups that effectively simulate release in nature (Tukey, 1969; Putnam and Duke, 1978; Rice, 1979) Conclusive evidence for the cause/effect relationship is lacking (Fuerst and Putnam, 1983; Harborne, 1977). Classes of Allelochemics and Factors Affecting Activity. A1 lelopathy appears to be a complex phenomenon involving chemicals that may be sequestered and/or maintained in an inactive form when in the donor plant; and then released into the soil system to perhaps undergo possible radical changes before reaching the receptor plant (Tukey, 1969; Putnam and Duke, 1978; Rice, 1979). After eliminating non- biochemical parameters as the basis for plant interference, researchers have attempted to identify the chemicals involved (Borner, 1955; Muller, 1965; Muller et a1, 1968; Tang, 1982). The chemicals implicated in allelopathy are often placed in that nebulous group called secondary compounds because of their lack of an obvious role in primary metabolism of a plant (Harborne, 1972; Moreland et a1, 1966; Swain, 1977; Whittaker, 1970; Hhittaker and Feeny, 1971). Those implicated as effective allelopathic agents include simple phenolic: acids, coumarins, terpenoids, flavanoids, alkaloids, cyanogenic glycosides, and glucosinolates (Bode, 1940; Harborne, 1972; Moreland et a1, 1966; Swain, 1977; Vrany'et a1, 1962; Whittaker and Feeny, 1971) in addition to water-soluble organic acids and alcohols (Putnam and Duke, 1978; Rice, 1979; Harper and Lynch, 1982). While many of these secondary compounds have been reported to be involved in various protective or defensive mechanisms, it seems probable that they have more than one role or function for a plant (Sieglar, 1977; Swain, 1977; Whittaker and Feeny, 1971; Harborne, 1972; Whittaker, 1970). Several workers have suggested that secondary chemical production is influenced by the plant's genetics, development, biochemistry and physiology; and its interaction with, and response to, biotic and abiotic factors of the environment (Tukey, 1969; Ellis and McCal 1a, 1973; Levin, 1971; McKay, 1974, 1979). For example, plant introductions of oat (Avena sativa L") were assayed and several lines were found to inhibit weed growth (Fay and Duke, 1977). Several accessions from the germplasm collection of cucumber (Cucumis sativus l") and related Cucumis spp. were screened and allelopathic activity was demonstrated in sand culture (Putnam and Duke, 1974). Growth inhibitors were subsequently identified in seeds and plants of cucumber (Lockerman and Putnam, 1977). A plantfs age or stage of development may also influence chemical production. Koeppe et al (1970) found concentrations of scapolin (7- 91 ucoside of 6-methoxy-7-hydroxycoumarin) and chlorogenic acid (3-0- caffeoquuinic acid) decreased with age in tobacco (Nicotiana spp.) leaves, although total amounts of the compounds increased with age due to the increase in leaf area. Woodhead and Bernays (1978) found the phenolic acid content of sorghum (Sorghum bicolor Ln) seedlings to be inversely related to plant height, which in turn was related to age (days after emergence) in a constant manner. In addition to age, cultivars of sorghum varied in phenolic acid concentrations and levels increased when plants were damaged by insects or pathogens (Woodhead, 1981). Plant organs also vary in quantity and composition of secondary compounds. Steinberg (1984) found higher concentrations of phenolic compounds in reproductive grounds (SporOphylls) of an intertidal kelp (Alaria marginata) than in vegetative blades. The sporophyl ls were consumed by herbivorous snails at a lower rate than were the vegetative blades. The authors suggested that differential internal production of defensive compounds could significantly affect the pattern of herbivory on the plant. Koeppe et al (1971) have found differential levels of scopolin and chlorogenic acid root and stems of tobacco. Effects of other factors, including nutrient levels, UV light, and cold, have been examined and found to play a role in secondary chemical production by a plant. Increasing deficiencies of nitrogen resulted in accumulation of both scopolin and chlorogenic acid in tobacco (Koeppe et a1, 1971). In addition, tobacco grown under low UV light contained lower concentrations of these compounds relative to controls receiving moderate intensities of UV light and other treatments exposed to high UV light. Tobacco plants exposed to cold temperatures (5 C) also accumulated greater concentrations of chlorogenic acid and scapolin than those maintained at higher temperatures. 8 From a different perspective, Stowe and Osborn (1980) examined the interaction between phenolic phytotoxicity and nutrient deprivation. Two concentrations of p-coumaric acid and vanillic acid were added to nutrient solutions containing various quantities of nitrogen and phOSphorous. Solutions were tested on the dry weight accumulation of barley (Hordeum vulgare L.) in an effort to determine whether certain phenolics were rendered less inhibitory by the presence of certain nutrients. The phenolic compounds were uniformly inhibitory only at the lowest levels of nitrogen and phosphorous. At higher nutrient concentrations, the effects of phenolics were highly variable and often enhanced dry weight of barley. Others have postulated that plant injury or stress by mechanical, pathological, or physiological action also influences production and release of metabolities (Koeppe et a1, 1970; Lawlor, 1979; Rice, 1984; Tukey and Morgan, 1963). Increased phenolic biosynthesis is a typical response of plant tissue to both irradiation and infection which reflects stress conditions (Koeppe et a1, 1969). Others have found that leaching of carbohydrates from young bean (Phaseolis vulgaris L.) leaves directly paralleled the light intensity received by the plants (Tukey, Wittwer, and Tukey, 1957). In a study on the electrophoretic patterns of proteins in rye (Secale cereale L.) protoplasts, rye was cold hardened by cold acclimation or dessication stress. The two treatments did not induce similar protein pattern changes indicating different metabolic and physiological responses of the cells to these stresses (Cloutier, 1984). Reid (1974) examined the effect of specific levels of induced water stress on the movement of 14C-labeled compounds in ponderosa pine (Pinus ponderosa Laws.) seedlings and the resultant exudation of 14C from the roots. Assimilation of 14c02 by plants under stress and subsequent translocation of 14C label to roots were both inhibited by a decrease in substrate water potential. Further, as water potential decreased, sugars as a percentage of total exudate increased, organic acids decreased and amino acids showed a slight decrease. Foliar application of various chemicals have also been shown to affect the quality and quantity of chemicals produced, as well as influence the associated microbial population in the rhizosphere (Halleck and Cochrane, 1950; Jalali and Suryanarayana, 1970; Rovira, 1969; Stallworth, 1963; Vrany et al, 1962L For example, Jalali and Domsch (1977) found foliar application of 3 fungicides caused changes in amino acid quantity and quality by wheat (Triticum Lu) roots. Increased levels of sc0polin have been found in tobacco leaves, stems, and roots 30 days after application of 2,4-D [(2,4-dichlor0phenoxy) acetic acid] (Dieterman et a1, 1964). Several compounds have been identified in root exudates including amino acids, various organic acids and alcohols, and other carbohydrates (Jalali and Domsch, 1977; Katznelson et a1, 1955; Rovira, 1956; Tang and Young, 1982). If plant root exudates are to be implicated in allelopathy, experiments must be designed to eliminate possible interacting effects of toxins resulting from shoot leachates and residues. Bell and Koeppe (1972), reported that giant foxtail (Setaria faberi Hervnn) root leachates reduced growth of corn (lea mays L.) in solution culture. They used a stairstep method to recirculate nutrient solutions and reduce interplant competition. 10 More recently, Tang and Young (1982) have successfully identified inhibitory compounds from the undisturbed root system of Bigalta limpograss [Hemarthia altissima (Poir;) Stapf. and HubbJ in a system which allows for continuous trapping of quantities of extracellular chemicals through use of selectively'adsorptive XAD-4 resin. The major activity of exudates was attributed to 3-hydroxyhydrocinnamic, benzoic, phenylacetic, and hydrocinnamic acids. Since the root system was undisturbed and the recovery was highly efficient compared to conventional solvent extraction methods, this trapping system may prove to be useful for a wide range of studies into rhi205phere chemistry. Role of Allelopathy in Natural and Agroecosystems. Allelopathic interactions in the environment may be expressed in many ways. Higher plants may belallelopathic to other higher plants, or to microorganisms (Rice, 1979; Nickel 1 , 1960). Microorganisms may be a1 lelopathic to higher plants, or other microorganisms (White and Starrat, 1967; YanderMerwe et a1, 1967; Hattingh and Louw, 1969). Thus, chemical interactions are important in shaping both natural and agricultural ecosystems where plants and microorganisms coexist. In natural ecosystems, al lelopathy may play a role in patterning of vegetation (Muller, 1971) or old field succession (Rice, 1971). Whittaker (1975) proposes that al lelochemic interrelationships are a major basis of community organization, niche differentiation, and community niche Space. The concentration of secondary substances in plants is such that substantial quantities can often be released into the environment from either the living plant or by decomposition of the litter. Once in the soil, they may have significant effects on other vascular plants, 11 soil microorganisms, or even on the plant from which they were released (Whittaker, 1975L. Harlan feels that the phytochemicals may be an important component of resistance and confer an advantage to the plant species. Levin (1976) has suggested that since the production of secondary compounds places energy demands upon plants, their production is purposeful and specialized. According to Odum (1969), secondary plant metabolites may be extremely important in preventing populations from overshooting their equilibrium density, thereby reducing oscillations as an ecosystem develops stability. Allelopathic effects appear to be especially significant in natural communities where there is strong dominance of a single species (Muller, 1966; Muller, 1969; Whittaker, 1975). Since most agroecosystems consist of vast monocultures of craps, allelopathy may also play a role in these manipulated ecosystems. Many of the early investigations into allelopathy were a result of crap phytotoxicity problems observed in agriculture. McCalla and Duley (1948, 1949) published two papers on the effects of decaying wheat (Triticum vulgare L. var. Mida) residues on corn (Z_e_a_ may; L.) growth. These were in response to a widespread use of stubble mulch farming for soil erosion problems during the dust bowl era. In many instances, yields were reduced in stubble mulch farming, suggesting that the detrimental effect of crop residues might be due to a combination of toxins released from residues, and from microorganisms that were caused to grow more profusely by substances in the residues. Later, Norstad and McCalla (1963) isolated fungi from stubble mulched field plots which produced a compound patulin, which was extremely toxic to corn plants. The organism was later identified as 12 Pencillium urticae Bain, and was found to comprise 90% of the total soil fungal population (Ellis and McCal la, 1973). When patul in was applied to soil planted with 'Yee' Spring wheat, yields were decreased. Therefore, they concluded that a single exposure of patulin to growing wheat plants is enough to produce yield reductions in the field. It now appears tht many problems associated with the use of crop rotations, no-til lage (NT), and cover crapping may involve a1 lelopathy. Basic to most NT system is the use of a cover crop. Frequently called ”smother crops", they have often been planted to help suppress weed growth (Overland, 1966). Potential smother crops include barley, rye, sorghum, buckwheat (Fagopyrum esculentum Moench), sudangrass, sweet clover (Melilotus Spp), and sunflower (Helianthus annus L.). Overland attributed the weed growth reduction primarily to competition although allelopathic interferences were not excluded. Most of the cover crop Species listed by Overland have been reported to be al lelopathic to certain test species (Rice, 1979). Since surface residues are inherent to NT programs, alle10pathic chemicals from the residue may leach into the soil and affect the growth of other plants (Guenzi and McCal la, 1962; McCalla and Haskins, 1964; Guenzi et a1, 1967). Controversy Associated with Al 1el opathy. Although a1 lelopathy has been frequently cited and implicated in plant/plant interactions, there are many incongruities and shortcomings to previous experiments. Bioassays must be carefully designed to test for toxicity on appropriate species and to include suitable controls for other forms of plant interference. Perhaps one of the most difficult obstacles to overcome involves the separation of competitive and allelopathic forms of interference. Most 'competition' studies fail to exclude the potential for chemical interference, while adequate controls for 13 competitive or physical effects hinder most of the allelopathy studies. For example, velvetleaf’(Abutilon theOphrasti Medic.)and soybean [Glycine max.(L.) MerrJ interference has been examined by using additive and substitive designs, in addition to individual plant yield- plant p0pu1ation functions (Dekker and Meggitt, 1983 a, b). Although the designs are widely used and generally accepted, none of the experiments would in this case exclude the potential.a11elopathic activity demonstrated by exudates of trichomes on the stems and petioles of velvetleaf (Sterling and Putnam, 1984). On the other hand, Bell and Koeppe (1972) examined the al lelopathic effects of seedling and mature giant foxtail (Setaria Igbgriyflerrhn) on the growth of corn using a "stairstep method" of bioassay. In this study, the 'treatment' consisted of the two species grown in sand in alternate pots, while the 'control' was a similar set- up where only corn was grown. Although mature foxtail reduced corn height by 20% and reduced both fresh and dry weights by about 35%, conclusions are limited to discussions of foxtail leachates on corn relative to corn leachates on corn. The stairstep method was an improvement over earlier leachate studies as it allowed for control of many 'competitive' factors of interference, such as physical space and light. Although nutrient levels were monitored by measuring the conductivity and pH of the nutrient solution at intervals, the possibility for differential uptake of nutrients between 'treatment' andi'control' would not necessarily be detected by these types of nutrient measurements. In many instances, it is the plant residues or decomposing tissues which contain the greatest phytotoxicity. In these instances, one must separate a1 lelopathy from the indirect effects of interference. In ti..- :4 ..-—..— —.—.___ 14 particular, very little is known about the physical and chemical changes associated with incorporation of residues into soil. There is the potential for alteration ofinicrobial activity and/or nutrient availability both of which may influence growth. The lack of suitable controls for these changes, and those associated with the mulch at the soil surface (temperature, light, moisture), necessitates caution during interpretations of results. Numerous reports of alleged a1 lelopathy are based on phytotoxicity by'aqueous extracts of ground plant tissue as such methods offer the advantage of being experimentally simple and relatively rapid. However, high concentrations of aqueous extracts can result in inhibition due to increases in the osmotic potential alone (Bell, ‘1974). The assumption implicit in studies with aqueous extracts is that the same extracted substances will be released from the leaves and/or litter of the plant under natural rainfall conditions. This assumption has not been verified. Further. Stowe (1977) compared bioassays of aqueous extracts with the distribution of plants in the field and fOund that “.uSpecies which were highly toxic to each other in bioassays were nevertheless growing right next to each other in the field...“ In conclusion, controversy over the role and importance of allelopathy hicontrolling plant growth and distribution within ecosystems has encouraged additional research on the phenomenon. Due to the nature of allelopathic interference and the complexity of chemicals produced by a plant, most studies do not clearly implicate a single chemical responsible for the toxicity. In cases where chemicals have been identified, questions still exist regarding their significance and advantage for one plant relative to another. 15 Whittaker (1975) has suggested that alle10pathic substances more likely evolved as defenses against animals and bacterial and fungal pathogens and that their phytotoxicity may only be a by-product of the other defensive roles. RYE (Secale cereale L.) Growth and Develop-ent. Winter rye is an annual grass species that has proven useful as a rotational species in a variety of cropping systems (Benoit et a1, 1962; Overland, 1966; Shear, 1968) and has been found to contribute organic matter, reduce soil erosion, and enhance water penetration and retention (Benoit et a1, 1962; Blevins et a1, 1971). It germinates well in untilled soils, and tolerates a wide range of soil moisture levels, pH, and fertility conditions (Nuttonson, 1958). Also, it germinates at low temperatures and is winter hardy. The root system of a single rye plant at the milky ripe stage has been found to extend downward as much as 1.5 m, with a lateral spread of over 0.9 m (Nuttonson, 1958). This extensive root system improves soil tilth and can extract considerable soil moisture (Nuttonson, 1958). ‘The branching, slender, and fibrous adventitious roots are functional throughout the life of the plant. With such an extensive root system permeating throughout the soil horizons, production or release of organic compounds may prove to be a significant factor in the microecology of the soil environment. This unique environment, which iS'under the influence of plant roots, is called the rhizosphere. Within this zone, interactions between plants and microorganisms can 16 greatly affect cr0p production and soil fertility'(Loehwing, 1971; Rovira, 1956, 1965, 1969, 1971, 1973; Richards, 1974). Winter rye produces considerable biomass early in the growing season. For this reason it has often been used as a green manure crop in sandy or low fertility soils. In a comparison of residue production from cereals, rye out-produced wheat, oats, and barley from 21 to 70% (Phillips, 1973). The massive production of residue by rye has the potential to influence the growth of succeeding plant species through release of allelopathic chemicals from residues. Prior Evidence for Allelopathy in Rye. There are numerous papers which indicate that rye influences the growth of other plants. As early as 1925, Cubbon found a rye crop to inhibit growth of grape (Vitis vinifera) plants, and suggested that a chemical might be implicated. Hill (1926) found that the addition of green rye to heavy soils depressed corn growth, while its growth was increased in lighter soils. Roots were more toxic than tops. Faulkner (1943), in Plowman's £911 , suggested seeding the land to rye to help eliminate weeds. Osvald (1953) found rye root exudates reduced germination of wild oats (Avena fatua L.L. Nuttonson (1958) noted that rye has been used to suppress wild oats and many other weeds. Another report (Phillips, 1973) indicated that a rye crOpping program helped control both dandel ions (Taraxacum officinale Weber.) and other broadleaf weeds. Robertson, et a1 (1976) found rye residues suppressed weed growth when compared to sod or conventional tillage plots. These and other similar observations have provided the basis for most of the research involving alleged a1 lelopathy by rye. A Spring-planted cover crop of living rye reduced total weed biomass by 90% over unplanted controls (Barnes and Putnam, 1983). 17 Greenhouse experiments were initiated to further separate the components of interference demonstrated by a living cover of rye. A modified "stairstep" bioassay, where direct interplant competition for space and light was reduced, enabled testing for effects of root leachates from living rye. Root leachates from 2 cultivars reduced lettuce (Lactuca sativa L.) shoot and total biomass by 25% and total biomass of tomatoes (Lyc0persicon esculentum Mill.) by 18% (Barnes and Putnam, 1983). 111a similar study, rye root leachates were found to reduce tomato root biomass by 20-32% and total biomass by 25 to 30%. Shoot growth was differently affected by two cultivars of rye and by the age of rye at time of planting. The greatest reductions in tomato biomass occurred in treatments where rye was 10-30 days old at the time tomatoes were planted. Unconcentrated solutions of rye root leachates had no effect on seed germination of cress (Lepidium sativum L.) barnyardgrass (Echinochloa crusgalli L. Beauv.) and common lambsquarters (Chenopodium album L.) (Barnes, 1981 ). These studies in quartz sand under greenhouse conditions indicated rye root leachates were more inhibitory to growth of tomato than tomato root leachates were on themselves. The interference observed by a living cover of rye might also arise from toxic compounds released by leachates of shoots. Shoots of different aged rye were misted and leachates were collected for bioassay. Shoot leachates had no effect on germination of lettuce, barnyardgrass, cress, and tomato or seedling growth of tomatoes (Barnes, 1981 ). Instances of poor germination and seedling growth were reported by growers in the Salinas Valley, where lettuce was planted too soon after turning in barley or rye cover craps. Patrick et al (1963) initiated 18 experiments to determine whether severe phytotoxicity could be detected when the cultural practice was repeated in the greenhouse with soil and plant residue obtained directly from the field. Lettuce root lengths and fresh weights were reduced in treatments where rye residue was present. Rye extracts with marked phytotoxicity were obtained after residues had been decomposed for 10-25 days. Toxicity of residues declined as the decomposition period increased, until by the 30th day, little or no phytotoxicity was observed. Patrick and Koch (1958) found decomposing rye residues to inhibit respiration in tobacco seedlings. In a later experiment, Patrick (1971) sampled tobacco fields which contained decomposing rye residues previously plowed under as a green manure. He found extracts of decomposing residue fragments to delay germination and reduce root growth of lettuce and tobacco. Similar phytotoxicity was exhibited with extracts of soil which were in contact with decomposing rye residues. No phytotoxicity was obtained with extracts of soil from which all recognizable decomposing rye residues had been removed. Ether extraction and gas chromatographic analysis identified several acids, including acetic, butyric, benzoic, and phenylacetic acid, to be the phytotoxic compounds” It‘was not determined whether phytotoxic substances were synthesized by soil microorganisms using the plant material as a substrate, or were the breakdown products inherent in the plant tissue. It may have been both, since phytotoxic compounds of significant potency were detectable only after the decomposing plant residue had been freed from most of the adhering soil, prior to extraction. Chou and Patrick (1976) later identified nine acids from ether extracts of decaying rye residues in soil. Phenylacetic, 4- 19 phenylbutyric, and salicyclic acids inhibited lettuce root growth at concentrations between 25 and 100 ppm. Other compounds identified included vanillic, ferulic, p-coumaric, p-hydroxybenzoic, o-coumaric and salicycladehyde and were active at ca 100 ppm. Although phytotoxicity and symptoms of injury obtained with a volatile bioassay were consistent with Patrick et a1 (1963), the extraction methods and sample preparation would have excluded identification of volatile compounds. Further, their results were based on three assumptions of questionable validity: the phytotoxic compounds in decomposing residues of rye were (1) released as volatiles; (2) polar in nature and (3) of a phenolic origin. As a result, Chou and Patrick identified only polar, known phenolic compounds for which standards could be obtained. The decomposition of rye residues, or any organic substrate, is a continuing process which requires rapid and sensitive assay methods to detect phytotoxic compounds during their short interval of production and disappearance (Patrick, 1971 ). This is evident from the various contradictory results often obtained during decomposition of similar plant residues. The ephmeral nature of such products, and their relatively rapid transition from one type of physiological activity to another, may be reasons why their occurrence is frequently missed. Plant injury is dependent on the frequency of a chance encounter with a growing root system, or with fragments of plant residues, at a time when decomposition is favorable for toxin production. Sholte and Kupers (1977, 1978) investigated the causes of the lack of self-tolerance of winter rye grown on light sandy soils. In an 18 yr rotation of rye, they found yield of seed and straw decreased 30% and 10% respectively, when compared to rye following other crops. 20 Nematodes and foot rot fungi were excluded as possible causes. In a later study, they provided circumstantial evidence that the soil microflora was involved in the self-intolerance. Kimber (1973) found cold water extracts of several grasses, including slightly green rye straw, that had rotted from periods up to 21 days, to inhibit growth of wheat grown under aseptic conditions. Sterile conditions were used to eliminate the possible interactions of pathogens and microbial products. The degree of inhibition varied from one species to another and also with the length of the rotting period. He found slightly green straw to be more toxic than fully matured residue. The most phytotoxic materials were from extracts of rye straw which had rotted for 4 days. Putnam and DeFrank (1979) screened numerous cover crops for weed suppressing activity. They found fall-planted cover crops to reduce both weed populations and biomass in the next growing season. Fal 1- killed 'Balboa' rye reduced weed biomass by 84% over no-residue controls, while Spring-killed rye residues were less toxic. Phytotoxicity of water soluble compounds leached from several plant Species suggested that allelopathy was a factor contributing to their effectiveness. Additional field trials indicated that a variety of annual weed Species could be suppressed consistently with residues of immature cereals (including rye, corn, barley, wheat, sorghum and oats), whereas larger seeded vegetables, such as legumes, grew quite well in the NT systems (Putnam and DeFrank, 1983). Residues of 40 day-old spring-planted rye reduced total weed, density by 69% and total weed biomass by 32% when compared to a non-toxic control mulch of poplar (Populus) excelsior (Barnes, 1981). Fall-planted, Spring-killed rye residues also reduced total weed 21 biomass by 68 to 95% when compared to controls with no residue (Barnes and Putnam, 19831. Weed biomass was reduced an additional 35% when compared to the control mulch, further indicating that allelopathy was one component of the interference by rye on other plant Species. A greenhouse bioassay was develOped to detect potential toxicity in Situations Similar to the field. Studies indicated that two contact herbicides used to kill rye differentially affected residue phyto- toxicity when compared to other mechanical and physical methods of kill (Barnes, 1981). Thirty-five day old rye was killed with paraquat (l,1'-dimethyl-4,4”—bipyridinium ion) or glyphosate [N-(phosphono- methyl) glycine] 10 days prior to bioassay. Seeds were directed through the residue into the soil without disturbing the system. Residues of paraquat-Sprayed 'Wheeler' rye reduced emergence and biomass of barnyardgrass more than residues killed by other methods. In contrast, residues of glyphosate-Sprayed rye were more inhibitory to lettuce emergence and tomato Shoot growth. While exudation of glyphosate from rye roots could be responsible for the reductions in germination and growth, no symptoms of glyphosate injury were observed. The stress of the chemical treatment could have caused rye to produce or release more toxic natural products. Rye killed with paraquat reduced the emergence of lettuce, tomato, and barnyardgrass to about 50% of that under control mulch or soil controls Sprayed with paraquat (Barnes, 1981). Emergence of barnyardgrass was reduced as the rate of paraquat used to kill the rye was increased to 1.12 kg/ha. Where no paraquat was applied, residues of rye still reduced emergence by 17% over the controls. Although paraquat appeared to enhance phytotoxicity, plant growth was suppressed 22 more by the rye residues. Researchers at North Carolina State University have also examined the feasibility of using a rye mulch for weed suppression in no-till crapping systems (Shilling et al, 19851. They found that fall-planted, Spring-killed rye reduced above ground biomass of several weed Species, including redroot pigweed (Amaranthus retroflexus LJ, common lambsquarters, and common ragweed (Ambrosia artemisiifolia L.L Subsequent isolation of compounds with toxicity in aqueous extracts of field grown rye resulted in identification of two chemicals,e- phenyllactic acid (PLA) and B-hydroxybutyric acid (HBA). Both acids inhibited common lambsquarters root growth 20% at 2 mM. Redroot pigweed root growth was inhibited 59 and 39% at 2 M by PLA and HBA, respectively. Although there are numerous reports suggesting allelopathic activity by rye and its residues, few have followed the protocols for proof of1allelopathic interference, as suggested by Fuerst and Putnam (1983). The purpose of this research was to investigate the chemical interference phenomenon (allelopathy) in rye, with particular emphasis on crop and weed responses to its residues. The experimental plan was to identify the symptoms of the interference in assays designed to test for chemical effects, to isolate, identify, and quantify phytotoxicities of chemicals from the residues, and to assess the importance of the chemicals in the observed interference. LITERATURE CITED Anonymous. 1984. International Chemical Congress of Pacific Basin Societies. Abstract of papers. Honolulu, Hawaii. Barnes, J. P. 1981. Exploitation of rye (Secale cereale Lu) and its residues for weed suppression in no-tillage crapping systems. Thesis for the M.S. degree, Michigan State University. 128 pp. Barnes, J. P. and A. R. Putnam. 1983. Rye residues contribute weed suppression in no-tillage cropping systems. J. Chem. Ecol. 9:1045-1057. Bell, P.11 and Koeppe. 1972. Noncompetitive effects of giant foxtail on the growth of corn. Agron. J. 64:321-325. Bell, A. A. 1974. Biochemical basis of resistance of plants to pathogens. Pp. 403-461 in ”Biological Control of Plant Insects and Diseases“. FG. Maxwell and F.S. Harris, (edS.) pp. 403-461 Univ. Press of Mississippi, Jackson. Benoit, R. E., Willits, N. A., and Hanna, W. J. 1962. Effect of rye winter cover craps on soil structure. Agron. J. 54:419. Belvins, R. L., Cook, 0., Phillips, 5. H., and Phillips, R. E. 1971. Influence of no-tillage on soil moisture. Agron. J. 63:593-596. Bode, H. R. 1940. Leaf excretions of wormwood (Artemisia absinthium) and their effect upon other plants. Plants: Arch. f. HiSS. Bot. 30:567-589. 23 10. 11. 12. 13. 14. 15. 16. 17. 18. 24 Borner, H. 1950. The role of toxic substances in the interactions of higher plants. Bot. Rev. 16:51-65. Borner, H. 1955. Untersuchungen uber Phenolische verbindungen aus Gefreidestroh and Getreiderukstanden. Naturwissenschaften. 42:583-4. Chou, C-H, and Z. A. Patrick. 1976. Identification and phytotoxic activity of compounds produced during decomposition of corn and rye residues in soil. J. Chem. Ecol. 2:369-387. Cloutier, Y. 1984. Changes of protein patterns in winter rye following cold acclimation and desiccation stress. Can. J. Bot. 62:366-371. Cochran, V. L., L. F. Elliott, and R. I. Papindick. 1977. The production of phytotoxins from surface crop residues. Soil Sci. Soc. 41:903-908. Cubbon, M. H. 1925. Effect of rye crop on the growth of grapes. Amer. Soc. Agron. 17:568-577. Dekker, J. H. and W. F. Meggitt. 1983a. Interference between velvetleaf (Abutilon the0phrasti) and soybean (Glycine max): I. Growth. Weed Res. 23:91-101. Dekker, J.1L and W.1fl Meggitt. 1983b. Interference between velvetleaf (Abutilon the0phrasti) and soybean (Glycine max): 11. Population dynamics. Weed Res. 23:103-107. deCandol le, M. A. P. 1932. Physiologie Vegetale III. Bechet Jeune. Lib. Fac. Med. p. 1474. Paris. Dieterman, L. J., G. Y. Lin, L. M. Rohrbaugh, V. Thiesfield, and S. H. Wender. 1964. ID and quantative determination of scopolin and Sc0poletin in tobacco plants treated with 2,4- dichlorOphenoxyacetic acid. Anal. Biochem. 9:139-145. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 25 Ellis, J.1L.and T. M. McCalla. 1973. Effect of patulin and method of application on growth stages of wheat. Appl. Microbiol. 25:562-566. Faulkner, E. H. 1943. Plowman's Folly. University of Oklahoma Press. 156 pp. Fay, P. K. and W. B. Duke. 1977. An assessment of alle10pathic potential in Avgng germplasm. Weed Sci. 25:224-228. Fisher, R. F. 1978. Juglone inhibits pine growth under certain moisture regimes. Soil Sci. Soc. Amer. J. 42:801-803. Fuerst, E. P. and A. R. Putnam. 1983. Separating the competitive and a1 lelopathic components of interference: theoretical principles. J. Chem. Ecol. 9:937-944. Grummer, G. and H. Beyer. 1960. The influence exerted by species of Camelina on flax by means of toxic substances. Pp 153-57 in [Th2 Biology gjflygggs. Oxford: Blackwell. 256 pp. Guenzi, W. 0. and T. M. McCalla. 1962. Inhibition of germination and seedling development by crop residues. Proc. Soil Sci. Am. 26:456-58. Guenzi, W. 0., T. M. McCal 1a, and F. A. Norstadt. 1967. Presence and persistance of phytotoxic substances in wheat, oat, corn, and sorghum residues. Agron. J. 54:164-65. Halleck, F. E. and V. W. Cochrane. 1950. The effect of fungistatic agents on the bacterial flora of the rhizoSphere. Phytopathol. 40:715-718. Harborne, J. B. 1972. Phytochemical Ecology. London: Academic Press. 22 pp. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 26 Harborne, J. B. 1977. Introduction 39 Ecological Biochemistry. Academic Press, London. 243 pp. Harper, J. L. 1977. P0pulation Biology 21 E13353, Academic Press, New York. 892 pp. Harper, S. H. T. and Lynch, J. M. 1982. The role of water soluble components in phytotoxicity from decomposing straw. Plant and Soil. 65:11-17. Hattingh, M. J. and H. A. Louw. 1969. Clover rhizoplane bacteria antagonistic to Rhizobium trifolii. Can. J. Bot. 15:361-64. Heisey, R. M. and C. C. Delwiche. 1983. A survey of California plants for water-extractable and volatile inhibitors. Bot. Gaz. 144:382-390. Hill, H. H. 1926. Decomposition of organic matter in soil. J. Agr. Res. 33:77-99. Holm, L. 1971. Chemical interactions between plants on agricultural lands, Pp. 95-101. US National Commission for International Biological Program (eds). Biochemical Interactions Among Plants. National Academy of Science, Washington, DJ; Jalali, B. L. and K. H. Domsch. 1977. Effect of some fungitoxicants on the amino acid spectrum of wheat root exudates. Phytopath. Z. 90:22-26. Jalali, B. L. and 0. Suryanarayana. 1970. Biochemical nature of root exudates in relation to root rot of wheat. 1. Amino acid Shifts in response to foliar treatments. 2. Pflanzenkrankh. Pflanzenschutz. 77:438-442. Katznelson, H., J. W. Rouatt, and T. M. B. Payne. 1955. The liberation of amino acids and reducing compounds by plant roots. Plant Soil. 7:35-48. 39. 40. 41. 42. 43. 44. 27 Kimber, R. W. 1973. II. The effect of time of rotting of straw from some grasses and legumes on the growth of wheat seedlings. Plant and Soil. 38:437-361. Koeppe, 0. E., L. M. Rohrbaugh, and S. H. Wender. 1969. The effect of varying U.V. intensities on the concentration of scapolin and caffeoquuinic acids in tobacco and sunflower. Phytochem. 8:889-896. Koeppe, 0. E., L. M. Rohrbaugh, E. L. Rice, and S. H. Wender. 1970. The effect of age and chilling temperatures on the concentration of scapolin in caffeoquuinic acids in tobacco. Physiol. Plant. 23:258-266. Koeppe, 0. E., L. M. Rehrbaugh, and S. H. Wender. 1971. The effect of environmental stress conditions on the concentration of cafeoquuinic acids and scopolin in tobacco and sunflower, Pp. 102-108. U.S. National Commission for International Biological Program (edSJ. Biochemical Interactions Among Plants. National Academy of Science, Washington, 0. C. Kossanel, J. P., Martin, J., Annel 1e, P., Peinot, M., Vallet, J. K., and Kurney, K. 1977. Inhibition of growth of young radicles of maize by exudations in culture solutions and extracts of ground roots of Chenogodium album L. Pp. 77-86. in Interactions o_f Plants and Microorganisms lg Phytocenoses, (A.M. Grodzinsky, Ed.). Kiev. Kozel , P. C. and H. B. Tukey, Jr. 1968. Loss of gibberellins by leaching from stems and foliage of Chrysanthemum morifolium 'Princess Anne'. Amer. J. Bot. 55:1184-1189. 45. 46. 47. 48. 49. 50. 51. 52. 53. 28 Lawlor, 0. W. 1979. Effects of water and heat stress on carbon metabolism of plants with C-4 photosynthesis. Fun 303-326. in "Stress Physiology in Crop Plants", H. Mussel and R. C. Staples (eds.), Wiley, N.Y. Wiley, N.Y. Levin, 0. A. 1971. Plant phenolics: An ecological perspective. Am. Nat. 105:157-181. Levin, 0. A. 1976. The chemical defenses of plants to pathogens and herbivores. Ann. Rev. of Ecol. and Sys. 7:121-59. Lockerman, R. H. and Putnam, A. R. 1981 . Growth inhibitors in cucumber plants and seeds. J. Amer. Soc. Hort. Sci. 106:418-422. Loehwing, W. F. 1971. Root interactions of plants, Pp. 195-238. 0.5. National Commission for International Biological Programs (eds). Biochemical Interactions Among Plants. National Academy of Science, Washington, 0.C. Lovett, J. V. and J. Levitt. 1981. Allelochemicals in the future agriculture. Pp. 169-80. in Biolggigal Husbandgy, (B. Stonehouse, ed.). Butterworths, London. Lovett, J. V. 1982. Allelopathy and self-defense in plants. Australian Weeds. 2:33-36. McCalla, T. M. and F. L. Duley. 1948. Stubble mulch studies. Effect of sweet clover extracted on corn germination. Science. 108:163. McCalla, T. M. and F. L. Duley. 1949. Stubble mulch studies: III. Influence of soil microorganisms and crop residues on the germination, growth and direction of root growth of corn seedlings. Soil Sci. Soc. Amer. Proc. 14:196-199. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 29 McCalla, T. M. and F. A. Haskins. 1964. Phytotoxic substances from soil microorganisms and residues. Bacteriol. Rev. 28:181- 207. McKey, 0. 1974. Adaptive patterns in alkaloid physiology. Am. Nat. 108:305-320. McKey, 0. 1979. The distribution of secondary compounds within plants. Pp. 55-133 in Herbivores, (G. A. Rosenthal and 0. H. Janzen, eds.). Academic Press. N.Y. Molisch, H. 1937. Der Einfluss einer pflanze auf die audere- allelopathie. Jena:Fisher. Moreland, 0. E., G. H. Egley, A. 0. Worsham, and T. J. Monaco. 1966. Regulation of plant growth by constituents from higher plants. Adv. Chem. 53:112-41. Muller, C. H. 1964. Volatile growth inhibitors produced by Salvia species. Bull. Torrey Bot. Club. 91:327-330. Muller, C.1L 1965. Inhibitory terpenes volatilized from Sglylg Shrubs. Bull. Torrey Bot. Club. 92:38-45. Muller, C. H. 1966. The role of chemical inhibition (a1 lelopathy) in vegetational composition. Bull. Torrey Bot. Club. 93:332-351. Muller, C. H. 1969. Allelopathy as a factor in ecological processes. Vegetatio. 18:348-357. Muller, C. H., P. Lorber, and P. Haley. 1968. Volatile growth inhibitors produced by Sal via leucophyl la: Effect on seedling growth and respiration. Bull. Torrey Bot. Club. 95:415-22. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 30 Muller, C.1L 1971. Phytotoxins as plant habitat variables, Pp. 64-71. 1L5. National Commission for International Biological Program (edsJ. Biochemical Interactions Among Plants. National Academy of Science, Washington, DJ; Nickell, L. G. 1960. Antimicrobial activity of vascular plants. Econ. Bot. 13:281-318. Norstadt, F. A. and T. M. McCal 1a. 1963. Phytotoxic substances from a species of Penicillium. Science. 140:410-411. Nuttonson, M. Y. 1958. Rye-climate relationships on the use of phenology in ascertaining the thermal and photo-thermal requirements of rye. Amer. Inst. of Crop Ecology. 219 pp. Odum, E. P. 1969. The strategy of ecosystem develOpment. Science. 164:262-270. Osvald, H. 1953. An antagonism between plants. Intern. Congr. Bot. (7th) Proc. Stockholm (1950). Overland, L. 1966. The role of allelopathic substances in the “smother crop” barley. Amer. J. Bot. 53:423-432. Patrick, 2. A. and L. W. Koch. 1958. Inhibition of respiration, germinatnnu and growth by substances arising during the decomposition of certain plant residues in the soil. Can. J. Bot. 36:621-647. Patrick, 2. A., T. A. Toussoun, and W. C. Snyder. 1963. Phytotoxic substances in arable soils associated with decomposition of plant residues. PhytOpathology. 53:152-61. Patrick, Z. A. 1971. Phytotoxic substances associated with the decomposition in soil of plant residues. Soil. Sci. 111:13-18. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 31 Phillips, S. H. and H. M. Young, Jr. 1973. No-til lage farming. Reiman Asso. Mi1., Wis. 224 pp. Putnam, A. R. and W. W. Duke. 1974. Biological suppression of weeds: Evidence for allelopathy in accessions of cucumber. Science. 185:370-372. Putnam, A. R. and W. B. Duke. 1978. Allelopathy in agroecosystems. Ann. Rev. Phytopathol. 16:431-51. Putnam, A. R. and J. DeFrank. 1979. Use of alle10pathic cover crops to inhibit weeds. Proc. IX Int. Cong. Plant Prot. pp. 580- 582. Putnam, A. R., J. DeFrank. 1983. Use of phytotoxic plant residues for selective weed control. Crop Prot. 2:173-181. Putnam, A. R. 1985. Weed allelopathy. p. 131-155 in Vol.1 of Weed Physiology, (Stephen 0. Duke, ed.) CRC Press, Inc. Boca Raton, Fla. Reid, C. P. P. 1974. Assimilation, distribution, and root exudation of 14C by ponderosa pine seedlings under induced water stress. Plant Physiol. 54:44-49. Rice, E.lu 1971. Some possible roles of inhibitors in old-field succession, Pp. 128-131. U.S. National Commission for International Biological Programs (eds.). Biochemical Interactions Among Plants. National Academy of Science, Washington, 0.C. Rice, E. L. 1984. AlleloLathy, 2nd edition. Academic Press, Inc. 422 pp. Richards, B. N. 1974. Introduction £2 the Soil Ecosystem. Longman, Inc. New York. 266 pp. 84. 85. 86. 87. 88. 89. 90. 91. 92. 32 Robertson, W. K., H. W. Lundy, G. M. Prine, and W. L. Currey. 1976. Planting corn in sod and small grain residues with minimum tillage. Agron. J. 68:271-74. Rovira, A. 0. 1956. Plant root excretions in relation to the rhizOSphere effect. I. The nature of root exudate from oats and peas. Plant Soil. 7:178-194. Rovira, A.[L 1965. Interactions between plant roots and soil microorganisms. Ann. Rev. Microbiol. 19:241-66. Rovira, A. 0. 1969. Plant root exudates. Bot. Rev. 35:35-59. Rovira, A. 0. 1971. Plant root exudates, Pp. 19-23. U.S. National Commission for International Biological Programs (edsJ. Biochemical Interactions Among Plants. National Academy of Science, Washington, 0.C. Rovira, A. 0. 1973. Zones of exudation along plant roots and spatial distribition of micro-organisms in the rhizosphere. Pestic. Sci. 4:361-366. Scholte, K and L. J. P. Kupers. 1977. The causes of the lack of self-tolerance of winter rye, grown on light sandy soils - influences of foot rots and nematodes. Neth. J. Agric. Sci. 25:255-262. Scholte, K. and L. J. P. Kupers. 1978. The causes of the lack of self-tolerance of winter rye, grown on light sandy soils - influences of phytotoxins and soil microflora. Neth. J. Agric. Sci. 26:250-266. Shear, G. M. 1968. The develOpment of the no-tillage concept in the U.S. Outlook Agric. 5:247-251. 93. 94. 95. 96. 97. 98. 99. 100. 33 Shilling, 0. G., R. A. Liebl, and A. 0. Horsham. 1985. Rye (Secale cereale L.) and wheat (Triticum aesti vum L.) mulch: The suppression of certain broadleaf weeds and the isolation and identification of phytotoxins. Pp. 243-271, in The Chemistyy g: Allelopathy, N. C. Thompson (ed.). Siegler, 0. S. 1977. Primary roles for secondary compounds. Biochem. Syst. Ecol. 5:195-199. Silverstein, R. M. and J. B. Simeone, eds. 1983. North American Symposium on Allelopathy. J. Chem. Ecol. 9:937-1291. Stal lworth, H. 1963. Some effects of 2.4-0 on sweet corn (Z_e_a gays) with emphasis on yield, tillering, root development and exudation of electrolytes from roots and stems. Diss. Abstr. 23:2653. Steinberg, P. 0. 1984. Algal chemical defense against herbivores: Allocation of phenolic compounds in the kelp M2 ma_rginata. Science. 223:405-406. Sterling, T. M. and A. R. Putnam. 1984. Phytotoxic exudates from vel vetleaf trichomes and their possible ecological role. Abstract in 1984 Proceedings of North Central Weed Control Conf. p. 78. Stotzky, G. 1974. Activity, ecology, and population dynamics of microorganisms in soil. Pp. 57-135 in Microbiol Ecology, A. Lankin and H. Lechevalia (eds.). CRC Press Inc. Cleveland, Ohio. Stowe, L. G. 1977. Allelopathy and its influence on the distribution of plants in an Illinois old field. Ph.D. Thesis, Univ. Chicago. 89 pp. 101. 102. 103. 104. 105. 106. 107. 110. 111. 34 Stowe, L. G. and Osborn, A. 1980. The influence of nitrogen and phOSphorus levels on the phytotoxicity of phenolic compounds. Can. J. Bot. 58:1149-1153. Swain, T. 1977. Secondary compounds as protective agents. Ann. Rev. Plant Physiol. 28:479-501. Tang, C-S and C-C Young. 1982. Collection and identification of allelopathic compounds from the undisturbed root system of bigalta limpograss (Hemarthia altissima). Plant Physiol. 69:155-160. Thompson, A. C., ed. 1985. The Chemistry o_f_ Allelopathy. Based on a Symposium by the division of pesticide chemistry of the American Chemical Society. 470 pp. Tukey,1L B.,.ha 1966. Leaching of metabolites from above ground plant parts and its implications. Bull. Torrey Bot. Club. 93:385-401. Tukey, H. 8., Jr. 1969. Implications of allelopathy in agricultural plant science. Bot. Rev. 35:1-16. Tukey, H. B., S. H. Wittwer, and H. B. Tukey, Jr. 1957. Reaction of carbohydrates from plant foliage as related to light intensity. Sci. 126:120-121. Tukey, H. B. and Morgan, J. V. 1963. Injury to foliage and its effect on the leaching of nutrients from above ground plant parts and its implications. Physiologia Plantarium. 16:557-565. Vandermerwe, K. J. P.P. Van Jaarsueld, M. J. Hattingh. 1967. The isolation of 2,4-diacety1-phloroglucinol from a Pseudomonas spp. S. Afr. Med. J. 41:1110. 112. 113. 114. 115. 116. 117. 118. 35 Vrany, J., V. V. Vancoura, and J. Macura. 1962. The effect of foliar applications of some readily metabolizable substances, growth regulators,and antibioticscnirhizoSphere microflora. Folia Microbiology. 7:61-70. White, G. A. and A. N. Sterratt. 1967. The production of a phytotoxic substance by Alternaria zinnie. Can. J. Bot. 45:2087-2090. Whittaker, R. H. 1970. The biochemical ecology of higher plants. Pp. 43-70. in Chemical Ecology, E. Sandheimer and J. B. Simeone (eds.). Academic Press, New York City. 336 pp. Whittaker, R. H. and P. P. Feeny. 1971. Allelochemics: Chemical interaction between Species. Science. 171:757-770. Whittaker, R. H. 1975. Communities and Ecosystems, 2nd ed. Macmillan Pub., Inc. New York. 358 pp. Woodhead, S. and E. A. Bernays. 1978. The chemical basis of resistance to Sorghum bicolor to attack by Locusta migratoria. Ent. Exp. Appl. 24:123-144. Woodhead, S. 1981. Environmental and biotic factors affecting the phenolic content of different cultivars of sorghum bicolor. J. Chem. Ecol. 7:1035-1046. CHAPTER 2 EVIDENCE FOR ALLELOPATHY BY RESIDUES AND AQUEOUS EXTRACTS OF RYE (Secale cereale L.) ABSTRACT Under Simulated no-till conditions in the greenhouse, rye (Secale cereale L. 'Wheeler') residues reduced emergence of lettuce (Lactuca sativa L. 'Ithaca') and proso millet (Panicum miliaceum L.) by 58% and 35% respectively, over that obtained with a wood shaving control mulch. Rye shoot tissue inhibited lettuce germination 52% more than root tissue. Petri dish bioassays of residue in soil confirmed phytotoxicity on cress (Lepidium sativum L. 'Curly'), lettuce, barnyardgrass (Echinochloa crusgglli L. Beauv.), and proso millet. Radicle elongation was a more sensitive measure of phytotoxicity than germination Egg 5;. Phytotoxicity increased as the distance from seeds to rye residues decreased. While rye Shoot residues caused 40% greater inhibition of cress radicle elongation in sterile vs. non-sterile soil, barnyardgrass was equally inhibited in both soil regimes. In no case was phytotoxicity enhanced by a non-sterile soil. Phytotoxic compounds in Shoots were water-extractable. Cress and barnyardgrass responded similarily to both aqueous rye extracts and to residues added to soil. 36 INTRODUCTION Rye is a winter annual grass Species often grown in the northern latitudes of the world which has proven useful aS a rotational species in many cropping systems (3, 10). It has been found to contribute organic matter, reduce soil erosion, and enhance water penetration and retention (3, 4, 201. It is common to plant rye in the fall, allow it to grow during the fall and early spring, and then plow'and disc it under before conventional planting in the Spring. An alternative to this practice is a reduced tillage cr0pping system where rye is chemically killed in the Spring. The residue remains at the soil surface where it may modify the environment both physically and chemically during seed germination and plant growth. Chemical interference with plant growth is termed alle10pathy and appears to be a viable strategy for weed suppression in agroecosystems (2, 13, 14, 18). Allelopathy has been identified as an important component of plant interference in both natural and agroecosystems (5, 15, 16, 17, 201. Detrimental weed and crop interference has traditionally been attributed to differential utilization of Space, light, water, and nutrients. While plants undoubtedly compete, their ability to chemically interfere may also be important. Levin (9) has suggested that production of secondary compounds places energy demands upon plants and hence must be purposeful and Specialized. The ability of plants to produce and release chemicals that interfere with the 37 38 growth of other plants may be more common than previously realized. Allelopathic chemicals produced by plants may confer a unique adaptive advantage to those Species (8, 21). Living winter rye reduced early season biomass of common lambsquarters (ChenOpodium album L.) by 98%, large crabgrass (Digitaria sanguinalis L. Scop.) by 42%, comnon ragweed (Ambrosia artemiisifolia L.) by 90% and total weed biomass by 94% over controls where no rye was present (1). Both competition for water, light, and nutrients and allelopathy may contribute to these reductions. In addition, residues of Spring planted rye killed after 40 days of growth, reduced total weed density by 69% and total weed biomass by 32% over a control mulch of paplar (Populus) excelsior (1). P0p1ar excelsior (PE) has been used to stabilize grass seedings along roadsides and was previously demonstrated to be a suitable control for the 'mulch' effect associated with residue on the surface. Residues of fall-planted, Spring-kil led rye reduced total weed biomass by 68 to 95% when compared to controls with no residue (2). Weed biomass was reduced an additional 35% when compared to the PE mulch, further indicating that al lelopathy was one component of the interference by rye on other plant Species (2). Rye residues appeared to reduce plant growth more than germination or emergence. Researchers at North Carolina have also found rye mulch to reduce above ground biomass of several weed species including redroot pigweed (Amaranthus retroflexus L.). common lambsquarters, and conmon ragweed (18). In addition to residue phytotoxicity, root leachates of living rye were also inhibitory in transfer studies where solutions were manually 39 collected and applied to test species (1). Total biomass of lettuce and tomato (lLyc0perSicon esculentum Mill.) was reduced 25% and 18%, respectivelyg by root leachates that had passed through two pots of rye. This indicated that root leachates from rye were more inhibitory to tomato and lettuce growth than were leachates from other tomato and lettuce plants. This evidence, in conjunction with previous experiments with rye residues, suggests potential allelopathic activity in winter rye (2). The purpose of our work was to further characterize the alle10pathic potential of rye residues and extracts. MATERIALS AND NETHODS General. 'Wheeler' rye for all experiments was grown under metal halide lights (500 uE-m'Z-s'l; 14 h daylength) in the greenhouse and watered with a soluble fertilizer (1.0 g/l of 20%N, 8.8%P, 16.6%K) solution every third day. The plant Species used as indicators were 'Ithaca' lettuce, 'Petoearly' tomato, 'Curly cress', barnyardgrass and proso millet. Soil used in all cases was a Spinks loamy sand (Psammentic hapludalf, sandy, mixed, mesic) with 1% organic matter (pH 6.5). For petri dish assays, rye shoots cut from the soil surface 35 days after planting (DAP), or roots rinsed free of soil, were dried at 50 C, cut into segments approximately 2 cm long, and weighed. Aqueous extracts were obtained from 35 DAP dried shoots that had been ground through a 40 mesh screen in a Wiley mill. Double distilled and deionized water (000W) was used in.all petri dish bioassays. In those assays where activity in sterile soil was tested, soil was autoclaved 40 (120 C; 23 psi) for 1 h (3 X), at 48 h intervals. Sterility was confirmed by plating two dilutions per soil sample on potato dextrose agar (19.5 g PDA/L 000W). Plates remained sterile for at least 72 h or the duration of the experiments. Square petri dishes (100 by 100 by 15 mm) with a surface area of 90.25 cm2 were filled with 150 g oven-dried soil. Dried rye residues were uniformly Spread over the surface of the dish at a constant depth after which 35 m1 of 000W were added to each dish. Whatman #1 filter paper was placed on top of residue and soil and indicator seed were placed on top of the filter paper. Petri dishes were covered and incubated vertically to induce geotrophic root growth. Seeds were considered germinated if the radicle emerged from the seed coat. All petri dish experiments were incubated for 72 h at 27 C in a growth chamber. Each dish contained all indicators tested. The number of seeds bioassayed per Species varied depending on the germination of that Species. The average root length for each species per dish is considered one replication per treatment. Analysis of variance for each experiment was based on a randomized complete block with four replications. Experiments were repeated twice and data presented is the results of one representative experiment. Inhibition by Rye Residues. Rye grown in 25 by 25 cm plastic flats was killed chemically 35 DAP with 1.1 kg ai/ha of the isopropylamine salt of glyphosate [N-(phosphonomethyl) glycine] or 1.1 kg ai/ha paraquat (1,1'-dimethy1-4,4'-bipyridinium cation) plus 0.5% (v/v) x-77 surfactant (alkylaryl polyoxyethylene glycol, free fatty acids, isopropanol). Chemical treatments were applied at 337 L/ha. To evaluate potential residual phytotoxicity by chemicals, tissue was also killed by freezing at -12 C for 15 h. The effects of shoot 41 tissue, root tissue, or both root and Shoot tissue compared to a PE mulch control. PE mulch was applied to control flats and those receiving rye roots after planting, at a rate equivalent to the average biomass of four flats of rye Shoot tissue (9.4 9). Seven days after Spraying or freezing the flats, 30 seeds of each indicator Species (lettuce, tomato, barnyardgrass, and proso millet) were planted into rye residues for bioassay. Percent emergence was recorded 14 DAP. An initial petri dish study was done to determine the effect of residue placement relative to seed. Treatments included a no residue control and 1.0 g rye Shoot tissue placed 0, 5, 10, and 15 mm away from seed. Adding soil in layered, 50 9 increments allowed for accurate placement of residue in the dish. Cress, lettuce (5 seeds each), barnyardgrass and proso millet (10 seeds each) germination and root length were expressed relative to the no-residue control. To evaluate the effect of plant part and quantity of residue on root elongation of the same number and types of indicator Species mentioned above, roots, Shoots and their combination, were assayed at 0.0, 0.25, 0.5, 1.0, and 2.0 g/dish placed 5 mm away from seed. Paper towel chopped into 2 cm fragments were also used as control S. Where both root and Shoot tissues were applied, the total weight of tissue was equal to the other treatments but was comprised of 50% root tissue and 50% shoot tissue. Within each species, the no residue and all paper towel controls were similar, and therefore, averaged. The treatment root length for each Species was expressed relative to this average. A final bioassay of residues evaluated activity of shoot tissue in sterile soil. Treatments consisted of the above rates of shoot 42 residues and towel controls placed 5 mm from seed in autoclaved and non-autoclaved soil. The plant residues and paper toweling for all treatments were sterilized with pr0pylene oxide (1 m1) injected into sealed pint mason jars. After 24 h, the propylene oxide was evacuated and the plant residues were stored for later use. Ten seeds of both cress and barnyardgrass were bioassayed. The treatment root length for each species was expressed relative to the average control for that species. Inhibition by Aqueous Extracts. Aqueous extracts of dried shoot tissue were obtained by mixing 50 g with 1 L 000W for 24 h at 4 C. After cold water extraction, the green aqueous solution was filtered through cheesecloth and centrifuged for 25 min at 9514 x g. 'The brown supernatant was lyophilized to preserve its chemical components prior to bioassay Hisoil; lyophilized crude extracts were rehydrated and diluted with 000W. Dilutions were suction sterilized through a (L45 micron filter and were transferred to sterile glass bottles for storage. Several rates of aqueous crude extract (0, 6, 13, 25, 50, and 100 mg/dish) in 6 m1 000W were applied to petri dishes (15 by 60 mm) filled with 25 g of autoclaved and non-autoclaved, oven-dried soil. Ten seeds of cress or barnyardgrass were placed on the soil surface and petri dishes were covered prior to dark incubation. Root lengths were measured in mm and expressed relative to the 000W control. RESULTS AND DISCUSSION Inhibition by rye residues. Emergence of the assay Species through rye residues varied with the source of the residue and with the 43 species tested. The methods used to kill rye did not Significantly affect plant emergence through its residues allowing averaging of data over the three killing methods. Emergence of both lettuce and proso millet was Significantly reduced by rye shoots and both roots and shoots when compared to PE mulch (Table 1). In contrast, rye had little effect on emergence of tomato or barnyardgrass. Emergence in root tissue plus PE mulch was Similar to the PE mulch control for all Species. Lettuce was the most sensitive indicator tested. It frequently appeared chlorotic upon emergence and its subsequent growth was stunted. Some plants appeared to escape injury. Although nutrient supplies were superoptimal, there were individuals of all species Showing chlorosis during emergence through rye residues. To confirm toxicity of residues in soil and the potential for dilution of activity by soil, an initial experiment examined the effect of residue placement relative to seed using a petri dish bioassay (Figure la, b, c, d). Rye residues had no effect on germination of barnyardgrass and proso millet at any distance. In contrast, residue placed directly next to seed significantly reduced cress and lettuce germination. Overall, as distance between residue and seed increased, phytotoxicity decreased. The primary phytotoxic effect by residues on all species was the inhibition of root growth. While soil diluted the phytotoxicity, residues still Significantly reduced root growth of all Species when placed 15 mm from the seeds. Lettuce was particularily sensitive to rye residues. Hhen placed <:losest to residue, the seeds imbibed water but turned black and failed to grow. In cases where lettuce did germinate, the hypocotyls appeared swol len and the apical root meristems were discolored. Growth was Stabsequently inhibited. These symptoms of injury to lettuce are 44 Table 1. Percent emergence of four indicator Species in residues of greenhouse grown 'Wheeler' rye.a Emergence Residue Lettuce Tomato Barnyard- Proso grass Millet ------------------- (%)----------------------------- Shoots 38 81 70 48 Roots/poplar excelsior 90 83 78 7O Shoots/roots 25 73 61 43 Poplar excelsior 83 80 71 78 L50 5% 24 NS NS 23 1% 32 -- -- 31 aTreatments within columns were averaged across the kill methods and subjected to analysis of variance. Each number represents the average of 12 data points. I 45 Figure 1. Effect of rye residue (1.0 g per 150 9 soil) placement relative to seed in soil on germination and root growth of four indicator species. Figure _a_. cress, b. lettuce, g. proso millet, _q. barnyardgrass. PERCENT OF CONTROL PERCENT OF CONTROL 46 O 341 CRESS so a I WRWNA'HON 32 I ROOT LENGTH 23 :‘ LETTUCE 1.505! I ”MINNDON 39 I ROOT LENGTH ‘16 ""0T iiI 16 1} §- BARNYARDGRASS L30 5: I GERHINATION "5 R I ROOT LENGTH 39 8‘ 8‘ 3‘ 8‘ ° ' o i 1 o S :6 :6 DISTANCE FROM RESIDUE (mm) D 6 IO 15 DISTANCE FROM RESIDUE (mm) 47 consistent with previous reports of rye residue toxicity (11, 12). There was a Significant interaction between the source and the quantity of rye residue present for cress, lettuce, and proso millet (Figures 2a, 2b, 2c). High rates of shoot tissue were more inhibitory to these species than high rates of root tissue. In contast, activity in this bioassay from low rates of roots or shoots was Similar. Lettuce and proso millet differed in their response to residues of different tissues. Shoots are more phytotoxic to lettuce as evidenced by equal inhibition with 1.0 9 Shoot tissue or 1.0 g of both tissues combined (roots and shoots). Imicontrast, roots plus Shoots resulted in greater inhibition of proso millet than either tissue by itself at a Similar rate. Proscinillet root lengths were 83% of control with 1.0 9 root tissue and 59% of control with 1.0 9 Shoot tissue. When they were combined, root length was only 27% of control indicating synergism. Colby's test (7) indicated an expected value of 51% for the combination, confirming a synergistic effect. With barnyardgrass (Figure 2d), in contrast to the other indicators, there was no Significant interaction between rye plant part and rate of residue (Figure 2d). The main effects of plant part and rate of residue were both highly Significant. Shoot tissue was much more inhibitory than root tissue. When root and Shoot tissue were applied together, activity was similar to that obtained with Shoots only: While these results implicate Shoot tissue as the primary source of inhibitors, it also suggests that both root and shoot tissue can act together in the field to injure selected species. Previous field studies have estimated rye surface residues at 4900 kg/ha (1, 2). This is equivalent to 4.0 9 tissue per petri dish, which 48 Figure 2. Effect of rye root and shoot residue in soil on root growth of four indicator Species. Figure _a_. cress, _b. lettuce, _c_. proso millet, g. barnyardgrass. 49 g_ CRESS A— o ROOTS 5‘ u SHOOTS o: . BOTH 1.. 5.- oh '5 L50 No- W E 2 Sn- L. o o o: o I T l f 0.0 O 6 1.0 I 6 2.0 3 ° PROSO MILLET A- in ROOTS 5‘ :- SHOOTS E ‘ BOTH ow OF '5 L50 1: NO-T W E z Els- '— o o m °o.o :0 0.5 i'.o 1.6 RESIDUE (0/150 0 SOIL) °o.o 016 11.0 1T5 20 g. a BARNYARDGRASS I ROOTS I SHOOTS . BOTH T ois Jo do RESIDUE (G/150 o SOIL) 50 resulted in 100% inhibition of germination of all Species. Subsequently, lower rates were assayed to allow for comparative evaluation of treatments. Since Zilg Shoot tissue almost totally inhibited germination of the more sensitive indicators (cress and lettuce), the rates of rye residue found under field conditions are probably sufficient to reduce germination and growth of many different plant species. Similar results occurred when the residues were evaluated for weed control (1, 2, l4, 18). It is important to determine if phytotoxicity associated with residues in soil is of plant or microbial origin. Plant residues may inherently possess phytotoxic properties or toxins may be formed during their decomposition (6,'11). In our study, there were no instances where the residues were more toxic in the presence of soil microorganisms (Figure 3). Using rye and relatively Short term bioassay durations, microbes (including possible pathogens) appear to detoxify rather than produce toxins. Even under conditions favorable for decomposition, the concentration of inhibitors from rye may be maintained at a level high enough to exert a depressing effect on plant growth. This is demonstrated in our study by the Significant phytotoxicity observed at high rates of residue on both cress and barnyardgrass. Root growth was inhibited greater than 50% when 2.0 g of shoot tissue was incorporated in both sterile and non-sterile soil. Cress and 'barnyardgrass differed in their response to residues in sterile and non-sterile soil. The magnitude of the differential response was particularly evident when 1.0 g of Shoot tissue was assayed. Sterile soil conditions resulted in 40% more inhibition of cress root length than occurred in non-sterile soil, but no Significant 51 Figure 3. Root growth of cress and barnyardgrass (as a % of control) in response to varying rates of rye shoot residue in sterile and non-sterile soil. 35%. o onKov mamfimm 3.8 o omiov ”Seam _ _ L o. C . . . . . _N o 00 o_N w.“ 0.“ m_o O 00 HO m 1% 1% I. fil 3 w I._ H 1% inn/w O 11. O . L O MJENFMIZOZ i WARNS-1202 fl MW wi—EMHM a ”...—KNEW B {a mmzmzm et.al (l98l) found both 2,4-dihydroxy-7-methoxy-l,4- benzoxazin-3-one (DIMBOA) and its glucoside extracted from maize, inhibited both cyclic and non-cyclic photophosphorylation in spinach 117 Figure 4. Response of Clamydomonas rheinhardtii Dangeard to various concentrations of DIBOA and BOA based on absorbance at 652 "Ill. 125 100 1 118 Chlomydomonos rheinhardtii C O L. ....) c: O 0 m LSD 5% 1% '45 I‘d u N v E 3‘ c: N " I!) mod (0 N I— <1: 23' ° ‘9 E DIBOA m “3 It BOA 95 i“ (1) CD < o w—l I ID ‘7 T T T l T T T -e -7 -5 -4 -3 -2 -1 MOLARITY (log 10) 119 (gpinacea oleracea L.) chlorOplasts. Inhibition of coupled electron transport was attained at concentrations of l and 4 mM, respectively. Several years ago, BASF Co. manufactured and evaluated a benzoxazinone related herbicide, Benzazin (BASF l7OOH), with photosynthetic activity. Moreland and Hill (l963) proposed that the H-N-C=0 fragment found in many polycyclic urea herbicides was related to activity. This fragment is present in BOA and reduced forms of DIBOA. Chlorosis was a symptom of injury by rye residues on several indicators (Barnes and Putnam, l983), and may result from the effects of DIBOA and BOA on photophosphorylation and electron transport. Activity of DIBOA and BOA in Soil. Uptake of allelochemicals from rye residue may be by direct contact with tissue fragments or through the soil (Barnes and Putnam, 1985). 'Therefore, the pure compounds, DIBOA and BOA were applied to soil for evaluation of activity on emergence of three indicator species. All rates of DIBOA and the higher rates of BOA completely inhibited emergence of both cress and lettuce 4 days after application (Table 4). Barnyardgrass emergence was less sensitive to the chemicals although high rates of both compounds significantly reduced emergence relative to controls. Barnyardgrass seedlings which did emerge appeared to be‘stunted and chlorotic. Two weeks after chemical application, emergence of lettuce and cress was still inhibited completely by the highest rates of DIBOA and BOA (Table 5). Although still reduced relative to control, some cress and lettuce germinated and emerged in the lower rates of DIBOA after l4 days. In general, emergence of barnyardgrass at l4 days was similar to that at four days. 120 Table 4. Emergence of barnyardgrass (BYGR), cress, and lettuce (expressed as a percent of control) 4 days after spray application of DIBOA and BOA to soil. Chemical Rate BYGR Cress Lettuce (kg/ha) ------------ % of control ------------ DIBOA 25 88 O 0 FTT‘ so 20 o o i“ 100 5 o o ‘ BOA 25 ll2 2l 72 h ‘ 50 63 O O lOO 15 O O LSD 5% 3l I9 20 Table 5. Emergence of barnyardgrass (BYGR), cress, and lettuce (expressed as a percent of control) l4 days after spray application of DIBOA and BOA to soil. Chemical Rate BYGR Cress Lettuce (kg/ha) ----------- 1 of control ------------- DIBOA 25 65 ll lO 50 l9 0 O lOO 7 0 0 BOA 25 Bl 51 72 50 61 9 l6 100 32 O O LSD 5% 29 19 24 121 The concentrations of benzoxazinones applied to soil in this study are considerably higher than the potential calculated for production in field situations if the data from fall-planted, spring-harvested rye (Table 3) is representative. In field studies where residue biomass was 4.9 MT/ha (rye killed at pre boot stage), the potential hydroxamic acid concentration is ca 0.5 kg/ha. This figure does not take into account the relative contribution from root tissue. Calculations of hydroxamic acids in residues of 35 day-old rye were based on 4.7 MT/ha [biomass of 40 day-old spring planted rye (Barnes, 198l)] and indicate a potential for ca 13.5 kg/ha from shoots and ca 0.8 kg/ha from roots. None of the calculations account for the quantity of BOA present, which also shows activity. When the pure compounds were bioassayed at rates lower than 10 kg/ha, there was no inhibition and possible stimulation by the chemicals (data not included). The stimulatory effects of hydroxamic acids at lower concentrations may result from their ability to chelate iron (waid, l975; Lewin, l984). Tipton and Buell (l970) have determined the stability constants for ferric iron complexes with two hydroxamic acids in maize [DIMBOA (log K= 21.3); DIBOA (log K= l9.4)] and found them to be much greater than that of citric acid (log K = ll.9) which is reported to function in microbial iron metabolism (Lewin, l984) and absorption and tranSport in higher plants (Sillen and Markee, 1964). Tipton and Buell concluded that at the concentrations (lO‘5 M to lO‘3 M) present in young maize plants, a high proportion of ferric iron must be bound as complexes of the hydroxamic acids. Argandona et al (1980) have similarily determined the concentration of hydroxamic acids in leaf extracts of different age 122 greenhouse grown rye. In order to transform their concentrations for 34 day-old rye to kg hydroxamic acid/ha, the following assumptions have been made: (l) lO kg fresh weight is equivalent to l kg dry weight; (2) the predominant hydroxamic acid in rye is DIBOAlwith a weight of l8l g/mole; and (3) a similarily aged Spring grown rye produces ca 447 MT dry tissue/ha in the field. According to the concentrations presented by Argandona et al (l980), 34 day-old rye contains l.9 mmole hydroxamic acids/kg fr wt or a potential of l6.2 kg hydroxamic acids/ha. This calculation is somewhat greater than the data presented, but further supports the potential for high levels in rye. Although no data is available on the concentration of benzoxazinones in field grown, spring planted, rye, it is logical to assume that the trend of decreasing hydroxamic concentrations with increasing age, determined by Argandona et al (l980), holds true. Two major differences between spring and fall planted rye are age and the exposure to cold. Both of these factors may influence the quantity and quality of chemicals produced by rye, as distinct developmental changes including emergence, shooting, heading, and flowering occur in winter rye as it matures. Temperature has the greatest influence on the duration of each stage in a given locality-~light, moisture, and nutrients being other important factors (Nuttonson, l958). As both Spring planted and greenhouse grown ryes lack the significant exposure to cold, concentrations in similarily aged greenhouse tissue may provide a rough approximation of the field tissue. The potential levels of 13.5 and 16.2 kg hydroxamic acids/ha calculated from concentrations determined by the authors and Argandona et al (l 980), respectively, could be considered at least ten times greater than most 123 rates of commercial herbicides used today. They are, however, about lO-lOO times less active than most synthetic herbicides. Hhile our major field observations for proof of alle10pathy have been based on phytotoxicity by residues of fall-planted, spring-killed rye, there is evidence for toxicity by Spring planted rye (Barnes, 198l). Residues of 40 day-old Spring-planted rye reduced total weed density by 69% and total weed biomass by 32% when compared to a PE control mulch. As age may be an important variable and could be managed by timing the kill, the potential exists for maximizing benzoxazinone concentrations in the field. Additional studies which quantify and compare concentrations in both fall and Spring planted rye killed at different ages would provide data useful in this regard. Ideally, the actual release of benzoxazinones into soil, and uptake by plants, perhaps using radiolabeled material, should also be documented for conclusive proof of their role in allelopathy. while phytotoxicity by residues in non-sterile soil was Significant after incubation for 72 h (Barnes and Putnam, l985), benzoxazinones may be easily degraded by microorganisms. Undoubtedly, several compounds, in addition to the solvent extractable benzoxazinones, contribute toward toxicity by rye residues. This is evidenced by the fact that almost 50% of the initial aqueous extract activity was associated with the protein precipitate and final aqueous fractions (Chapter 3). The identification of benzoxazinones and their subsequent implication in allelopathy by rye, has been based on steps directed toward isolation of the most active chemicals. Although the ether extract accounted for only l2% of the potential toxicity, it was ‘ll to 25 times more active than the protein and final aqueous fractions. 124 Additional toxicity by residues may also result from phytoactive degradation products. Chou and Patrick (1976) found significant toxicity with residues of rye which had decomposed for up to 30 days. BOA is the major chemical breakdown product of DIBOA and in our studies shows greater activity against germination of all dicot Species tested. Although the FeCl3 method precludes quantitation of BOA and subsequent determination of its significance in rye residue toxicity, levels should presumably increase as DIBOA is broken down. Monitoring the breakdown and resultant toxicity in the soil over time should provide evidence for the persistance of the chemicals. In conclusion, the importance of allelochemicals produced in response to injury or infection is indicated by the benzoxazolinone series of compounds which occur naturally in many grain plants (Beck and Reese, 1976). ‘Their role and significance in resistance to insects and disease organisms have been investigated more thoroughly than their role in al lelopathy. With regard to plant activity, the greenhouse bioassays provided evidence fbr toxicity on whole plants in simulated no-till systems (Barnes, 198l; Barnes and Putnam, 1983). The Similar injury symptoms noted where residues andpure compounds were applied support the premise of allelopathic interference by rye with benzoxazinones playing a prominant role. "“T 1‘ LITERATURE CITED Argandona, V. H., J. Luza, H. M. Niemeyer, and L. J. Corcuera. l980. Role of hydroxamic acids in the resistance of cereals to aphids. Phytochem. 19:1665-l668. Argandona, V. H., H. M. Niemeyer, and L. J. Corcuera. l98l. Effect of content and distribution of hydroxamic acids in wheat on infestation by the aphid Schizaphis Lraminum. Phytochem. 20:673- 676. Barnes, J. P. l98l. Exploitation of rye (§_e_ga_l_e_ cereale L.) and its residues for weed suppression in vegetable crOpping systems. M.S. Thesis, Michigan State University. l28 pp. Barnes, J. P. and A. R. Putnam. l983. Rye residues contribute weed suppression in no-till crOpping systems. J. Chem. Ecol. 9:1045-57. ’ Barnes, J. P. and A. R. Putnam. l985. Evidence for al lelopathy by residues and aqueous extracts of rye (§e_c_a_l__e_ cereale). Heed Science (Submitted). Beck, 5. D. and J. C. Reese. l976. Insect - Plant Interactions: Nutrition and metabolism. p. 4l-92 in recent advances in phytochemistry. Vol. 10 in Biochemical Interactions between plants and insects. Ed. J. H. Hal lace and R. L. Mansel l. Plenum Press, N.Y. 125 10. 11. 12. 13. 14. 15. 16. 126 Benoit, R. E., N. A. Hillits, and H. J. Hanna. 1962. Effect of rye winter cover crop on soil structure. Agron. J. 54:419. Blevins, R. L., D. Cook, S. H. Phillips, and R. E. Phillips. 1971. Influence of no-tillage on soil moisture. Agron. J. 63:593-596. Chou, C-H and Z. A. Patrick. 1976. Identification and phytotoxic activity of compounds produced during decomposition of corn and rye residues in soil. J. Chem. Ecol. 2:369-387. Corcuera, L. J., M. O. Hoodward, J. P. Helgeson, A. Kelman, and C. D. Upper. 1978. 2,4-dihydroxy-7-methoxy-2flyl,4-benzoxazin-3(4fl)- one, an inhibitor from 133 may; with differential activity against soft rotting Erwinia Species. Plant Physio. 61:79l-795. Cubbon, M. H. 1925. Effect of a rye crop on the growth of grapes. J. of Amer. Soc. of Agron. 17:568-577. Fuerst, E. P. and A. R. Putnam. 1983. Separating the competitive and allelopathic components of interference: Theoretical principles. J. Chem. Ecol. 9:937-944. Hamilton, R. H. 1964. Tolerance of several grass Species to 2- chloro-s-triazine herbicides in relation to degradation and content of benzoxazinone derivatives. J. Ag. and Food Chem. 12:14-17. Harborne, J. B. 1977. Introduction 39 Ecological Biochemistcy. Academic Press. London. 243 pp. Hess, F. D. 1980. A Chlamydomonas algal bioassay for detecting growth inhibition herbicides. Heed Science. 28:515-520. Hill, H. H. 1926. Decomposition of organic matter in soil. Jour. Agr. Res. 33:77-99. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 127 Hofman, J. and 0. Hofmanova. 1969. l,4-benzoxazine derivatives in plants sephadex fractionation and identification of a new glycoside. J. Biochem. 8:109-112. Kimber, R. H. 1973. II. The effect of rotting of straw from some grasses and legumes on the growth of wheat seedlings. Plant and Soil. 38:347-361. Lewin, R. 1984. How microorganisms transport iron. Science. 225:401-402. Long, B..Ln G. M. Dunn, and D. G. Routley. 1974. Rapid procedure for estimating cyclic hydroxamate (DIMBOA) conc. in maize. Crap Science. 14:601-603. Lovett,, J. V. 1982. Al lelopathy and sel f-defence in plants. Australian Heeds. 2:33-36. Moreland, D. E. and K. L. Hill. 1963. Inhibition of photochemical activity of isolated chloroplasts by polycyclic ureas. Heeds. 11:284-287. Muller, C. H. 1966. The role of chemical inhibition (Allelopathy)in vegetational composition. Bull. Torrey; Bot. Club. 93:332-351. Nuttonson, M. Y. 1958. Rye-climate relationships on the use of phenology in ascertaining the thermal and photo-thermal requirements of rye. American Inst. of Crap Ecology. 219 pp. Osvald, H. On antagonism between plants. Proceedings of the 7th Intern. Bot. Congress. Stockholm 1950 Osvald & Aberg, Eds. Almquist and Hiksell - Stockholm 1953.;L 167-171. Overland, L. 1966. The role of allelopathic substances in the “smother crop” barley. Amer. J. Bot. 53:423-432. 27. 28. 29. 30. 31. 32. 33. 34. 35. 128 Patrick, 2. A. and L. H. Koch 1958. Inhibition of respiration, germination, and growth of substances arising during the decomposition of certain plant residues in the soil. Can. J. Bot. 36:621-647. Patrick. 1971. Phytotoxic substances associated with the decomposition in soil of plant residues. Soil Science. '11:13-18. Putnam, A. R. and J. DeFrank. 1982. Use of phytotoxic plant residues for selective weed control. Crop Protection. 2:173-181. Putnam, A. R., J. DeFrank, and J. P. Barnes. 1983. Exploitation of allelopathy for weed control in annual and perennial crOpping systems. J. of Chem. Ecol. 9:1001-1009. Queirolo, C. B., C. S. Andreo, R. H. Val 1ejos, H. M. Niemeyer, and L. J. Corcuera. 1981. Effects of hydroxamic acids isolated from Gramineae on adenosine 5'-triphophate synthesis in chloroplasts. Plant Physiol. 68:941-943. Rice. 1974. Allelopathy. Academic Press. N.Y. 353 pp. Shilling, O. G., R. A. Liebl, and A. O. Horsham. 1985. Rye (Secale cereale L.) and wheat (Triticum aestivum L.) mulch: The suppression of certain broadleaf weeds and the isolation and identification of phytotoxins. In. N.C. Thompson (ed.) The Chemistry o_f Allelopathy. pp. 243-271. Sil len, L. G. and A. E. Martel 1. 1964. Stability Constants o_f Metal Iron Complexes, 2nd ed.,'The Chemical Society, London. Sullivan, S. L., V. E. Gracen, and A. Ortega. 1974. Resistance of exotic maize varieties to the European corn borer Ostrinia nubilalis (Hubner). Environ. Entomol. 3:718-720. 36. 37. 38. 39. 40. 41. 129 Swain, T. 1977. Secondary compounds as protective agents. Annu. Rev. Plant Physiol. 28:479-501. Tipton, C. L. and E. L. Buell. 1970. Ferric iron complexes of hydroxamic acids from maize. Phytochem. 9:1215-1217. Haid, J. S. 1975. Hydroxamic acids in soil systems. 1 Soil Biochemistry, ed. Paul and McLaren, NY, NY. p. 65-101. Hhittaker, R. H. and P. P. Feeny. 1971. Allelochemics: Chemical interaction between species. Science. 171:757-770. Hoodword, M. 0., L. J. Corcuera, H. K. Schnoes, J. P. Helgeson, and C. D. Upper. 1979. Identification of l,4-benzoxazin-3-ones in maize extracts by GLC and M.S. Plant Physiol. 63:9-13. Zuniga, G. E., V. H. Argandona, H. M. Niemeyer, and L. D. Corcuera. 1983. Hydroxamic acid content in wild and cultivated gramineae. Phytochem. 22:2665- 2668. HI CHIGQN STRTE UNIV LI BRRRI ES ll1lllll|9ll Ill lllzlll7l lo I