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J » It ”I'- ._ < - . 4.‘ "r,‘ A 7 ..‘ -‘( (421:"{p1;7'7 '3’ - I ,. 77.75! '1’“: 57:4. . {55“} .7 ’23:. h, I' H ‘ J25.’V'p}¢;g #1:, f' 7” cr ’ ' J' r j; ":2” 7 {6:45 ,’y’ 'n 77f:,44;': 50"! ‘7 ffl‘u'l. v” "-71 "g,',‘... ‘ .. :"370'7'0. 7. an 104 , |r97u #9762392" . “777% a 1'6)?" fir ”@777, ...., ae- Jfifg (trig. g3; IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 293 00563 3197 3l.1lBRARY Michigan State University This is to certify that the thesis entitled MICROBIAL TRANSFORMATION OF RYE (Secale cereale L.) RESIDUES: A SOURCE OF POTENT ALLELOCHEMICALS presented by Curt James Whitenack has been accepted towards fulfillment of the requirements for Master of Sc1ence degree in Department of Hort1cu1ture ! 1 I Major professor Date 5/25/88 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES -_ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. IECIOBIAL IBAISFORHIIIOI OP’RIB (Secale cereaLe L.) RESIDUES: A SOURCE OF POIEITIALLBDOCEEHICALS BY Curt James Whitenack A.!EESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of HISIER.OP SCIENCE Department of Horticulture 1988 SROObfix ans-mm mum man 0? m (Secale cereale L.) mrnuns: A scum: a your means By Curt James Whitenack Stable diazoperoxides were isolated from soil and characterized after L11 vitro microbial degradation of the benzoxazinones, the allelochemicals previously reported from rye. 2,3-benzoxazolinone (BOA) was shown to be transformed in the soil to 2,2'-oxo-l,l'-azobenzene (Compound 4) , while 6-methoxy-2,3-benzoxazolinone (MBOA) resulted in production of mono- and dimethoxy analogs of the above compound. Leaching experiments with rye residue E gi_tr_g showed the presence of both BOA and 2,4-dihydroxy-l,4(211)-benzoxazin-3-one (DIBOA) in the soil, but failed to detect any of the above compounds. Bioassays of the compounds showed strong herbicidal activity, 8-10 times more toxic than the previously reported allelochemicals, the benzoxazinones. These results may explain an additional source of toxicity from rye residues that has not been accounted for by the benzoxazinones. Nematicidal assays indicated that the LCSO of Compound 4 was between 1 and 10 ppm. 100$ mortality was observed after four hours exposure to 10 ppm. Dedication This work is dedicated to my Aunt Caroline, and my mother, Lois, who made it possible in so many ways, and also to Brenda, because without her guidance, I would probably still be wondering if I could get into graduate school. ACINOILBDGEHENIS After nearly four years in Michigan, it occurs to me that I have recieved a great deal of help, from a lot of people, and while it is not possible to recognize all of them, I would like to mention the major players. I would like to thank the members of my advisory committee, Dr. Stanley Ries, Dr. James Miller, and especially my major professor, Dr. Alan Putnam, for their advice and guidance, and my "unofficial” committee member, Dr. Muralee Nair, for his extroardinary patience and teaching ability. Without their input, there would be no thesis. I would also like to thank Jackie Schartzer, for her assistance in the preparation of this manuscript. And finally, I would like to thank Brenda for her encouragement and belief, and my friends here in Michigan, who kept me sane and were always willing to help blow off steam. There's only one thing to say to you guys: “What a long, strange trip it's been.“ - The Grateful Dead iv PIGS LIST OF FIGURES.............. OOOOOOOOOOOOOOOOOO OOOOOOOCCOCOOOOOOCIO v11 CHAPTER 1: LITERATURE REVIEW INTRODUCTION...................................................... Use of Natural Products for Pest Control......................... Microbial Pesticides............................................. Microbial herbicides............................................. Allelopathy...................................................... Benzoxazinones................................................... Chemistry of Benzoxazinones...................................... Biological Activity of Benzoxazinones............................ \DQQ-wal-‘H LITERATURE CITED.’IOOOOOOOOOOOOOOOOOOOOOO0.00.00.00.000.0.0.0000... 19 CHAPTER 2: ISOLATION AND CHARACTERIZATION OF PRODUCTS ARISING FROM MICROBIAL OXIDATION OF BENZOXAZINONES PRODUCED BY CEREAL CROPS mmTOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. ..... OOOOOOOOOOOOOOOOOOOO 23 mDUCTIONOOOOOOIOOOOOOOOO0.0.0.0000...0.0.0.0....OCOOOOOOOOOOOOC 24 MATERIALS AND METHODS.............................................. 25 General experimental.............................................. 25 Spray Reagents for Thin-layer Chromatography...................... 26 Isolation of 6-methoxy-2,3-benzoxazolinone (MBOA), Compound 2..... 26 Isolation of 2,4-dihydroxy-l,4(ZH)-benzoxazin-3—one (DIBOA) compound 3........................................................ 27 Isolation of 2,2'-oxo-l,l'-azobenzene, Compound 4................. 28 Isolation of 4-methoxy-2,2'-oxo-1,l'-azobenzene, Compound 5, and 4,4'-dimethoxy-2,2'-oxo-l,l'azobenzene, Compound 6............ 30 Leaching Experiments with Rye Residue............................. 31 RESULTS AND DISCUSSION............................................. 32 Structural Identification of Compound 4........................... 32 Structural Isolation of Compounds 5 and 6......................... 37 Proposed mechanism for Production of Compounds 4, 5, and 6........ 37 Inhibition of Bio-transformation Reaction in the Presence of Rye Residues................................................... 42 LITERATURE CITEDooooooooooooooooooooooo0.0.00.0.0.000.000.000000000044 TIBLE OE’CUIIEITB continued: PAGE CHAPTER 3: BIOLOGICAL ACTIVITY OF 2,2'-OXO-1,l'-AZOBENZENE: AN ALLELOCHEMICAL ARISING THROUGH MICROBIAL OXIDATION OF 2,3- MZOXAZOLINONE (BOA)O...0.0...COOOOOOOOOCOOOOOOOOOOOOOOOCCCO...... 45 ABSMToooooooooooooooooooa0000000000000...aaooaaoaoooooooooooooo 45 mDUCTIONOOOOO0......O0.0...0...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 46 MATERIALS AND METHODS............................................. 47 Herbicidal Bioassay Format....................................... 47 Seed Scarification Treatments.................................... 48 Antifungal bioassay Format....................................... 48 Insecticidal Bioassay Format..................................... 49 Nematicidal Bioassay Format...................................... 50 RESULTS AND DISCUSSION............................................ Sl Herbicidal Activity of Compound 4................................ 51 Comparison of Herbicidal Activity of Compound 4 and Parent Benzoxazinones................................................... 61 Results of Anti-Fungal Assays.................................... 72 Results of Insecticidal Assays................................... 73 Results of Nematicidal Assays.................................... 73 Conclusions...................................................... 74 LITERATURE CITEOOOOIOOOOOO00......OOOOOOOOOOOOOOOOOO00.000.00.00. 76 vi FIGURE LIST OF FIGURES PAGE CHAPTER 1 Chemical breakdown scheme for benzoxazinone compounds produced by cereal crops (Virtanen and Hietala, 1960).... CHAPTER 2: Structures of parent benzoxazinones, Compounds 4, 5, and 6....0.0.0.0...C...OOOOOOOOOOOIOOOOOOOOOO00.00.00.000 Proposed structure of Compounds 4, 5, and 6 arising from microbial oxidation of 2,3-benzoxazolinone in the soil"...0.00.00...OCIOOOOOOOOOOOOOOOO00.000.00.00...O... Proposed structure of intermediates I and II, in the formation of Compounds 4, 5, and 6....................... Benzoxazinone breakdown scheme of Virtanen amended to include microbial metabolites of benzoxazinones.......... CHAPTER 3 Response of garden cress seedling root elongation to increasing concentrations of Compound 4................... Response of barnyardgrass seedling root elongation to increasing concentrations of Compound 4................... Promotion of rye root elongation by Compound 4............ Response of proso millet seedlings root elongation to increasing doses of Compound 4............................ Inhibition of tomato seedling root growth by Compound 4..OOOOOOOOOOOOOOOOOOOOOOOO00.00.000.000...OOOOOOOOOOOOOOO Inhibition of redroot pigweed seedling root growth by increasing concentrations of Compound 4................... Inhibition of velvetleaf seedling root elongation by increaSj-ng doses Of commund 4.0.0.0000000000000.0.0.0.... Relative toxicities of Compound 4 and the parent compounds, BOA and DIBOA against garden cress............. vii 34 36 39 41 53 55 58 60 63 65 67 69 LISI'OP FIGURES continued: 9 Relative toxicities of Compound 4 and the parent compounds, BOA and DIBOA against barnyardgrass............ 71 viii In recent years, increased public concern over the environmental impact of pesticide use has caused a re-evaluation of the place of synthetic chemicals in agricultural pest control. Even chemicals such as atrazine and alachlor, once regarded as relatively safe, are being scrutinized because of their presence in ground water. Others, such as aldrin, dieldrin, and DDT, are still detectable in Great Lakes fish, even though the use of these chemicals was banned several years ago. It seems obvious that the public will not allow continued use of synthetic crop protection chemicals at current levels, thus suitable alternatives should be developed. An attractive alternative to synthetic chemicals is the use of natural products from higher plants or microorganisms. Duke (1986) lists several advantages to the development of natural products as crop protection chemicals. First, because many biologically active oanpounds are produced by pathogens, these compounds may have a built- in specificity, allowing the development of highly selective pesticides. Natural products also may provide a basis for new chemical groups of pesticides. Specifically, the synthetic chemist can use the molecular structure of the natural product as a “template" to develop analogs that affect a specific site of action. This may be especially 1 . a...‘ ~A" ‘_ I 2 helpful in controlling resistant weeds with altered target sites. Finally, since chemicals of plant and microbial origin consist primarily of carbon, hydrogen, oxygen, and nitrogen, and have been present in the environment for many years, it is likely that microorganisms have evolved that can easily degrade them. In contrast, many synthetic compounds, such as the halogenated hydrocarbons, have extremely long half-lives in the soil. Natural products also have disadvantages. They may be extremely toxic to mamals. For example, Botulinum toxin, a natural product of Clostridium botulinum, is among the most toxic chemicals known. Scale- up from laboratory to pilot plant to full production may be prohibitively expensive and time consuming. There are, however, numerous examples where natural products have been used successfully for agricultural pest control. A classic example of the use of a natural compound to control a pest is the use of pyrethroid insecticides. Since ancient times, the powdered flowers of Chrysanthemum cinerariaefolium Vis. (gyrethrum cinerariaefolium Trev.) have been used as an insecticide (Matsui and Yamamoto, 1971). The insecticidal components are called pyrethrins, and while they were once used extensively in the field, their use is now limited mainly to control of domestic insects. Originally, the crushed flowers were extracted with an organic solvent to partially purify the pyrethrins, but when this became too costly, the toxins were characterized and produced synthetically. licrobial Pesticides. Microbially produced antibiotics have been used in human medicine for many years, but several also have applications in the management of plant pathogens. -Some are registered for 3 agricultural use in Japan, the most widely used being streptomycin, for the control of various bacterial diseases of fruit trees and vegetables. Blasticidin S and kasugamycin, registered in l962 and 1965, respectively, are used to control rice blast, a fungal infection of rice (0112a sativa) (Misato, 1982). Microbial phytotoxins are an attractive potential source of new herbicides and new herbicide chemistry. Cycloheximide, a glutarimide antibiotic produced by several Streptomyces species, is reported to control barnyardgrass (Echinochloa crusgalli L.) and pond weed (Potamegeton sp.) in paddy-grown rice without adversely affecting the rice seedlings (Sekizawa and Takematsu, 1982). Anisomycin, a metabolite of a Streptomyces sp., is toxic to barnyardgrass and crabgrass species (Digitaria sp.), but has no effect on tomato (mopersicon sculentum Mill.) seedlings (Yamada, et. al., 1974). Using the structure of anisomycin as a starting point, Japanese chemists developed the synthetic herbicide methoxyphenone, now used comercially in Japan to control barnyardgrass in rice. Bialaphos (2-amino-4-methylphophinoyl-butylyl)-alanylalanine), a new microbial herbicide produced by Streptomyces viridochromogenes is related to the commercial herbicide glyphosate. While it is slower acting than paraquat, it is substantially faster than glyphosate (Sekizawa and Takematsu, 1982) . Beisey and Putnam (1987) examined the herbicidal properties of geldanamycin and nigericin, two antibiotics produced by Streptomyces hygrosccmicus. Geldanamycin is a member of the ansamycin group of antibiotics and has strong phytotoxic activity in vitro. In petri dish germination bioassays, the authors reported inhibition of cress (Epidium sativum L.) radicle elongation to 50% of control at 1-2 ppm. 4 Nigericin, a polyether antibiotic, showed equal activity. Mishra, et. a1. (1988) screened 906 microbial isolates for phytotoxic properties. These included 796 actinomycete isolates, 70 fungi, and 40 non-actinomycete bacteria. Of all the isolates tested, 72, or about 8% caused significant inhibition of radicle elongation in cress germination bioassays. The most frequently active genera were Streptomyces and the novel actinomycete Actinoplanes. Of these genera, 18% and 13%, respectively, were active. Specific phytotoxins isolated in this study were reported by Heisey, et. a1. (1988). This work indicates that microbial phytotoxins are fairly widespread in nature, and that continued screening in this area could lead to the development of new herbicides . WP” Metabolites of higher plants also exhibit phytotoxic properties, as evidenced by the numerous reports of the adverse effects of both living plants, and decaying plant residues on crops. This influence may be the result of phytotoxins leaching directly from the residues or production of phytotoxins by microorganisms associated with the residues. Allelochemicals are compounds that are produced by higher plants or microorganisms and that affect the growth of other plants. The overall mediation of one plant's growth by another through chemical means is known as allelopathy (Whittaker and Peeny, 1971; Rice, 1974) Cubbon (1925) reported that a rye (Secale cereale L.) crop, grown simultaneously with a grape crop, reduced the grape's growth relative to a control. Nutrient and water supply were carefully monitored to ensure that these factors did not become limiting. The high nutrient 5 level of the soil and abundant water supply suggested that competition was not the cause of the reduced grape growth. Rather, a chemical agent produced by the rye caused this effect (Cubbon, 1925). Phytotoxins released by the microbial breakdown of plant residues can be leached into the soil and influence the germination and growth of susceptible plants. For many years, the problem of establishing peach trees on the site of an old peach orchard was attributed to pathogens, nutrient deficiencies, or spray residues, none of which seemed to completely explain the cause. Proebsting and Gilmore (1939) investigated the hypothesis that after tree removal, either the roots left in the soil, or their breakdown products were toxic to the new trees. They found that when they combined peach roots with soil and planted peach seedlings in this soil, there was a severe inhibition of seedling root growth. Eventually, they refined this to show that the majority of the toxicity was contained in the root bark. They noted that when peach bark is heated with water, it gives off the odor of benzaldehyde. The authors took this to indicate the presence of amygdalin, a cyanogenic B-glycoside. To test the possibility that amygdalin was the toxic agent, amygdalin was added to soil in which a peach seedling was grown. This treatment caused no injury, indicating that amygdalin itself is not toxic. If a trace of emulsin, an enzyme that catalyzes the hydrolysis of amygdalin to glucose, benzaldehyde, and hydrogen cyanide was added along with amygdalin, severe injury resulted. Patrick (1955) examined the involvement of microorganisms in the production of toxicity from peach root bark and found that many microorganisms capable of hydrolyzing amygdalin could easily be isolated from the soil. Based on this, he proposed that the toxic effects of peach root bark in the soil were caused by hydrogen cyanide 6 and benzaldehyde produced by microbial hydrolysis of amygdalin. Patrick and Koch (1958) examined the effects of aqueous extracts of decaying residues of timothy (Phleum puatense L.), rye, corn (Egg ggyg_L.), and tobacco (Nicotiana tabacum L.) on the respiration, oxygen uptake, and growth of tobacco seedlings. They found that as the residues decomposed, the aqueous extracts became more acidic, and that there was a high correlation between extract acidity and toxicity. The most highly toxic extracts were always acidic. When the pH of toxic extracts was adjusted to a more neutral range, toxicity decreased only slightly, suggesting that the inhibition of tobacco seedling respiration was caused by toxic substances, rather than the acidity of the extract alone. Additionally, they compared the toxicity of extracts of decomposed residues to extracts made by mixing macerated tissue with soil and extracting before decompostion could occur. In all cases, they found no toxicity unless microbial degradation of the residues had occurred. They concluded that the (plants themselves contained no compounds inhibitory to the respiration of tobacco seedlings, but all toxic compounds were formed during the microbial degradation of the residues. Phytotoxic chemicals produced by higher plants that are contained in the intact plants may be released upon disruption of the tissue during the decomposition of the residues. Chou and Patrick (1976) studied the phytotoxic compounds released during the degradation of corn and rye residues. Plant material was combined with soil and allowed to decompose for up to 30 days. In the aqueous extracts of dBCONPOBing corn residue, they detected eighteen phytotoxic compounds: mostly low molecular weight organic acids such as benzoic, phenylacetic,and 4-pheny1butyric acids, along with cinnamic acid 7 derivatives such as ferulic, caffeic, o—coumaric, p-coumaric, trans- cinnamic, and vanillic acid. The cinnamic acid derivatives are frequently implicated in allelopathy by many species, and are one of the fundamental groups of allelochemicals (Rice, 1984). Weston and Putnam (1986) showed that aqueous extracts of quackgrass (mmron repens L. Beauv.) severely inhibited radicle elongation in alfalfa (Medicago sativa L.), cress (Lepidium sativum L.), soybeans (Glycine max (L.) Merr.), and navy beans (Phaseolus vulgaris L.). These extracts did not affect the growth of Rhizobia comonly associated with legumes, indicating that inhibition of legume growth by quackgrass was not the result of inhibition of the growth of the rhizobial symbiont. Subsequently, a flavone (tricin) was isolated that was particularly inhibitory to root growth, specifically the formation of root hairs (Weston, et al, 1987). Although this compound also had no affect on rhizobia, the point of infection in the legume- rhizobium symbiosis is the root hair. Therefore, inhibition of root hair formation results in inhibition of the symbiosis. Barnes and Putnam (1983, 1986) examined the allelopathic potential of residues and aqueous extracts of rye. Comparisons between a bare ground control and a living cover of spring planted rye showed a 94% reduction in total weed biomass under the rye. When residues were planted in the spring and killed after 40 days, there was a 69% reduction in total weed biomass, relative to a bare ground control. To separate the physical and chemical components, comparisons were also made to a control consisting of a poplar (Populus tremuloides L.) excelsior cover. The poplar excelsior simulates the physical effects produced by the mulch, such as shading, moisture retention, etc. In this case, the killed residue reduced weed biomass 32% relative to the 8 poplar excelsior control, indicating that there was a chemical component to the weed supression. Later work (Barnes et al., 1987) implicated the benzoxazinones, cyclic hydroxamic acids produced by cereal crops, as the toxic agents in allelopathy by rye. Bum: 130.114 Chemistry. In 1955, Virtanen, et. a1. isolated a cyclic hydroxamic acid, or benzoxazinone, from rye seedlings. Chemical analysis identified the compound as 2,3-benzoxazolinone (BOA). The same authors later isolated 6-methoxy-2,3-benzoxazolinone (MBOA) from wheat and corn (Virtanen et al., 1957) simultaneously with Loomis (1957). These compounds were isolated and purified based on their biological activity against fungi (Fusarium nivale) and European corn borer (Ostrinia nubilalis). When the structures were confirmed, the authors first believed they had isolated and identified the active principle. Virtanen, however, noted that while the benzoxazolinone could be extracted from crushed tissue with boiling water, direct extraction of the crushed tissue with diethyl ether failed to extract any benzoxazolinone. This suggested the presence of a precursor in the tissue. To isolate this precursor, intact tissue was boiled to inactivate all enzymes prior to extraction. Using this method, they isolated a glucoside precursor, 2-O-glucosy1-l,4(ZH)-benzoxazin-3-one (GDIBOA) from rye, and a methoxylated analog, 2-O-glucosyl-7-methoxy- 1,4-benzoxazin-3-one (GDIMBOA) from corn and wheat. Treatment of the glucoside with a crude enzyme preparation from rye caused hydrolysis to glucose and a second precursor, 2,4-dihydroxy-l,4(28)-benzoxazin-3-one (DIBOA) in rye and 7-methoxy-2,4-dihydroxy-l,4(ZB)-benzoxazin-3-one 9 (DIMBOA) in corn and wheat (Virtanen and Hietala, 1960) (Hietala and Virtanen, 1960). Hofman and Hofmanova (1969) showed that when intact tissue was carefully extracted, using liquid nitrogen as a fixative to prevent all enzyme activity, only the glucoside derivatives were extracted. This indicated that there was no free aglycone or benzoxazolinone in living, intact tissue. Upon injury to the plant, B- glucosidases are released that rapidly hydrolyze the glucoside to the aglycone, which in turn decomposes in water to form the benzoxazolinone (Figure l). Naturally occurring benzoxazinones have also been reported from two additional plants. Job's tears (Coix lachryma jobi) contains methoxylated derivatives (Koyama, 1955) and bears breech (Acanthis mollis L.) contains GDIBOA in the seeds (WOlf et al. 1985). These are the only known instances of 1,4-benzoxazinone production in higher plants, although production by microorganisms is fairly common (Tipton, et. al., 1967). Biological Activity of Bensosazinnnes Activity against insects. Loomis (1957) investigated the resistance of corn to the European corn borer (Ostrinia nubilalis). At that time, it was established that different cultivars of corn had varying degrees of susceptibility to corn borer attack. Despite years of research aimed at developing a highly resistant cultivar, no one was able to clearly define the mechanism of resistance. Loomis succeeded in isolating and purifying a compound he termed “resistance factor A“ (RFA). Incorporation of RFA in an artificial diet completely inhibited the growth of the corn borer larvae. Chemical analysis indicated that RFA was, in fact, MBOA. Virtanen felt that, because the derivative present 10 Figure 1: Chemical breakdown scheme proposed by Virtanen and Rietala (1960) for benzoxazinone compounds produced by cereal crops. 11 (3 II N o C) o"""--gll.i 'Enzymatic HYdrOlysis TH III rq::][iTC) R O OH Chemical Rear"armament 40:“; Figure 1 p n. ‘-r‘f§)?l: 12 in intact tissue is the glucoside (GDIMBOA), it would be highly unlikely that the borer would be exposed to the benzoxazolinone. Based on the rapidity of the enzymatic hydrolysis of the glucoside upon injury to the tissue, it is more likely that the feeding insect would be exposed to the aglucone (DIBOA) . To differentiate between the possible derivatives, Klun and Brindley (1966) attempted to correlate the BOA content of 11 inbred lines of corn with the observed resistance of those lines in the field to corn borer attack. They found that a linear relationship existed between BOA recovered from 100 g of tissue and resistance to attack. The most highly resistant lines had approximately ten times as much BOA in the tissue as the most highly susceptible lines. However, because MBOA is directly derived from DIBOA, and ultimately from GDIBOA, the amount of BOA present is actually a measure of the DIBOA or GDIBOA initially present. Therefore, a strong correlation between BOA concentrations and resistance can only be interpreted as a correlation between generic benzoxazinone concentrations and resistance. To attribute a greater level of importance to a single derivative, the differential toxicities must be evaluated. When the authors bioassayed BOA against corn borer larvae, they found that it significantly decreased pupation of the larvae. They were unable to show any toxicity or increased mortality caused by BOA, reinforcing the idea that the active compound was actually DIBOA. Klun, Tipton, and Brindley (1967) investigated this possibility by examining the activity of purified DIBOA in an artificial diet. They found an inhibition of pupation similar to that caused by BOA. They also found that DIBOA caused a 25% mortality rate . Long and co-workers (1977) examined the role of DIBOA in the 13 resistance of corn to the corn leaf aphid (Rhopalosiphum maidis Fitch.). Incorporation of DIMBOA into the diet at a concentration of 0.5 mg/ 1.0 g of diet resulted in 20.8% mortality. These authors also showed an inverse linear relationship between the benzoxazinone concentration and the resistance to aphid infestation in the field. Subsequent analysis of the chemical basis for this resistance by Beck (1983) strengthens the idea that the dominant chemical factor in determining resistance is the benzoxazinone content. of the plant. These authors examined the relationship between resistance and the concentrations of hydroxamic acids, total phenols, and orthodihydroxyphenol concentrations in corn tassels. They were unable to establish any significant correlation to resistance other than that involving hydroxamic acids. Argandona (1980) compared the resistance of wheat, rye, and barley to infestation by the cereal aphid (Metopolophium dirhodum). Six days after applying aphids to plants, researchers recorded the extent of infestation and extracted and quantified the benzoxazinones. Barley, the most severely infested species, contained no detectable benzoxazinones. The three lines of wheat included in the study had intermediate levels of infestation. Rye exhibited the highest degree of resistance to infestation and the highest concentration of benzoxazinones. To confirm the role of DIBOA in resistance to this aphid, the authors immersed freshly cut barley leaves in a solution of DIBOA and allowed the leaves to take up the solution. The leaves were exposed to aphids. After five days, the aphids were counted to determine the extent of infestation, and the DIBOA was quantified. Again, they found a very high coefficient of determination (0.94) between the quantity of DIMBOA present and the degree of aphid 14 infestation. Additionally, as the plants aged, the concentration of benzoxazinones decreased, as did their resistance to infestation. When aphids were fed an artificial diet containing DIBOA or MBOA, significant mortality resulted, but the concentration of BOA required to kill 50% of the population (LD50) was 15 times the concentration of DIMBOA required for the same effect. Concentrations of 500 ppm deter the feeding activity of both European corn borer and African army worm (godoptera exempta) in leaf disk feeding bioassays (Rubo and Kamikawa, 1983) . Anti-microbial activity. Virtanen (1957) first isolated 2,3- benzoxazolinone from rye seedlings as an anti-Fusarium factor. His observations of the difference in resistance of rye varieties to infection by Fusarium nivale led to an investigation of the possibility that rye contained a chemical factor that conferred resistance to infection. Extraction and purification of the active substance yielded 2,3-benzoxazolinone. Bioassays of BOA against Fusarium nivale indicated that BOA was especially active in vitro, causing complete inhibition of fungal growth at concentrations above 500 ppm. Further bioassays showed BOA to inhibit Penicillium roquefortii and Sclerotinia trifoliorum. El Naghy and Linko (1962) investigated the role of benzoxazinones in the resistance of wheat to stem rust and demonstrated an inverse linear relationship between the concentration of GDIBOA and extent of injury. The most strongly resistant cultivars had high concentrations of GDIBOA, while the least resistant cultivars had barely detectable levels of GDIBOA. Hypersensitivity is a cannon characteristic of resistant cultivars. Upon infection, host cells in the immediate area 15 of infection die rapidly, resulting in small areas of necrotic tissue. The rust pathogen (Puccinia graminis), being an obligate intracellular parasite, is unable to spread beyond the dead cells. Both the aglycone and the benzoxazolinone caused a high degree of phytotoxicity at the levels at which the purified compounds were inhibitory to the rust. Based on this, the authors proposed that the mechanism of resistance depends on this phytotoxicity. Upon infection by the pathogen, tissue damage results in the release of B—glucosidases that rapidly cleave the glycoside, releasing the phytotoxic aglycone. The aglycone causes the death of host cells at the infection loci, preventing the spread of the parasite. The aglycone also inhibits germination of the uredospores. Later work (Knott and Rumar, 1972) cast serious doubt on the dominance of GDIBOA in resistance of wheat to stem rust. While these authors demonstrated a strong relationship between GDIBOA concentration and resistance for extremely sensitive and extremely resistant varieties, the relationship did not hold at all for the varieties with intermediate resistance. Therefore, while it appears that the benzoxazinones are involved in resistance to stem rust, there are clearly other, possibly more important, mechanisms involved. A stronger case exists for the involvement of benzoxazinones in the resistance of corn to Helminthosporium turcicum, the causal agent of northern corn leaf blight. Corn has two main chromosomal loci that are involved in resistance to _H_. turcicum. The Ht locus is a single dominant gene conferring resistance to infection. The Bx locus is also a single, dominant gene that determines production of benzoxazinones. Couture (1971) compared corn varieties with a bxbx, or benzoxazinone- deficient genotype to those with a BxBx, or benzoxazinone-normal genotYPe, with dominant and recessive Ht genotypes, to assess the ' “ “warms? 16 relative importance of the two loci. In all cases, the benzoxazinone deficient genotype was the most susceptible to infection, regardless of the Rt genotype, indicating the importance of the benzoxazinones in this resistance. Bioassays of DIBOA on germinating spores of g. turcicum indicate a very high level of fungitoxic activity. Concentrations above 6 ppm almost completely inhibited spore germination. Spores that germinated had shorter germ tubes than controls. These results indicate that DIBOA has two main effects on g. turcicum infection. First, it inhibits germination of fungal spores, and secondly, it restricts the mycelial growth of any spores that do germinate. Long (1975, 1978) demonstrated a significant linear correlation between DIBOA concentration and resistance to g. turcicum. Detoxification of s-triazines. Tolerance of corn to the pre-emergence herbicide simazine (2-chloro-4,6-bis(ethylamino)-s-triazine) may be based, in part, on the nonenzymatic dechlorination of simazine to form 2-hydroxysimazine. Hamilton and Moreland (1961) used radiolabelling techniques to show this conversion in vivo, and recover hydroxysimazine from the plant tissue. They also demonstrated the mediation of this reaction in vitro by both GDIBOA and DIBOA. Monitoring the reaction by ultraviolet absorption showed that the hydroxamic acid was not degraded or altered in the course of the reaction, indicating that it plays a catalytic role in the process. Later work by Hamilton (1964) indicated that while the benzoxazinone-catalyzed degradation of s- triazines may by involved in resistance, it is not the dominant factor. Hamilton demonstrated a linear relationship between benzoxazinone content and ability of excised roots of several cereal crops to degrade simazine. Sorghum, however, contains no benzoxazinones, yet is more 17 tolerant than wheat or rye. Palmer and Grogan (1964) compared two lines of corn that are essentially isogenic, except for a single recessive gene that rendered one sensitive to atrazine and simazine. Although both lines contained GDIBOA, the resistant line contained nearly 2.5 times a much at each age tested. The sensitive line converted more atrazine per unit weight than the resistant line, suggesting that the resistance is based on a different mechanism than benzoxazinone-catalyzed dechlorination. Phytotoxic Activity. The first report of phytotoxic activity by benzoxazinone derivatives was by Virtanen (1957), when he noted that 2,3-benzoxazolinone (BOA) inhibited the germination of oat seeds at a rate of 0.5 to 1.0 mg per seed. El Naghy (1962) also noted that BOA exhibited phytotoxic activity on wheat, as did the aglucone, DIBOA, in the hypersensitivity reaction to Puccinia graminis infection. The most thorough investigation of the phytotoxic properties of the benzoxazinones produced by rye was conducted by Barnes, et al (1987a a b). The greatest phytotoxic activity contained in aqueous extracts of rye herbage was associated with two compounds later identified as DIBOA and BOA. Of the two compounds, DIBOA exhibited a higher degree of toxicity in petri dish bioassays. Inhibition of cress (Lgpidium sativum L.) root elongation to 50% of control was achieved with DIBOA at a concentration of 0.37 mM, while a concentration of 1.05 m was required for the same effect with BOA. Additional bioassays demonstrated the phytotoxic effects of DIBOA and BOA on a wide range of nonocotyledons and dicotyledons, including tomato, barnyardgrass, proso millet (Panicum miliaceum L.), large crabgrass (Digitaria sanguinalis L. Scop.), redroot pigweed (Amaranthus retroflexus L.). In general, 18 dicotyledonous species were approximately 30% more sensitive to these compounds than the monocotyledononous species tested. Benzoxazinones are thought to cause phytotoxicity by inhibiting photophosphorylation in the chloroplast (Quierolo, et a1, 1981). 5 M and BOA at 1.0 x 10"3 M caused Barnes showed that DIBOA at 7.5 x 10- a 50% inhibition of chlorophyll production by the green alga Chlamydomonas rheinhardtii. This observation provides support for the idea that the site of action of the benzoxazinones is the chloroplast, specifically, chlorophyll production. Although the benzoxazinone derivatives tested are highly phytotoxic to most plants tested, Barnes calculated that only about 12% of the potential toxicity of the aqueous extract was present in the benzoxazinone fraction (Barnes and Putnam, 1987), suggesting the presence of additional phytotoxins. Clearly, some of this additional activity is caused by cinnamic acid derivatives and volatile acids and aldehydes (Chou and Patrick, 1976). The toxicity of rye residues in the field appears to be the result of this combination of toxicants, but a detailed study of the toxic constituents of the leachate from rye residues has not been conducted. An additional source of toxins is from microbial metabolic activity on compounds in the leachate. The great diversity of soil microorganisms presents limitless possibilities for metabolism of rye-produced compounds, either attenuating or enhancing the total toxicity. The objectives of this study were to assess the effects of microbial transformation on benzoxazinones produced by rye, to isolate and identify any such transformation products, and to determine their biological activity. Literature Cited Argandona, V.H., J.G. Luza, H.M. Niemeyer, and L.J. Corcuera. Role of hydroxamic acids in the resistance of cereals to aphids. Phytochem. 19:1665-1668 (1980) Barnes, J.P. and A.R. Putnam. Rye residues contribute weed suppression in no-tillage cropping systems. J. Chem. Ecol. 9(8):1045-1057 (1983) Barnes,J.P. and AWR. Putnam. Evidence for allelopathy by residues and aqueous extracts of rye (Secale cereale L.) Weed Sci. 34:384-390 (1986) Barnes,J.P. and AuR. Putnam. Role of benzoxazinones in allelopathy by rye (Secale cereale L.). J. Chem. Ecol. 13(4):889-905 (1987) Barnes, J.P., A.R. Putnam, B.A. Burke, and A.J. Aasen. Isolation and characterization of allelochemicals in rye herbage. Phytochem. 26(5):1385-1390 (1987) Back, D.L., G.M. Dunn, D.G. Routley, and J.s. Bowman. Biochemical basis of resistance in corn to the corn leaf aphid. Crop Sci. 23:995-998 (1983) Chou, C.H. and z.A. Patrick. Identification and phytotoxic activity of compounds produced during decomposition of rye and corn residues in soil. J. Chem. Ecol. 2(3):369-387 (1976) Couture, R.M., D.G. Routley, and G.M. Dunn. Role of cyclic hydroxamic acids in monogenic resistance of maize to Helminthosporium turcicum Physiol. Plant Path. 1:515-521 (1971) Cubbon, MPH. Effect of a rye crop on the growth of grapes. J. Amer. Soc. Agron. 17:568 (1925) Duke, 8.0. in: The Science of Allelgpathy, Putnam, A.R., and C.S. Tang eds. Wiley and Sons, New York 1986 El‘Nahgy, M.A., and P. Linko. The role of 4-O-Glucosyl-2,4-dihydroxy-7- nethoxy-l,4-benzoxazin-3-one in resistance of wheat to stem. rust. Physiol. Plant. 15:764-771 (1962) Hamilton, R.H., and D.E. Moreland. Simazine: degradation by corn seedlings. Science 135:373-374 (1962) Hamilton, R.H. Tolerance of several grass species to 2-chloro-s- triazine herbicides in relation to degradation and content of benzoxazinone derivatives. Agric. Food Chem. 12:14-17 (1964) 19 v. -..-...‘m-n_nanr a! “a" * it"s? 20 Heisey, R.M. and A.R. Putnam. Herbicidal effects of geldanamycin and nigericin, antibiotics from Streptomyces hygroscopicus. J. Nat. Prod. 49(5):859-865 (1986) Heisey, R.M., 3.x. Mishra, A.R. Putnam, J.P.. Miller, C.J. Whitenack, J.E. Keller, and J. Huang. Production of herbicidal and insecticidal metabolites by soil microorganisms. Am. Chem. Soc. Symp. Ser. (In press) Hietala, P.R. and A.I. Virtanen. Precursors of benzoxazolinone in rye plants: precursor I, the glucoside. Acta Chem. Scand. l4(2):502-504 (1960) Hofman, J., and O. Hofmanova. 1,4-Benzoxazinone derivatives in plants: sephadex fractionation and identification of a new glucoside. Euro. J. Biochem. 8:109-112 (1969) Honkanen, E., and A. Virtanen. On the reaction of 2,4—dihydroxy-l,4- benzoxazin—3-one to 2,3-benzoxazolinone. Acta Chem. Scand. 15(1):221—- 222 (1961) Klun, J.A., and T.A. Brindley. Role of 6-methoxybenzoxazolinone in inbred resistance of host plant (maize) to first-brood larvae of european corn borer. J. Econ. Ent. 59(3):711-718 (1966) Rlun, J.A., C.L. Tipton, and T.A. Brindley. 2,4-dihydroxy-7-methoxy- 1,4-benzoxazin-3-one (DIBOA) , an active agent in the resistance of maize to the european corn borer. J. Econ. Ent. 60(6):1529-1533 (1967) Rnott, 0.1!. and J Rumar. Tests of the relationship between a specific phenolic glucoside and stem rust resistance in wheat. Physiol. Plant Path. 2:393-399 (1972) Koyama, T., M. Yamato, and K. Kubota. J. Pharm. Assoc. (Japan)76:1077 (1956) Kubo, I., and T. Ramikawa. Identification and efficient synthesis of 6- methoxy-Z-benzoxazolinone (BOA), an insect antifeedant. Experientia 39:359 (1983) Long, B.J., G.M. Dunn, J.S. Bowman, and D.G. Routley. Relationship of hydroxamic acid content in corn and resistance to the corn leaf aphid. Crop Sci. 17:55-58 (1977) Loomis, R.S., S.D. Beck, and J.P. Stauffer. The european corn borer (Pyrausta nubilalis Hubn.) and its principal host plant. V. A chemical study of host plant resistance. Plant Physiol. 32(5):379-385 (1957) Matsui, M., and I. Yamamoto. Pyrethroids. in Naturally Occurrirg Insecticides, Jacobson, M. and D.G. Crosby, eds. Marcel Dekker, New York (1971) x- 2....-- sun-“h"_al 7:." “if—'1‘. "" 21 Misato, T., Recent status and future aspects of agricultural antibiotics, in Pesticide Chemistry: Human Welfare and the Environment, Proceedings of the 5th International Congress of Pesticide Chemistry. Pergamon Press, New York (1982) Mishra, S.K., C.J. Whitenack, and A.R. Putnam. Herbicidal properties of metabolites from several genera of soil microorganisms. Weed Sci. 36(1) : 122-126 Palmer, R.D., and C.O. Grogan. Tolerance of corn lines to atrazine in relation to content of benzoxazinone derivative, 2-Glucoside. Weeds 13:219-222 (1965) Patrick, z.A. The peach replant problem in Ontario. II. Toxic substances from microbial decomposition products of peach root residues. Can. J. Bot. 33:461—485 (1955) Patrick, Z.A. and L.W. Koch. Inhibition of respiration, germination, and growth by substances arising during the decomposition of certain plant residues in the soil. Can. J. Bot. 36:621-647 (1958) Proebsting, E.L., and A.E. Gilmore. The relation of peach root toxicity to the re-establishing of peach orchards. Proc. Amer. Soc. Hort. Sci. 38:21-24 (1941) Rice, E.L. Allelopathy, 2nd edition. Academic, New York (1984) Sekizawa, Y. and T. Takematsu. How to discover new antibiotics for herbicidal use. In Pesticide Chemistry: Human Welfare and the Environment. Proceedings of the 5th Annual Congress of Pesticide Chemistry Pergamon Press, New York (1982) Tipton, C.L., J.A. Klun, R.R. Husted, and M.D. Pierson. Cyclic hydroxamic acids and related compounds from maize. Isolation and characterization. Biochem. 9:2866 (1967) Virtanen, A.I., P.K. Hietala, and O. Wahlroos. Antimicrobial substances in cereals and fodder plants. Arch. Biochem. Biophys. 69:486—500 (1957) Virtanen, A.I. and P.R. Hietala. Precursors of benzoxazolinone in rye plants: Precusor II, the aglucone. Acta Chem. Scand. l4(2):499-502 (1960) Weston, L.A., and A.R. Putnam. Inhibition of legume seedling growth by residues and aqueous extracts of quackgrass (Agrcmyron repens) . Weed Sci. 34:366-372 (1986) Weston, L.A., B.A. Burke, and A.R. Putnam. Isolation, characterization, and activity of phytotoxic compounds from quackgrass (Agropyron repens (L.) Beauv.). J. Chem. Ecol. 13(3):403-421 (1986) Whittaker, R.H. and P.P. Feeny. Allelochemics: chemical interactions between species. Science 171:757-770 (1971) .. :- B‘w-‘ .- ‘I-‘t-fi £3346. a, tamer 22 WOlf, R.B., G.F. Spencer, and R.D. Plattner. J. Nat. Prod. 48:59 (1985) Yamada, 0., S. Ishida, F. Futatsuya, K. Ito, H. Yamamoto, and K. Munakata. Agric. Biol. Chem. 38:1235 .. _ _...___,,_ _~._.————.a- . . . . ' n “M .' — v z».- mammmrmormmnmm oxrmrmorsmmzmmcmancmps Compound 4, 2,2'-oxo-l,1'-azobenzene, was isolated and characterized from soil supplemented with 2,3-benzoxazolinone (BOA). A parallel experiment with 6-methoxy-2,3-benzoxazolinone (BOA) yielded Compound 4, as well as it's monomethoxy- and dimethoxy- derivatives, Compounds 5, and 6, respectively. These compounds were produced only in the presence of soil microorganisms, via possible intermediates, I and II, which may dimerize, or react with the parent molecule to form the final products. In the case of MBOA, it was shown that the demethoxylation precedes the oxidation step. Although BOA and DIBOA were leached out of the rye residues, there were no detectable amounts of Compounds 4, 5, or 6 in the soil. When BOA was mixed with soil and rye residue, either under field conditions or in 33552, no 2,2'-oxo- 1,1'-azobenzene was detected. Levels of free BOA in the soil were greatly reduced by incubation. 23 mm Once an allelopathic system has been described in nature, it is desirable to elucidate the chemical processes involved. Fuerst and Putnam (1983) outlined four criteria, paralleling Koch's postulates, for proof of allelopathy. First, the adverse reaction of the recipient must be demonstrated and quantified, followed by isolation and purification of the chemical agent responsible for the inhibition and repeated demonstration of the inhibition using the pure compound(s). Finally, the compound should be recovered from the recipient's local environment. An investigation of the allelopathic activity of rye (Secale cereale L.) by Barnes, et. a1. (1987) confirmed that rye is highly allelopathic and implicated 2,3-benzoxazolinone (BOA), and 2,4- dihydroxy-l,4(2H)-benzoxazin-3-one (DIBOA) as the primary allelochemicals produced by rye. Experiments to detect BOA and DIBOA in the soil were not conducted. Once in the soil system, the benzoxazinones would be susceptible to 'microbial transformation by various soil microbes. For the benzoxazinones to be involved in long term allelopathic activity, they must be sufficiently resistant to such microbial transformations. Alternatively, if the parent compounds are metabolized, it is concievable that biologically active metabolites may be involved in the overal process of allelopathy by rye residues. The chemistry of the benzoxazinones is well documented. Originally isolated from rye by Virtanen (1957), BOA was subsequently shown to be the third step in a chemical breakdown scheme initiated upon injury to 24 -m.‘ -. Nth. 25 the plant (Virtanen and Hietala, 1960). Hofman and Hofmanova (1969) showed that when rye foliage was carefully extracted, so that no enzyme activity was allowed to take place, only the glucosyl conjugate of DIBOA was isolated. Earlier work (Honkanen and Virtanen, 1961) had clearly established the reaction of DIBOA to BOA. BOA is the most logical compound to choose for studies on the fate of benzoxazinones in the soil since it is the most likely to be present in the soil. Patrick and Koch (1958) conducted a study of the toxic substances arising as a result of microbial decomposition of plant residues, including rye. They found that unless decomposition of the residues occurred, no toxic substances were present in the soil extract. If, however, decomposition occurred, the extract of the soil—residue mixture was extremely toxic to the respiration, oxygen uptake and growth of tobacco seedlings. They did not determine whether the toxins were of plant or microbial origin. The objective of this work was to assess the impact of soil microbes on benzoxazinones, isolate and identify any bio-transformation products, and to monitor the levels of BOA and DIBOA in both residues and underlying soil over a period of time. W5!" 1 hperinental. Proton and carbon-l3 nuclear magnetic resonance ( H- and 13C -NMR) analyses were performed on a Varian XL-300 spectrometer, 300 MHz for proton and 75 MHz for carbon. Electron impact mass spectral (BI-MS) analysis was done on a Hewlett Packard model 5895 quadrupole mass spectrometer at 70 eV. Chemical ionization (methane) (CI-MS) and High Resolution (HRMS) mass spectra were generated on a 26 Jeol model Hx-llo mass spectrometer. Ultraviolet (UV) absorption analyses were performed on a Gilford Response II ultraviolet spectrophotometer and infrared (IR) spectra, on a Perkin Elmer model 1170 FTIR spectrophotometer. Melting points were determined on a Thomas model 40 Micro Hot Stage apparatus and are uncorrected. HPLC analysis was performed on a Waters Radial Pak 8 mm ID x 10 cm, radially compressed C18 column (Waters Assoc., Div. of Millipore, Inc. Milford, MA), using KH2P04 (0.01 M, pH 3.0)-acetonitrile (80:20 v/v). Unless specified, flash chromatography was performed on a silica gel column (Merck silica gel G, grade 60) and thin layer chromatography, on silica gel plates (Merck silica gel G F-254, 0.250 m layer). 2,3- benzoxazolinone (BOA), Compound 1, was obtained commercially from the Aldrich Chemical Co., Milwaukee WI. Soil used was a Spinks loamy sand (Psamentric, hapludalf, sandy, mixed, mesic), collected from a field site at Michigan State University for use in the greenhouse, and stored in large bins under dry conditions for approximately one year. Soil was sterilized when necessary by autoclaving (1 hr on three successive days) . Spray reagents for TIC detection. DIBOA was detected on thin layer plates with a spray reagent consisting of 5% FeCl3 in 95% ethanol, acidified with conc. HCl. A spray reagent consisting of 1% ceric sulfate in conc. H2804 was used to detect BOA and BOA. Isolation of 6—nethoxy-2,3-bensomazolinone (M) , Compound 2. Ten-day- old maize seedlings ('Pioneer 3737') were harvested and frozen at -20°C overnight (592 g). The thawed plant material was homogenized in a Waring blender with distilled water (1.4 L). This was kept at room temperature (1 hr) to ensure hydrolysis of the glucoside, 2-O-glucosyl- . .7. =7? *‘rr‘T “us; '.a a ",1 27 7-methoxy-l,4-benzoxazin-3-one (GDIMBOA) to 7—methoxy-2,4-dihydroxy- 1,4-benzoxazin-3-one (DIMBOA) and refluxed (2 hr), to cause the conversion of DIMBOA to MBOA. After cooling, the extract was strained through cheesecloth and the filtrate was acidified with cone. HCl (pH 1.0). The acidified extract was filtered through Whatman #4 and extracted with diethyl ether (5 x 300 ml). The ether fraction was washed once with distilled water (300 ml), dried over anhydrous M9804 and the solvent was removed in 22239. The crude extract thus obtained (720 mg) was initially purified by flash column chromatography (CHCl3- MeOH 5:1) followed by TLC check with ceric sulfate detection. The fractions positive to ceric sulfate were pooled and the solvent was removed _'1_q mag. This partially purified fraction (451.0 mg) was further chromatographed by TLC (CHCl3-MeOH 6:1). The ceric sulfate positive band was removed, eluted (CHCl 3-MeOH 2:1) , and the solvent was removed by rotary evaporation. The colorless compound (129.8 mg) thus obtained was recrystallized from hexane-acetone (-20°C), yielding needle-like crystals (70.8 mg), Compound 2. mp 144-1480C, UV (MeOH) 232.5 nm (9420), 290.0 nm (5050), IR (KBr) 3200, 1790, 1638, 1500, 131a, 1210, 1140, 1100, 1025, 970 cm'l, lH-NMR (CD300) a 3.56 (an, s, OCHB), 7.80 (1H, d, J - 8.2 Hz), 7.65 (13, d, J=2 Hz), 7.58 (13, d, J = 3.7 Hz), EI—MS, m/z: 165 (100, mi), 150 (so). Isolation of 2,4—dihydromy-1,4(2H)-bensoxasin—3-cne (DINA), W 3. Seedlings of rye ('Wheeler', 25-days-old) were lyophilized at 150 C (101.5 g), homogenized in a Waring blender with distilled water (1 L) and kept at room temperature (1 hr) to ensure enzymatic hydrolysis of the glucoside 2-O-g1ucosyl-l,4-benzoxazin-3-one (GDIBOA) to 2,4- dihydroxy-l,4-benzoxazin-3-one (DIBOA). After filtering through "-- ....- “i“ r" "“"."‘.""—‘I" 28 cheesecloth, the filtrate was heated in a water bath until the temperature of the extract reached 70°C. It was then cooled immediately in an ice bath. Coagulated components were removed by vacuum filtration using Whatman # 1 filter paper. The filtrate was acidified with conc. HCl (pH 1.0) and extracted with diethyl ether ( 4 x 300 ml). The ether fraction was washed once with distilled water (300 ml), and the solvent was removed _i_r_1_ m. The crude extract thus obtained (355.8 mg) was initially purified by column chromatography (CHCl3-MeOH 4:1), followed by TLC check with ferric sulfate detection. The fractions positive to ferric sulfate were pooled and the solvent was removed at reduced pressure. This partially purified fraction (187.2 mg) was further chromatographed by TLC (CHCl3-MeOH 6:1). The ferric sulfate positive band was eluted (CHCl3-MeOH 2:1) and removal of the solvent by rotary evaporation afforded a pale yellow residue (89.2 mg). ‘mis residue was titurated with diethyl ether and filtered through a sintered glass filter (fine). Recrystallization from ether-cyclohexane afforded colorless needles (22.4 mg) of Compound 3, mp 151-1550C, IR (KBr) 3400, 3200, 2380, 1670, 1590, 1280, 1220, 1040, 750 and, 1H - MIR (CD300) d 5.70 (13, S), 7.35 (13, dd, J = 1.0, 8.0 Hz), 7.10 (3H, m). HRMS, m/z: 181.0476 (“370410. M+) Isolation of 2,2'-omo—1,1'-asobenzene, (it-pound 4. Compound 1, 2,3- benzoxazolinone, (20 mg) was mixed thoroughly with soil (50 g) and the mixture was transferred to a 250 m1 erlenmeyer flask. Distilled water (5.0 ml) was added to the above and incubated at 26°C (10 days). Soil for sterile controls was autoclaved. BOA and filter-sterilized (0.22- um membrane) water (5.0 ml) were added aseptically to the soil, and the system was incubated at 26°C (10 days). "is- ,l’ '5]. TM." ' 29 The control and experimental soil as described above, were extracted with methanol (4 x 100 ml each). The methanol extract of the control soil was pale yellow, while the experimental extract was intensely orange. Both extracts were filtered, separately, through a sintered glass filter (fine) and the solvent was removed by rotary evaporation. A preliminary TLC analysis of the extracts indicated the presence of an orange band, only in the experimental soil. This orange compound (19.8 mg) was purified by TLC, (CHCl3-MeOH-HCOOH 20:1:1). Repeated purification was carried out by TLC (toluene-EtOAc-NH4OH 5:4:1) until the resulting dark red compound (1.8 mg) gave only one spot by TLC. Recrystallization from hexane-acetone (-20°C) yielded Canpound 4 as orange-red needles, mp. 223-228°C (decomp.), UV (MeOH) 237 nm (Ea-28268), 432 nm (Ea-23711), IR (KBr pellet) 760, 1574, 1587 curl, 1'3 mm (DMSO) a 7.75 (23, a, J - 8.4 Hz, H-6, H—6'), 7.55 (an, m, n-3, -4, -s and H-3', -4-, -5'), 13c mun (01430) a 99.87 (8, x2), 104.86 (d, x2), 117.25 (d, x2), 126.60 (d, x2), 129.19 (3, x2), 130.34 (3, x2), EI-MS, m/z: 212 (100, 14+), 185 (60), 184 (33); CI-MS, m/z: 212 (100, M1), 185 (28) 184 (18); HRMS, m/z: 212.0588 (C12H8N202, M+); FAB- MS (+) m/z: 213 04* + a) A similar experiment with compound 3, DIBOA, was carried out as in the case of compound 1 and the work up and purification yielded two canpounds that by analysis proved to be BOA and Compound 4, as well as unreacted Compound 3. Isolation of W 4 from Field Soil to Which NA was Added. A comercial sample of Compound 1 (1.0 g) was mixed with the top 1/2" of soil in a small plot (4 ftz) at the location from which soil used for _i_n_ vitro conversion of BOA to Compound 4 was obtained. A soil sample , —-—.. 1." P I _.._. _._.....__.——.....— r 9’. 30 (390 g) was taken from the center of the plot (7 days) and extracted with methanol as above (105.5 mg). Removal of the solvent at reduced pressure gave an orange residue. Analysis of this extract by TLC confirmed the presence of Compound 4. A control plot of the same size and location, but not supplemented with BOA was also extracted for comparison, and showed no benzoxazinones or diazoperoxides present. Isolation of 4-nethoxy—2,2'-omo-1,1'-azobenzene Cmpound 5, and 4,4'- di-etboxy-2,2'-omo-l,1'-asobenzene, W 6. The experiment with Compound 2 (BOA) was carried out as in the case of compound 1 and work- up and purification yielded a single component. Recrystallization from hexane-acetone afforded Compound 5 as red-orange needles, mp 218 - 220°C (decomp.)3 0v (neon) 231 nm (E a 23591), 450 m (E - 21122); IR: 2860, 2830, 1650, 1H -NMR (DMSO) (1 3.70 (3H, s, we), 7.74 (1H, d, J I 8.4 Hz), 7.64 (1H, d, J =- 8.8 Hz), 7.52 (3H, m), 7.11 (1H, J n 2 Hz), 7.01 (13, J =- 6 Hz), EI-MS m/z: 242 (100, M+); mas—us m/z: 243 m“) A small amount of 4,4'-dimethoxy-—2,2'-oxo-l,l'-azobenzene, 4. compound 6, was detected by both EI- and FAB-MS at 272 04*) and 273 (M + H), respectively. An additional experiment was carried out in which a 1:1 mixture of Compound 1 and Compound 2 was added to soil and incubated as above. Work-up, purification and analysis by EI-MS gave compounds 4, 5, and 6. EI-MS m/z: 212 (72%), 242 (26%), and 272 (1.5%). Microbial Ecology of Transformation Reaction. Several attempts were made to isolate the organism(s) responsible for the biotransformation of BOA. Soil (1 g) was mixed with physiological saline (0.85% NaCl, 9 ml) and vortexed. Serial dilutions (1:10) were then plated on Nz-amine _,——I. r . . , 31 agar (NZ-amine A, 39; agar 18 g, distilled water 1L) . The plates were sealed in a polyethylene bag and incubated at 25°C. After 72 hr, individual colonies were removed and inoculated into screw-capped test tubes containing sterile soil (5 g), and BOA (2 mg). These were wet with sterile distilled water (0.5 m1) and incubated as above. Leaching Experiments with Rye Residue. Rye seedlings ('Wheeler', 35- days-old) were harvested at the soil level (900 g), placed on a flat (25 cm x 50 cm) of soil and watered over the top at 72 hr intervals. Residue and underlying soil samples were taken at 0, l, 2, 3, 6, 10, 17, and 27 days, respectively. Methanol extracts (4 x 100 ml) of the soil samples were stored at -20°C. The lyophilized residue samples were hmogenized in a Waring blender with distilled water (300 m1), kept at room temperature (1 hour), and strained through cheesecloth. The pH of the extract was adjusted to 1.0 with conc. HCl. The acidified extract was filtered through Whatman 41 under vacuum and extracted with diethyl ether (4 x 100 ml). The solvent was removed E mg and the dried samples were stored at -20°C. Several experiments were also conducted in which lyophilized rye residue (10 g) was cut into 2 cm pieces and mixed into dry soil (1 kg) with distilled water (100 m1). Three such systems were set up in 2 L beakers, each comprising a sample, that was extracted at 24, 72, and 240 hr respectively. Soil and residue were separated after drying at 45°C (1 hr). Methanol extraction of soil (3 x 500 m1) and removal of the solvent yielded dry samples that were stored at -20°C. The lyophilized residue was homogenized in a Waring blender with distilled water (200 ml), kept at room temperature (1 hr), strained through cheesecloth and acidified with conc. HCl (pH 1.0). The extracts were ,.___.__ _..___.._.-_p—......4.1: . s u 3' 32 processed as above and stored at -20°C. All samples were analyzed simultaneously. Preliminary analysis of the samples was carried out by TIC (CHC13-MeOH 5:1). DIBOA and BOA were detected with the ferric chloride and ceric sulfate spray reagents, respectively. Samples that did not contain BOA or DIBOA by TLC were not analyzed further. Samples containing BOA or DIBOA were partially purified by TLC (CHC13- MeOH 5:1) and analyzed by HPLC. Both compounds were quantified respective to external standards by monitoring at 230 nm. An additional experiment was conducted in which BOA (10 mg), rye tissue (5 g fresh), and distilled water (2.5 ml) were added to soil (25 g) and incubated at 26°C (10 days). An identical system, lacking residues, was used as a control. mums AND DIME! Compound 4, 2,2'-oxo-l,l'-azobenzene, isolated as a bio- transformation product of 2,3-benzoxazolinone (BOA) (Figure 1) from non-sterile soil, gave the molecular formula of C12H8N202 by HRMS. the doublet at 7.75 ppm, integrated for 2 protons was assigned to the 6, and 6' hydrogens ortho to the nitrogen in the aromatic ring. The only other signal observed in the H NMR spectrum was a multiplet at 7.55 ppm, integrated for 6 protons. The 13C -NMR spectrum of compound 4 gave only 6 signals for 12 carbon atoms, indicating a symetrical structure for this molecule. The strong IR absorption at 760 cm-1, indicative of an aromatic system with four adjacent hydrogens, was in full agreement with the NMR data and confirmed the proposed structure (Figure 2). Incorporation of DIBOA, compound 3, into soil also resulted in the '1_- [_t.‘ ‘ao'm. Q‘m" a; ‘O-O‘M -. n . '4; 4 “1.44.! 33 Figure 1: Structure of parent benzoxazinones, compounds 1, 2, and 3. 34 ii / N O n \ 0 R = H = Compound 1 = 2,3-Benzoxazolinone (BOA) R = OMe = Compound 2 = 6-methoxy-2,3-benzoxazolinone (MBOA) OH N O O OH Compound 3 2,4-dihydroxy-l,4(2H)-benzoxazin-3-one (DIBOA) Figure 1 3 l ' V w“, 35 Figure 2: Proposed structures of compounds 4, 5, and 6, arising from microbial oxidation of 2,3-benzoxazolinone in the soil. 36 R 0—0 a N=N R1 3 32 = H 8 Compound 4 Rl .- 3, R2 = OMe 8 .Compound 5 R1 = 32 = OMe = Compound 6 Figure 2 37 formation of compound 4. Addition of methoxylated BOA, compound 2, gave compounds 5 and 6, characterized by spectral methods (Figure 2). It is interesting to note that the most abundant product of MBOA transformation is compound 5 (95%), with compound 6 present as a minor species, as evident by mass spectral data. Trace amounts of compound 4 were also present in the above mixture. Formation of compound 4 in the soil could be the result of an intermediate (I, Figure 3), produced enzymatically from BOA, which in turn reacts with another molecule of BOA. Alternatively, this intermediate could undergo dimerization to afford compound 4. In the case of MBOA, the major product was compound 5, a monomethoxy analog of Compound 4. This indicated that prior to the enzymatic oxidation leading to intermediate II (Figure 3), there is a demethoxylation step involved. That is, an enzymatic demethoxylation, followed by enzymatic oxidation would also yield intermediate I, which in turn would react with BOA, to give compound 5. The dimethoxy derivative, compound 6, should result from the reaction of intermediate II with MBOA, or from dhmerization of intermediate II. The experiment with equal amounts of M and BOA gave compound 4 as the major product (72%) , compound 5 (26%), and compound 6 in very small quantity (1.5%). Therefore, it is evident that intermediate II is less abundant than intermediate I, and that the demethoxylation occurs prior to the enzymatic oxidation. Based on these results, it is possible to extend the breakdown scheme for benzoxazinones proposed by Virtanen et. a1. (1960) (Figure 4). Addition of BOA or BOA into sterile soil did not produce compounds 4, 5, or 6, suggesting that these compounds are produced by soil microbes. A saline extract of the soil, to isolate the total microbial population was added to soil containing BOA, and resulted in 38 Figure 3: Proposed structures of intermediates I and II in the formation of compounds 4, 5, and 6 cowcmu_xo _a_eeee_z . co_cmpxxo;um5mo _a_eeee_z . :01 Z .5... s . . a . sp.,-20.4134!!! all» all It. .0. 3.; nose: .HH A eecbaetxe Pa_eeeeaz eeaeae_xe _eaeeeaaz 2::::c> _1 Figure 3. 40 Figure 4: Chemical breakdown scheme of Virtanen amended to include Compounds 4, 5, and 6. 41 CH4 hi :::[if!) R C) Fl \\\‘ I (D”/L\‘cn4 Chemical Rearrangement Home Microbial Oxidation R1 0".0D/R2 N 2N / DU 50 I,” II :0 I 30' N lu N 30 ll 0 3 (D Figure 4 42 production of compound 4, after incubation for 7 days. Experiments to isolate the organism(s) responsible for the transformation were inconclusive. None of the colonies isolated were capable of transforming BOA. It appears as if the transformation reaction does not provide a selective advantage to those microbes capable of carrying it out. That is, the reaction is probably not an energy yielding reaction, but perhaps instead, is catalyzed by an extracellular oxidase enzyme. Production of compound 4 was also demonstrated in the field, using commercial BOA, indicating that these organisms are present in the environment. Although trace amounts of BOA and DIBOA were isolated from the soil in laboratory leaching experiments using rye residue, Compound 4 was apparently not in this soil. In order to verify this result, an 13 gitgg experiment with rye residue and BOA was conducted and the products were quantified. Only trace amounts of compoumd 4 were observed by TLC from the soil containing BOA and fresh rye residue. It was also interesting to note that most of the added BOA in the residue sample (92%) had disappeared. The control sample containing no residue, produced the normal amount of compound 4 as mentioned earlier, yet contained approximately 9.7 times as much of the added BOA as did the residue sample. These results indicated that either some enzymatic inhibitors had leached from the rye residue into the soil, or that the leachate had killed the microbe(s) responsible for the conversion. Since the microbes responsible for the production of compounds 4, 5, and 6 have not yet been identified, it is difficult to argue that the leachate has killed these organisms. Chemical reaction between BOA and the leachate is a possible explanation for the diminished production of compound 4, as well as the disappearance of most of the added BOA. The above results pt. Ax. Ana.“.¢_a.n .a‘ 4.2.... 'I .- ' l. _ i u, 1" E,”” 43 were repeated in large scale experiments as well. Our results suggest a role for soil microorganisms in the overall process of allelopathy by rye. The primary allelochemicals produced by rye, BOA and DIBOA, have been shown to be present in the soil by in ggtgg_1eaching experiments. Once in the soil, these compounds undergo microbial transformation, resulting in the diazoperoxides, Compounds 4, 5, and 6. Compound 4 was not detected in field soil, or in the soil of in giggg experiments with rye residue. However, the biological activity of Compound 4 (Chapter 3) clearly demonstrates the potential role of these compounds in allelopathy by rye residues. -_- ”iufl‘s on: -n... _-N . . . r LITERATURECIE) Barnes, J.P., and A.R. Putnam. Evidence for allelopathy by residues and aqueous extracts of rye (Secale cereale L.) Weed Sci. 34:384-390 (1986) Barnes, J.P., and A.R. Putnam. Role of benzoxazinones in allelopathy by rye (Secale cereale L.) J. Chem. Ecol. 13(4):889-905 (1987) Barnes, J.P., A.R. Putnam, B.A. Burke, and A.J. Aasen. Isolation and characterization of allelochemicals in rye herbage. Phytochem. 26(5):l385-1390 (1987) Feurst, E.P. and A.R. Putnam. Separating the competitive and allelopathic components of interference. Theoretical principles. J. Chem. Eccl. 9:937-944 (1983) Hofman, J. and O. Hofmanova. 1,4-Benzoxazinone derivatives in plants: Sephadex fractionation and identification of a new glucoside. Euro. J. Biochem. 8:109-112 (1969) Bonkanen, E. and A. Virtanen. On the reaction of 2,4-dihydroxy-l,4- benzoxazin-3-one to 2,3-benzoxazolinone. Acta Chem. Scand. 15(1):221- 222 (1961) Patrick, LA. and L.W. Koch. Inhibition of respiration, germination and growth bysubstances arising during the decomposition of certain plant residues in the soil. Can. J. Bot. 36:621-647 (1958) Virtanen, A.I., P.R. Hietala and O. Wahlroos. Antimicrobial substances in cereals and fodder plants. Arch. Biochem. Biophys. 69:486-500 (1957) Virtanen, A.I. and P.R. Hietala. Precursors of benzoxazolinone in rye plants: Precursor II, the aglucone. Acta Chem. Scand. 14(2) :499 - 502 (1960) 44 M3 BIOWICAL acrrvn'! OP 2,2'-0X0-1,1'-Am-, AH MGR!- ARISIIG THROUGH MICROBIAL.OIIDATIOI OP 2,3-BEHIOXAIOLIIUIB.(BOA) ABSTRACT Compound 4, 2,2'-oxo-l,l'-azobenzene, an allelochemical from rye, isolated previously and shown to be a product of microbial oxidation of 2,3-benzoxazolinone, was evaluated in terms of it‘s biological activity. The compound exhibited strong phytotoxic activity. Root elongation of tomato (Lycopersicon esculentum Mill.) was strongly inhibited (ICSO - 5-7 ug/ml), as were barnyardgrass (Echinochloa crusgalli L.), redroot pigweed (Amaranthus retroflexus L.), velvetleaf (Abutilon theophrasti L.), and garden cress (Lepidium sativum L.). Radicle elongation of rye (Secale cereale L.) was promoted 59% relative to control at 6.25 ug/1.5 ml. Proso millet (Panicum milaceum L.) radicle elongation was promoted 66% relative to control at 6.25 ug/l.5 m1, but was inhibited 90% at 200 ug/1.5 m1. In comparative assays using cress and barnyardgrass, Compound 4 was shown to be 7.5 times as toxic as BOA, and 10 times as toxic as DIBOA. Antifungal assays indicated no activity against Aspergillus flavus, Rhizoctonia solani, and Fusarium ogspgrum f. sp. asparagi. Insecticidal activity was evaluated with mosquito larvae, Aedes triseriatus Say) and showed limited activity at 200 ppm. The compound showed strong nematicidal activity (Panagrellus redivivus) with 100% mortality after 24 hours at 10 ppm. 45 “I Benzoxazinones are reportedly involved in several of the chemical defense systems of cereal crops. Originally isolated by Virtanen (1957) as chemical factors in the resistance of rye to Fusarium infection, these compounds were eventually implicated in the resistance of corn to Helminthosporium turcicum infection (Couture, 1971), and in the resistance of wheat to stem rust (El Naghy and Linko, 1962). In addition to antifungal activity, benzoxazinones were shown to be involved in the resistance of corn to attack by european corn borer (Loomis, 1957), corn leaf aphid (Long et. al., 1977) and cereal leaf beetle (Argandona, et. al., 1980). Recently, Barnes, et. a1. (1987) demonstrated the allelopathic potential of benzoxazinones produced by rye, but they were unable to account for all of the potential toxicity with benzoxazinones alone. Apparently, these compounds did not account for all of the observed activity. The wide range of biological activity, along with the inability of previous authors to account for all of the observed toxicity in terms of the benzoxazinones, led to the investigation of the possibility that the metabolites arising from microbial metabolism of the parent benzoxazinones might also lend activity. A stable diazoperoxide was isolated from soil in laboratory experiments on the microbial metabolism of benzoxazinones (Chapter 2). The obvious