Rye (Secale cereale L.) and Wheat (Triticum aestivum L.) Mulch: The

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Rye (Secale cereale L.) and Wheat (Triticum aestivum L Mulch: The Suppression of Certain Broadleaved Weeds and the Isolation and Identification of Phytotoxins 1

DONN G. SHILLING , REX A. LIEBL, and A. DOUGLAS WORSHAM Crop Science Department, Weed Science Center, North Carolina State University, Raleigh, NC 27695-7627

Research over the years has indicated improved control of c e r t a i n annual broadleaf weeds in no-till cropping systems when mulches are left on the soil surface. Therefore, we i n i t i a t e d studies i n no-till corn, soybeans, sunflower, and tobacco to evaluate and separate the effects of small grain mulches and t i l l a g e on broadleaf weeds. Planting corn no-till into a desiccated green wheat cover crop reduced Ipomoea spp. biomass 79% compared to a non-mulched, tilled treatment. Elimination of t i l l a g e i n non-mulched treatments was as e f f e c t i v e i n reducing weed biomass as replacing mulch on tilled soil. In double-crop soybeans, there was no mulch effect; t i l l a g e , however, greatly increased Ipomoea spp. biomass. In no-till tobacco, elimination of t i l l a g e and presence of a rye mulch reduced biomass of Amaranthus retroflexus L., Chenopodium album L., and Ambrosia a r t e m i s i i f o l i a L. by 51, 41, and 73%, respectively. Rye mulch reduced C. album biomass i n both the tilled and the n o n - t i l l e d systems by 60%. In full-season soybeans and sunflowers planted into desiccated green rye, the elimination of tillage and the presence of rye mulch reduced aboveground biomass of C. album, A. a r t e m i s i i f o l i a , and A. retroflexus 99, 92, and 96%, respectively. Rye mulch was as e f f e c t i v e as elimination of t i l l a g e i n reducing C. album and A. retroflexus biomass. A. a r t e m i s i i f o l i a was not s i g n i f icantly affected by rye mulch i n any of these cropping

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Current address: Monsanto Agricultural Products Co., 800 N. Lindbergh Blvd., St. Louis, MO 63167

0097-6156/85/0268-0243S08.25/0 © 1985 American Chemical Society

In The Chemistry of Allelopathy; Thompson, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.


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systems. A l l e l o p a t h i c suppression of several broadleaf weeds by the mulches is implicated i n these studies. In further studies to examine possible causes of weed suppression i n no-till cropping systems utilizing killed rye (Secale cereale L.) and wheat (Triticum aestivum L.) as a mulch, two phytotoxic compounds not previously implicated i n allelopathy, β-phenyllactic acid (βΡLΑ) and β-hydroxybutyric acid (βHBA), were i d e n t i f i e d from aqueous extracts of field-grown rye. The βΡLΑ and βHBA at 8 mM inhibited Chenopodium album L. hypocotyl growth 68 and 30%, respectively i n laboratory bioassays. Both acids inhibited C. album root growth 20% at 2 mM. Amaranthus retroflexus L. hypocotyl growth was inhibited 17% by βPLA at 0.8 mM and 100% at 8 mM with βΗΒΑ giving 27% i n h i b i t i o n at 8 mM. A. retroflexus root growth was inhibited 59 and 39% at 2 mM by βΡLΑ and βΗΒΑ, respec t i v e l y . The compound i d e n t i f i e d i n a l k a l i extracts from wheat having greatest i n h i b i t o r y effects on Ipomoea lacunosa L. seed germination was i d e n t i f i e d as f e r u l i c acid (4-hydroxy-3-methoxy cinnamic a c i d ) . At 5 mM, germination and root length was inhibited 23 and 82%, respectively. Sida spinosa L. germination and root length, with carpels present on the seed, was inhibited 85 and 82%, respectively. F e r u l i c acid was decarboxylated by a bacterium l i v i n g on the carpels of S. spinosa seed to a more phytotoxic styrene derivative, 4-hydroxy-3-methoxystyrene. These compounds plus other unidentified phytotoxic chemicals could, i n part, explain suppression of c e r t a i n weeds by rye and wheat mulches i n no-till crops.

C u l t i v a t i o n of soil has and w i l l continue to be an important means of c o n t r o l l i n g weeds (jL) · However, extensive soil c u l t i v a t i o n leads to various problems such as losses of soil, soil moisture and nutrients. This r e s u l t s i n water p o l l u t i o n by both the soil i t s e l f and pesticides and nutrients associated with i t (2, _3, 4_) . Minimum or n o - t i l l cropping systems can reduce these problems because various crop residues ( i . e . , mulch) are l e f t on the soil surface with a minimum of soil disturbance i n planting the crop. The presence of crop residues has been reported to both increase (5, 6) and decrease crop y i e l d s (_7) and not t i l l i n g to increase c e r t a i n d i f f i c u l t to control weeds (8). However, other reports indicate that the presence of c e r t a i n mulches can reduce the biomass of c e r t a i n weeds (9-15) and allow for higher crop y i e l d s (5, 6). Thus, under c e r t a i n conditions, mulches can suppress c e r t a i n weed species, but determining the reason(s) presents many l o g i s t i c a l problems, especially under f i e l d conditions. To determine the cause(s), the physical and chemical ( i . e . , allelopathy) effects of the mulch and the role of soil disturbance (or the lack of, as would be the case i n a n o - t i l l system) must be separated. Under n o - t i l l conditions, many parameters are affected i n such a manner as to a l t e r germination of weed seeds. Putnam et a l . (16) stated that "eliminating t i l l a g e reduces densities of many annual species presumably because numerous seeds are isolated from



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favorable s i t e s for germination." One major r e s u l t of soil d i s t u r bance is that i t provides the seed with l i g h t which is required i n many instances for germination (17, 18, 19). The elimination of t i l l a g e alone (without any mulch present) has been shown to reduce weed populations (9, 15, 16). N o - t i l l systems are also known to affect soil moisture, temperature and pH, a l l of which could a f f e c t the emergence and growth of p l a n t s — b o t h crops and weeds (4^, 20). Even with these problems, attempts have been made to demonstrate that mulches suppress weeds a l l e l o p a t h i c a l l y . Putnam and DeFrank (12) and Barnes and Putnam (39) used Populus wood shavings to separate chemical and physical e f f e c t s of mulches. Their work indicated that c e r t a i n mulches do possess a l l e l o p a t h i c p o t e n t i a l . L i e b l and Worsham (9) and S h i l l i n g and Worsham (14) placed mulch on t i l l e d soil, after t i l l i n g , i n an attempt to provide the weeds with an exposure to l i g h t . Their work also indicated that at l e a s t part of the suppression of weeds by wheat and rye mulch is a l l e l o p a t h i c . Thus, research to date indicates that both mulch and the lack of soil t i l l a g e contributes to the suppression of weeds i n n o - t i l l cropping systems. A l l e l o p a t h i c e f f e c t s are probably due to i n h i b i t o r y compounds being released d i r e c t l y from plants (or their residues) or microbial metabolites. In an early study, Guenzi and McCalla (21) showed that most crop residues contain water-soluble substances that can depress the growth of corn (Zea mays L . ) , wheat (Triticum aestivum L.) and sorghum (Sorghum b i c o l o r L.). Guenzi and McCalla (22) i d e n t i f i e d f i v e phenolic acids from a number of plant residues and showed that a l l i n h i b i t e d the growth of wheat. Chou and Patrick (23) i s o l a t e d and i d e n t i f i e d a number of phytotoxic compounds from soil amended with rye (23). Other studies have also indicated that compounds from plant residues can adversely a f f e c t plant growth (11, 23, 24, 25, 26). Shettel and Balke (27) have further demonstrated that a l l e l o p a t h i c compounds can s e l e c t i v e l y suppress c e r t a i n weed species. The implication that " l i v i n g crops" can a l l e l o p a t h i c a l l y suppress weeds has been made by Putnam and Duke (28) and Leather (29). These studies demonstrated that the p o t e n t i a l for suppression of weeds could be enhanced by crop selection. Putnam and Duke (28) suggested that there is p o t e n t i a l f o r breeding crops to better suppress weeds by u t i l i z i n g and improving a l l e l o p a t h i c c h a r a c t e r i s t i c s . Fay and Duke (30) evaluated the amount of scopoletin (6methoxy-7-hydroxy coumarin) exuded from 3,000 accessions of Avena sp. They suggested that the "wild types" of crop v a r i e t i e s could have once possessed a l l e l o p a t h i c p o t e n t i a l , but t h i s character has been inadvertently selected against over time. The p o s s i b i l i t y that microorganisms play a p i v o t a l r o l e i n the production and persistence of phytotoxic compounds has also been demonstrated (7_, 23, 2j>, 31, 32). Guenzi et a l . (7) found that aqueous extracts of weathered corn and sorghum residues were most phytotoxic to wheat growth after 4 and 16 weeks of decomposition, respectively. The greatest phytotoxicity exhibited by aqueous extracts of wheat and oat (Avena sativa L.) straw occurred at or near harvest time, with e s s e n t i a l l y a l l t o x i c i t y gone after four weeks of decomposition. Kimber (33) found that aqueous extracts of several grasses and legumes that had been r o t t i n g f o r periods of up to 21 days were i n h i b i t o r y to the growth of wheat. He also showed that straws cut while s t i l l green produced a higher l e v e l of t o x i c i t y



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than those cut when f u l l y mature. Toussoun et a l . (34) found that the production of water-soluble phytotoxins of barley (Hordeum vulgare L.) residue i n soil required a soil moisture content above 30% of the soil and residue dry weight. Extracts became toxic 7-10 days a f t e r incorporation into soil with phytotoxic a c t i v i t y reaching a maximum i n 3 weeks. Thus, microorganism can enhance and/or reduce the phytotoxicity of plant residues. Since the early 1960s, increased emphasis has been placed on the r o l e of soil microorganisms i n the production of phytotoxic substances from plant residues. Norstadt and McCalla (35) postulated that the i n h i b i t o r y e f f e c t s of crop residues might be due to a combination of toxins from plant residues and from microorganisms that are more p r o l i f i c where plant residues are present. A number of fungi have been isolated from soil obtained from n o - t i l l p l o t s . Many of the fungi produced substances toxic to higher plants (36). Patrick and Koch (37) reported that aqueous extracts of various plant residues had no e f f e c t on the r e s p i r a t i o n of tobacco (Nicotiana tabacum L.) seedlings. They did, however, demonstrate that substances formed during the decomposition of plant residues i n the soil markedly inhibited the r e s p i r a t i o n of tobacco seedlings. In later studies, Patrick (38) and Chou and Patrick (23) isolated and i d e n t i f i e d a wide range of toxic compounds from decomposing rye (Secale cereale L.) and corn residues. In another study, Cochran et a l . (32) found that plant residues produced wheat seedling root i n h i b i t o r s only a f t e r conditions became favorable f o r microbial growth. The purposes of the studies reported here were to: (1) charact e r i z e the e f f e c t s of rye and wheat mulch and soil disturbance on c e r t a i n weed species, (2) determine i f allelopathy was involved, (3) determine i f rye and wheat straw extracts were phytotoxic to c e r t a i n weed species, (4) i s o l a t e , characterize, and i d e n t i f y i n h i b i t o r y compounds, and (5) determine quantitatively the phytot o x i c i t y of i n h i b i t o r y compounds that were i d e n t i f i e d .

MATERIALS AND METHODS Rye Mulch Studies F i e l d Research. In 1982 and 1983, experiments were conducted to evaluate the e f f e c t s of primary t i l l a g e ( i . e . , soil t i l l e d at time of planting) and above- and below-ground rye residue [ i . e . , shoot (mulch) versus root residue] on weed populations i n three cropping systems (flue-cured tobacco, soybeans and sunflowers). No r e s i d u a l herbicides were used i n any of the following experiments. These tests were conducted at the Central Crops Research Station near Clayton, NC (Johns sandy loam soil type) and Lower Coastal P l a i n Research Station near Kinston, NC (Goldsboro sandy loam soil type). In October of 1981 and 1982, f i e l d s were disked and rye (Secale cereale L. 'Abuzzi') planted at 188 kg/ha. Two no-rye treatments were also established at t h i s time and maintained plant free by applications of paraquat (1,1'-dimethyl-4,4'-bipyridinium ion) at O.6 kg/ha as needed. Each test was a completely randomized block design replicated four times. In A p r i l of 1982 (flue-cured tobacco) and 1983 (soybean and sunflower), treatments were set up as follows: (1) mulch removed;


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(2) mulch removed and soil t i l l e d ; (3) mulch cut; (4) mulch removed, soil t i l l e d and mulch replaced; (5) mulch desiccated with 3.36 kg/ha of glyphosate; (6) mulch desiccated with O.6 kg/ha of paraquat; (7) no rye, soil t i l l e d ; and (8) no rye, n o - t i l l . Once these treat ments were established, the tests were treated with 3.36 kg/ha of glyphosate [N-(phosphonomethyl) glycine] to desiccate weeds and l i v i n g rye. In 1982, tobacco (Nicotiana tabacum L. 'McNair 944 ) was transplanted approximately 10 days a f t e r treatment and i n 1983 soybean (Glycine max L. 'Essex ) and sunflower (Helianthus annuus L.) were planted on the same day. During the growing season data were collected to determine the e f f e c t s of soil t i l l a g e and rye mulch on % weed control, weed biomass, weed density, soil temperature and incident l i g h t . Weed ratings were determined by v i s u a l l y com paring treatment plots to the no rye, t i l l e d plot ( i . e . , t h i s t r e a t ment represented 0% weed c o n t r o l ) . A completely randomized design was used with four r e p l i c a t i o n s . 1


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Growth Chamber Experiment. The experiment was conducted to evaluate the a l l e l o p a t h i c potential of rye. This was accomplished by water ing common lambsquarters with an aqueous extract of rye. F i e l d grown rye was collected i n A p r i l of 1983 (at anthesis) and a i r dried for seven days. The plant material was extracted with d i s t i l l e d water for s i x hours with shaking. The extract was obtained by f i l t r a t i o n . The extract was then diluted to obtain the following concentrations: 1 g/20 ml, 1 g/30 ml, 1 g/60 ml. A l l extracts were adjusted to pH 5.5 with IN KOH. Osmotic potential of each extract was determined and appropriate controls established using polyethyl ene g l y c o l . Common lambsquarters (O.052 g) was seeded into 1 L pots containing quartz sand and watered with nutrient solution. Starting on the second day of the experiment, pots were watered with 20 ml of the appropriate s o l u t i o n . A t o t a l of s i x applications of the extract were applied. The plants were grown for 28 days at 25°C with a 12 hr day length. At this time the number of plants per pot was determined. A completely randomized block design was used with four r e p l i c a t i o n s . Data were subjected to analysis of variance and regression analysis using the general l i n e a r model procedure of the S t a t i s t i c a l Analysis System (40). Means were compared using Waller-Duncan procedure with a Κ r a t i o of 100. Polynomial equations were best f i t t e d to the data based on significance l e v e l of the terms of the equations and R2 values. Wheat Mulch Studies F i e l d Studies. At a l l locations, the area planted i n small grains was divided into two parts. One part was planted i n corn the follow ing spring, and the other part planted i n soybeans ('Ransom') follow ing small grain harvest. Study 1. Wheat ('McNair 1813') was planted at the Central Crops Research Station near Clayton (Lynchburg sandy loam) and the Tide water Research Station near Plymouth (Bayboro loam), North Carolina, at a rate of 101 kg/ha i n October of 1980. The following spring or early summer, plots were set up i n which a green wheat cover crop or wheat straw and stubble remaining after wheat harvest was: (1) l e f t

American Chemical Society Library 1155 16th St., N.W.

In The Chemistry of Allelopathy; Thompson, A.; ACS Symposium Series; Washington, American Chemical Washington, DC, 1985. D.C.Society: 20036

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i n t a c t , (2) mowed with a s i c k l e bar mower and wheat mulch or straw removed, (3) mowed, removed and soil t i l l e d with a disk, or (4) mowed, removed, t i l l e d and the wheat mulch or straw replaced following t i l l a g e . Following either residue removal or t i l l i n g , but p r i o r to replacement, corn or soybeans were planted using a John Deere 2-row Max-Emerge n o - t i l l planter. Planter adjustments were made to obtain a uniform planting depth i n the various treatments. Paraquat at O.56 kg ai/ha and alachlor at 2.2 kg ai/ha were applied following planting or mulch/straw replacement. Herbicides were applied as a tank mix using a C0 back sprayer with a c a r r i e r volume of 171.2 L/ha. Treatments within each crop were arranged i n a randomized block design with four r e p l i c a t i o n s . Plots consisted of four 96-cmwidth rows 15 m long. At both locations, morningglory was the predominant weed species. During the growing season, morningglory plant counts and biomass data were obtained to evaluate the effects of wheat residues and t i l l a g e on morningglory growth.


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Study 2. This study was conducted at the Upper Coastal P l a i n Research Station near Rocky Mount (Norfolk loamy sand) and the Lower Coastal P l a i n Research Station near Kinston (Goldsboro loamy sand), North Carolina, to investigate the e f f e c t s of various small grain residues on weed growth i n n o - t i l l corn and soybeans. Wheat, oats (Avena sativa L. 'Brooks'), barley (Hordeum vulgare L. 'Keowee') and rye ('Abruzzi') were planted at rates of 101, 72, 108, and 94 kg/ha, respectively, i n October of 1980. Two treatments were also included which were not planted i n small grains. Bare soil plots were kept weed-free during the winter and early spring months with applications of paraquat (O.56 kg ai/ha). The following growing season, small grains were either k i l l e d with paraquat i n early spring and planted n o - t i l l i n corn or harvested i n June and soybeans planted n o - t i l l i n stubble and residue remaining. Prior to corn or soybean planting, one of the bare soil treatments was t i l l e d with a disk to simulate a conventional seedbed. The other bare treatment remained undisturbed and was planted n o - t i l l . Herbicide treatment, s t a t i s t i c a l design and plot size were i d e n t i c a l to those of Study 1. Throughout the growing season, v i s u a l weed ratings were obtained to evaluate the effects of the four small grain residues and t i l l a g e on weed growth. Isolation and I d e n t i f i c a t i o n of Phytotoxins Rye Mulch Studies. Extraction Procedure. A flow chart of the i s o l a t i o n and i d e n t i f i c a t i o n procedure is presented i n Figure 1. Field-grown rye ('Abruzzi', harvested at early flowering stage on March 24, 1983, from the Central Crops Research Station, Clayton, NC) was a i r - d r i e d for 7 days. The tissue (150 g) was extracted with 3 L of d i s t i l l e d water for 10 hr with agitation. The extract was f i l t e r e d through cheesecloth and then centrifuged at 28,000 x g for 20 min. The supernatant was reduced i n volume to 300 ml i n vacuo at 50°C. Sixty ml of the concentrated aqueous extract was dried i n vacuo » the residue extracted with 20 ml of methanol and f i l t e r e d . The methan o l i c extract was stored at 0°C u n t i l use.


S H I L L I N G ET A L .

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PLANT MATERIAL


AQUEOUS EXTRACT

*

DRIED EXTRACT

PARTITION

MEOH SOLUBLE

ORGANIC PHASE BIQASSAYED

I T L C O F ORGANIC PHASE

(ACTIVE)

MEOH SOLUBLE BIOSSAYED

BIOASSAYED

(ACTIVE)

ACTIVE REGION (RF = O.4-O.52)

ION EXCHANGE CHROMATOGRAPHY

Ï ANIONIC ~T~

BIOASSAYED (NOT ACTIVE)

1

HPLC

Τ

ACTIVE FRACTION (TIME = 7-9, 9-11 MIN) SILYLATION GLC

Τ GLC-MS

Figure 1.

Flow diagram of separation technique.



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The remaining 240 ml of concentrated aqueous extract was adjusted to pH 2.5 with 2 N HC1 and partitioned three times with 240 ml of d i e t h y l ether and then 240 ml of ethyl acetate. The organic fractions were combined and dried for 2 hr over MgSO^ and then f i l t e r e d , dried i n vacuo and reconstituted with 80 ml of diethyl ether:ethyl acetate (1:1). The components i n the organic phase were separated f i r s t using thin-layer chromatography (TLC). The organic phase (80 ml) was streaked onto 20 plates of 1000 ym s i l i c a GF (Analtech, Inc., Newark, DE) and developed using toluene-ethyl formate-formic acid [(5:4:1), 41]. Compounds were then located with u l t r a - v i o l e t l i g h t (UV) at 250 nm. The compounds were eluted from the s i l i c a gel with ethyl acetate-methanol (2:1) followed by f i l t r a t i o n with Whatman #1 f i l t e r paper. The f i l t r a t e was dried i n vacuo and the residue dissolved i n 80 ml of methanol. Thirty ml of the above methanol solution which contained b i o l o g i c a l l y active compounds (R = O.4-O.52) was f i l t e r e d (O.4 ym m i l l i p o r e f i l t e r ) and dried i n vacuo. This material was then dissolved i n 15 ml of 35% aqueous methanol and components separated using high performance l i q u i d chromatography (HPLC). The instrument used was a Waters Associates HPLC (Milford, MA; Model 6000A pumping system, Model U6K i n j e c t o r , Model 480 Lambda-max LC spectrophoto meter) with a yBonddapak column (7.8 mm χ 30 cm). The solvent (35% aqueous methanol) was delivered i s o c r a t i c a l l y at a flow rate of 1.5 ml/min and the column effluent monitored at 350 nm (O.5 aufs for 6-30 min). Effluent was collected every 2 min with a t o t a l run time of 30 min (Figure 1). Each of the 15 fractions was dried i n vacuo and residue dissolved i n 15 ml of methanol and stored at 0°C. An extract blank ( i . e . , d i s t i l l e d water only) was also included throughout the extraction procedure to ensure that a l l isolated compounds were of b i o l o g i c a l o r i g i n . f

Qualitative Analysis. Extract samples (200 y l ) which had been p u r i f i e d by TLC (R = O.4-O.52) and HPLC (7-11 min) were dried at 40°C under N2 gas. T r i m e t h y l s i l y l (TMS) derivatives of samples were formed by adding 25 y l of N,0-bis(trimethylsilyl)trifluoroacetamide (BSTFA, Pierce Chemical Co., Rockford, IL) and 25 y l pyridine to a 1-ml reaction v i a l and heating to 70°C for 30 min (42). Deuterated t r i m e t h y l s i l y l derivatives (d-TMS) of samples were prepared by r e action with Deutero-regisil (Regis Chemical Co., Morton Grove, IL). The derivatized samples (1-4 y l ) were injected ( s p l i t l e s s ) into a 30-m (0-35-mm i.d.) DB-5 fused s i l i c a c a p i l l a r y column [95% dimethyl(5%)-diphenyl poly-siloxane, J and W S c i . , Inc., Rancho Cordova, CA]. A Hewlett Packard GC/MS 5985B was used to obtain retention times (t ), electron impact (EI) and chemical i o n i z a t i o n (CI) mass spectra of the derivatized samples and standard compounds. Helium was used as the c a r r i e r gas (ca. 1 ml/min) and methane as the makeup gas (15 ml/min) for CI runs. Chromatographic conditions were as follows: i n j e c t i o n port temperature, 250°C.; detector temperature, 270°C.; column temperature, 50°C for 2 min and then 8°C/min programmed to 280°C. A l l mass spectra were acquired at 70 eV. A Varian GC 3700 (Varian Associates, Sunnyvale, CA) was used to corroborate i d e n t i f i c a t i o n s by c o - i n j e c t i o n of samples and standard TMS derivatives. Bioassays. B i o l o g i c a l a c t i v i t y of various isolated compounds was determined using Chenopodium album L. seed collected i n North f


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Carolina i n 1981. Each sample (1.4 ml of methanolic sample) was placed into a 3-cm p e t r i dish and the solvent evaporated under a laminar flow hood at room temperature. Seventy seeds (O.035 g) were then placed into the p e t r i dishes and 1.4 ml of s t e r i l i z e d (O.2 umf i l t e r ) 15 mM Mes [2-(N-morpholino)ethanesulfonic acid; Sigma Chemical Co.] buffer adjusted to pH 5.5 was added. The dishes were kept i n the dark at 25°C for 84 hr, exposed to 12-hr fluorescent l i g h t (250 μ einsteins/m /sec), and then placed back i n the dark for an additional 4 days (17). Percent germination, root and hypocotyl lengths were then determined. The following chemicals were obtained commercially (Sigma Chemical Co.) and bioassayed with C. album and Amaranthus retroflexus L. (seeds collected i n North Carolina i n 1980) following i d e n t i f i c a tion: DL-3-hydroxybutyric acid (DL-3-hydroxy-butyric acid as a Na s a l t ) and L-3-phenyllactic acid (L-2-hydroxy-3-phenyl-propanoic acid). Data were subjected to analysis of variance and regression analysis using the general linear model procedure of the S t a t i s t i c a l Analysis System (40). Polynomial equations were best f i t t e d to the data based on significance l e v e l of the terms of the equations and R.2 value.


2

Wheat Mulch Studies Extraction Procedure. Wheat ('McNair 1813') plant material used i n t h i s study was harvested from the f i e l d i n early spring after the wheat had t i l l e r e d but before heading. The plant material was dried at 50°C for 48 hr. Five-g samples of wheat plant material were soaked i n 150 ml of water for 10 hr a t room temperature. The mixture was f i l t e r e d and the f i l t r a t e used to germinate pitted morningglory (Ipomoea lacunosa L.) and ragweed (Ambrosia a r t e m i s i i f o l i a L.) seed. The effect of the extract on germination was tested i n the presence and absence of light. Extractions were done by the procedure of Guenzi and McCalla (22). Five-g wheat samples were hydrolyzed with 2 Ν NaOH f o r 4 hr at room temperature. The a l k a l i n e extract was f i l t e r e d and the f i l t r a t e a c i d i f i e d to pH 2 with concentrated HC1 and extracted with d i e t h y l ether. The concentrated ether f r a c t i o n was used to spot thin-layer chromatography (TLC) plates. S i l i c a g e l G obtained from Sigma Chemical Company was used. The plates were developed i n a benzene-methanol-acetic acid system (80:10:5 v/v/v). Compounds separated by one-dimensional TLC were detected by exposing the developed plates to long-wave (3360 A) UV l i g h t . The isolated compounds noted under UV l i g h t were i n d i v i d u a l l y scraped from the TLC plates and then bioassayed for morningglory seed germination inhibition. I d e n t i f i c a t i o n . One compound isolated by TLC was found to be very i n h i b i t o r y to morningglory seed germination and was i d e n t i f i e d using mass spectrometry. A LKB 2091 GC-MS was used f o r GC-MS analysis. In addition to GC-MS, the sample was also analyzed by d i r e c t probe.


THE C H E M I S T R Y OF A L L E L O P A T H Y

252


Bioassay for Toxicity of Inhibitory Compound(s). Since the i d e n t i t y of the i n h i b i t o r y compound was determined to be f e r u l i c acid (4-hydroxy-3-methoxycinnamic a c i d ) , f e r u l i c acid obtained from Sigma Chemical Company was used i n germination bioassays. F e r u l i c acid solutions of 5.0, 1.0, and O.5 mM buffered to pH 6.5 with sodium phosphate were used to germinate morningglory, p r i c k l y sida, ragweed, crabgrass ( D i g i t a r i a sanguinalis L . ) , corn, and soybean (Glycine max L. Merr.). The weed and crop seed were germinated i n 9-cm P e t r i dishes. To each dish, 10 ml of test solut i o n was added. At the termination of the bioassay (4 days), percent germination and root length data were taken.

RESULTS AND DISCUSSION Rye Mulch Studies F i e l d Research. Rye mulch (aboveground herbage) caused a s i g n i f i cant increase i n the percent control of grass species even when the soil was t i l l e d (cut mulch, glyphosate and paraquat desiccation and t i l l and replace mulch versus no rye t i l l or n o - t i l l (Table I ) . Rye root residue also caused a s i g n i f i c a n t improvement i n grass control although not up to the l e v e l provided by the addition of rye mulch. Soil disturbance did not s i g n i f i c a n t l y a f f e c t the l e v e l of grass control (no rye, t i l l versus n o - t i l l ) which is d i f f e r e n t than the response of broadleaf weeds. A l l three broadleaf weeds, redroot pigweed, common lambsquarters and Ambrosia a r t e m i s i i f o l i a (common ragweed) responded to t i l l a g e i n the same m a n n e r — t i l l a g e caused an increase i n density and biomass (Table II) and a decrease i n percent weed control [Table I (no rye, n o - t i l l versus t i l l ) ] . Many weed species are known to respond p o s i t i v e l y to soil t i l l a g e Ç9, 15, 16). At least part of the reason is that t i l l a g e provides a l i g h t stimulus which is a requirement f o r many species (18, 19). Common lambsquarters seed collected i n North Carolina were shown to respond p o s i t i v e l y to l i g h t both q u a l i t a t i v e l y and quantitatively (data not shown). The requirement of l i g h t for the germination of common lambsquarters has been reported previously (17). The effect of t i l l a g e ( i . e . , increase i n biomass, density and decrease i n percent weed control) was eliminated by placing rye mulch on t i l l e d soil—no rye t i l l versus t i l l and mulch replaced (Table II). The reduction i n biomass of common lambsquarters and redroot pigweed was greater than for common ragweed; 96, 78, and 39%, respectively. Also note that the density of only redroot pigweed was s i g n i f i c a n t l y reduced by replacement of the rye mulch. In other words, the mulch caused a tremendous decrease i n the size of common lambsquarters and ragweed but not a reduction i n numbers. The broadleaf weeds responded to t i l l a g e i n the same general manner when rye root residue was present, however, i t does appear that the growth of a l l the weeds was inhibited by the presence of rye root residue. This can be seen i n Table II by comparing r e s u l t s from the t i l l e d treatment with and without rye root residue. In the case of common ragweed, biomass was reduced 34% (178.3 g no rye, t i l l versus 118.3 remove mulch t i l l ) , but the density increased by 69% (140 p l a n t s / 2 . 2 m versus 280 p l a n t s / 2 . 2 m ) when rye root 2

2



33c 73a 43bc

80a 73a lOd 55b

60cd 84a 70bc

65bc 77ab lOf 32e

58ab 69a 63 ab

74a 79a 8c 41b

71bc

91a

82ab

84ab

81ab

28d

33d

Rated 49 days a f t e r planting.

c

^Sunflower and soybeans only.

Large crabgrass and f a l l panicum; rated 31 days a f t e r planting.

b

^ e a n s within a column followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t as determined by Waller-Duncan T-test (K r a t i o = 100).

68a

49d

55ab

60c

Remove mulch, no t i l l Remove mulch, till Cut mulch, no-till Remove mulch, t i l l , replace mulch Glyphosate, no-till Paraquat, no-till No rye, till No rye, no-till

Common Ragweed

Grass*

5

Q

Common Lambsquarters

% Weed Control'

Treatment

Redroot Pigweed

C,d

Table I. The Effect of T i l l a g e and Rye Residue (Above- and Belowground) on Weed Control Averaged Across Three Cropping Systems at Clayton, NC, i n 1982 and 1983



a

O.4c O.2c

2c

lc

5

2

41b

140a

5b

5b

120a

7b

132a

25b

(plants/2.2m )

Density*

Common Lambsquarters C

124.3b

199.9a

25.9d

44.3d

44.8d

32.4d

117.8bc

65.6cd

(g)

AGB 5

2

15c

140b

16c

9c

186ab

17c

236a

21c

(plants/2.2m )

Density*

Common Ragweed

AGB:

Aboveground biomass.

AGB and densities determined 49 days after test i n i t i a t i o n .

Sunflower and soybeans only.

C

34.7c

178.3a

12.3c

5.9c

108.2b

29.9c

118.3b

34.0c

(g)

AGB

^ e a n s within a column followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t as determined by Waller-Duncan T-test (K r a t i o = 100).

12.0ab

O.8bc

3bc

21ab

O.4c

2c

22.1a

O.8bc

4bc

26a

2.4bc

8 abc

(8)

Remove mulch, no-till Remove mulch, till Cut mulch, no-till Remove mulch, t i l l , replace mulch Glyphosate, no-till Paraquat, no-till No rye, till No rye, no-till

2

AGB°

(plants/2.2m )

5

Treatments

Density*

Redroot Pigweed

Table I I . The Effect of T i l l a g e and Rye Residue (Above- and Belowground) on Weed Biomass and Density Averaged Across Three Cropping Systems at Clayton, NC, i n 1982 and 1983



17.

SHILLING ET AL.

Rye and Wheat Mulch

255

residue was present i n the t i l l e d soil. Common lambsquarters responded to t i l l a g e i n the presence of rye root residue i n the same manner as common ragweed, i n that biomass was decreased but the density was unaffected. Redroot pigweed appears to be the most inhibited by rye root residue i n that both density and biomass were reduced i n the t i l l e d treatment. The growth of common lambsquarters and redroot pigweed was s i g n i f i c a n t l y reduced by rye root residue i n the n o - t i l l system as well (no rye, n o - t i l l versus remove mulch, n o - t i l l ) . Thus, both common lambsquarters and redroot pigweed are affected by both rye mulch and root residue, but common ragweed appears to be only moderately affected by rye residues. However, a l l weed species observed responded to soil disturbance. Overall, the elimination of t i l l a g e and the presence of rye (cut mulch, n o - t i l l versus no rye, t i l l ) caused a decrease i n the biomass of redroot pigweed, common lambsquarters and common ragweed of 96, 84, and 83%, respectively. The majority of the reduction i n common ragweed was due to the elimination of t i l l a g e and not the presence of rye. This is contrary to the findings of Putnam et a l . (13) who reported that common ragweed was inhibited by rye mulch. Growth Chamber Tests. The extraction of f i e l d grown rye with water was an i n i t i a l attempt to determine the a l l e l o p a t h i c potential of rye simulating a natural release mechanism ( i . e . , r a i n ) . Common lambsquarters was used because i t seemed to be the most sensitive weed species of the indigenous weeds observed. As shown i n Figure 2, common lambsquarters emergence (density) was s i g n i f i c a n t l y inhibited by aqueous rye extracts at the higher concentration. Low concentrations of known a l l e l o p a t h i c compounds are known to cause stimulation of weed species (26). The rye extract caused a s i m i l a r response from common lambsquarters. Another explanation f o r the stimulation at low concentrations could be that the extract provided additional nitrogen for the common lambsquarters. It should be pointed out that various measurements were taken i n the f i e l d during the two years of research to determine which growth parameters (light and soil temperatures) were affected by these treatments. The degree to which each parameter was affected was then used to e s t a b l i s h laboratory l i m i t s , within which their possible role i n explaining the observed effects was evaluated. Of those tested, l i g h t and allelopathy appear to be the most probable causes of the observed e f f e c t s . The implications of these studies are f i r s t that agro-ecosystems could be manipulated to b i o l o g i c a l l y reduce c e r t a i n weed pressures. And, second, chemicals present i n mulch and/or root exudates could be of p r a c t i c a l significance i n terms of new herbicide chemistry. Wheat Mulch Studies F i e l d Studies. Study 1. Morningglory plant populations and biomass were found to be greatly affected by both primary t i l l a g e and wheat mulch. In general, t i l l a g e greatly increased densities of morningglory while the presence of a wheat mulch had the tendency to reduce morningglory growth. Morningglory populations consisted of a mixture of pitted (I. lacunosa L.) and t a l l ( I . purpurea L. ) species.




256

—ι

1 1 1:60

1

1 1:30

1

ι

1

1:20

Concentration of rye extract (RE) (g/ml)

95°/o mean confidence intervals

a

"Emergence = 19.75 + 974.52 (RE)-54,295.51 (RE) + 562,102.0 (RE) 2

Figure 2.

The e f f e c t

3

of an aqueous extract of rye on the emergence

of Chenopodium album.



17.

SHILLING ET AL.

Rye and

257

Wheat Mulch

In the corn test (Table III), the non-mulched t i l l e d treatment accumulated an average of 302% more morningglory biomass than nonmulched n o n - t i l l e d plots. The t i l l a g e e f f e c t , however, was essent i a l l y eliminated when t i l l a g e was followed by replacement of the wheat mulch. Morningglory growth i n the t i l l e d and replaced plots was similar to that observed i n the n o n - t i l l e d treatments. In the n o n - t i l l e d treatments, there was l i t t l e effect of the wheat mulch on morningglory biomass. Enhanced soil moisture under the mulch may have compensated f o r the reduced numbers of morningglory plants i n i t i a l l y observed i n the n o n - t i l l e d mulch treatment as compared to the n o n - t i l l e d non-mulched plots. In the soybean test (Table IV), there was no e f f e c t of the wheat mulch on morningglory growth. Age differences of the mulch between the two tests may have accounted for the lack of weed control. Wheat at the time of treatment was f u l l y mature i n the soybean test and green i n the corn test. Researchers have shown that straw cut while s t i l l green produced a higher l e v e l of phytotoxicity than those cut when f u l l y mature. In addition, r a i n f a l l required to leach toxins from the mulch into the soil may have been lacking when soybeans were planted since the growing season i n North Carolina i n 1981 was generally dry. Although a mulch effect on morningglory growth i n the soybean test was not observed, t i l l a g e increased morningglory biomass 269% over n o n - t i l l e d treatments at the Clayton location (Table IV). In addition to morningglory, similar trends were observed f o r cocklebur (Xanthium pensylvanicum Wallr.). Higher cocklebur populations were observed i n t i l l e d plots than i n n o n - t i l l e d plots (data not shown). Table I I I . Effect of T i l l a g e and Wheat Mulch on Morningglory Populations and Biomass i n Corn a

Morningglory 2b Dry wt. gm/7 . 4 Plants/7.4m Clayton Plymouth Clayton Plymouth 2c

Remove mulch, n o - t i l l Remove mulch, t i l l Remove mulch, t i l l , replace N o - t i l l into mulch

4.0b 72.4a 3.2b 3.4b

245.7ab 346.8a 86.4b 31.7b

13.7b 157.0a 30.7b 37.5b

73.0b 192.2a 63.1b 34.6b

Within each column, values sharing the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at the O.05 l e v e l , according to Duncan's multiple range test. u

Counts made 25 days a f t e r planting. Biomass obtained 45 days after planting.

No deleterious effects of either the wheat mulch or t i l l a g e were observed on soybeans (Table V) or corn (data not presented). In general, crop growth was better i n mulched or n o - t i l l plots. Enhanced soil moisture i n the mulched treatments and reduced morningglory densities i n the n o n - t i l l e d treatments may have contributed to improved crop growth.


258


Table IV.

Effect of T i l l a g e and Wheat Mulch on Morningglory populations and biomass i n soybeans Morningglory 2b

Plants /7.4m Clayton Plymouth


Remove mulch, n o - t i l l Remove mulch, t i l l Remove mulch, t i l l , replace N o - t i l l into mulch

15.8ab 24.0ab 9.0b 28.0a

1.0c 71.5a 44.0b 1.5c

2c

Dry wt. gm/7.4 Clayton Plymouth 58.9b 253.7a 207.1a 65.8a

253, ,4a 194, .la 202, ,5a 262, .9a

Within each column, values sharing the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at the O.05 l e v e l , according to Duncan's multiple range t e s t . Counts made 30 days after planting. Biomass obtained 90 days after planting. Table V.

Soybean biomass as influenced by t i l l a g e and wheat mulch 2b

Soybean fresh wt. Kg/6.0m Clayton Plymouth Remove mulch, n o - t i l l Remove mulch, t i l l Remove mulch, t i l l , replace N o - t i l l into mulch

7.82b 7.91b 9.98ab 10.21a

11.93ab 10.57b 10.57b 12.93a

a

W i t h i n each column, values sharing the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at the O.05 l e v e l , according to Duncan's multiple range test. u

Biomass obtained 90 days after planting.

Study 2. The effects of wheat, oats, barley and rye mulches on three broadleaf weed species and crabgrass ( D i g i t a r i a spp.) are shown i n Table VI. Weed control data for the corn test at Kinston are not presented because of poor cover crop k i l l by the paraquat treatment. In both the corn and soybean tests, there were no appreciable differences i n the broadleaf weed control among the four small grain mulches. In addition, age of the small grain plant material at planting time had e s s e n t i a l l y no effect on weed control. The reduction i n weed control by the older wheat straw i n the soybean test of Study 1 was not observed here. Broadleaf weed control i n the small grain stubble and residue of the soybean test was similar to the control observed with the younger mulches present i n the corn t e s t . The reasons for the observed differences between the two studies are unclear. Perhaps i t is because weed densities are so greatly reduced by eliminating t i l l a g e the presence of a mulch would have l i t t l e influence. This is probably why i n the corn test of Study 1 a reduction i n weed growth by the mulch was only observed i n p l o t s that were t i l l e d followed by replacement of the wheat mulch.



84ab 83a-c 79a-c 66a -d 20e

41c-e 65a-b 61a-d 41c-e 90ab

81a-d 88ab 88ab 68a-d 49b-e

85a-c 85a-c 90ab 80a-c 96a

60c-e 53de 84a-c 50de 41e

81a 81a 89a 28 cd 3d

69ab 68 ab 88a 35c 74a

74a

77a

82a

39c

36c

Oats

Barley

Rye

No cover, n o - t i l l

No cover, t i l l e d

Ratings conducted 35 days a f t e r planting.

Ratings conducted 95 days after planting.

Corn test at Kinston abandoned due to poor cover crop k i l l with paraquat.

b

c

d

Within each crop, and within each location, values sharing the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at the O.05 l e v e l , according to Duncan's multiple range t e s t .

85ab 35de

93a

71a-d

68b-e

83a

Prickly Sida

76a

Corn Rocky Mount MorningCrabgrass glory

78a

Prickly Sida

Kinston SickleCrabgrass pod

Wheat

Small grain Mulch

Rocky Mount CrabMorninggrass glory

Soybean*

5

Table VI. Effect of Small Grain Mulch and T i l l a g e on Weed Control i n Corn and Soybeans



260


When broadleaf weed control ratings were averaged over location, crop, and weed species [morningglory, p r i c k l y sida (Sida spinosa L.), and sicklepod (Cassia o b t u s i f o l i a L>)], weed control i n the n o - t i l l mulched plots was 37 and 62% better than the control achieved i n the non-mulched n o n - t i l l e d and the non-mulched t i l l e d treatments, respectively (Table V I I ) . Of the three broadleaf weed species studied, the growth of p r i c k l y sida was influenced the most by mulch and t i l l a g e treatments. The finding that f e r u l i c acid leached from small grain straws is converted to a more phytotoxic styrene derivative by bacteria on the carpels of p r i c k l y sida seed may account f o r the greater reduction of this weed by small grain residues (26). Crabgrass control i n the mulched plots was generally lower than the control of broadleaf species, despite the alachlor treatment at planting time (Table VI). Poor crabgrass control i n the n o n - t i l l e d treatments may have been due to the low early season r a i n f a l l which reduced the effectiveness of alachlor. Table VII. Mean broadleaf weed control of mulched and unmulched treatments averaged over location, crop, and weed species a

Small grain mulch Wheat Oats Barley Rye No cover, n o - t i l l No cover, t i l l e d ^ e e d species include:

^ Weed control 81.0a 76.0a 76.1a 84.1a 50.0b 29.8c morningglory, p r i c k l y sida and s i c k l e -

pod. Values sharing the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at the O.05 l e v e l , according to Duncan's multiple range test. As i n Study 1, no adverse effects of either mulch or t i l l a g e were observed on corn or soybean growth. Small grain mulches studied here d i d suppress weed growth, but equally important is the role of t i l l a g e . Eliminating t i l l a g e reduced densities of morningglory, p r i c k l y sida and sicklepod. When a soil is t i l l e d , environmental factors w i l l change which may enhance weed seed germination. Of these factors, exposure of weed seed to l i g h t may be the most important (19), even though morningglory germination studied here was not influenced by l i g h t (data not shown). For the most part, weed control is achieved mainly through c u l t u r a l , mechanical, or chemical practices. Because weed control options are limited with the adoption of minimum t i l l a g e cropping practices, the d i f f i c u l t y of c o n t r o l l i n g weeds is often c i t e d as a problem i n n o - t i l l crop production (8). However, environmental and ecological differences which d i s t i n g u i s h n o - t i l l from conventional t i l l a g e may benefit growers by enhancing control of c e r t a i n weed species i n n o - t i l l cropping systems. With the proper choice and management of cover crops and plant residues, i t may be possible to supplement i f not reduce the number and amount of herbicides used


17.

SHILLING E T A L .

Rye and

Wheat Mulch

261

i n these cropping systems by eliminating t i l l a g e which r e s t r i c t s weed seeds to poor germination s i t e s and by u t i l i z i n g natural phyto toxic substances leaching from plant residues.


Isolation and I d e n t i f i c a t i o n of

Phytotoxins

Rye Mulch Studies. Extraction of the dried aqueous extract (Figure 1) with methanol gave a preparation which showed the greatest a c t i v i t y , giving 58, 80 and 86% i n h i b i t i o n of C. album hypocotyl length, root length and germination, respectively (data not shown). Therefore, methanol was used for a l l subsequent transfers. The a c t i v i t y i n the aqueous extract was also e f f e c t i v e l y p a r t i tioned into a d i e t h y l ether organic phase. At this step, r e l a t i v e i n h i b i t i o n increased compared to the methanol extract. The organic phase extract caused 96, 93, and 89% i n h i b i t i o n of C. album hypocotyl length, root length, and germination, respectively (data not shown). "Semi-preparative HPLC" was then used to further purify the extract. Instrumentation and techniques were the same as described previously. Active compounds were contained i n the 7-11 min f r a c t i o n (data not shown). 3-phenyllactic acid (3PLA) and 3-hydroxybutyric acid (3HPA) were i d e n t i f i e d i n the HPLC active f r a c t i o n by comparing retention time ( t ) and mass spectra with those of standard compounds. Chem i c a l i o n i z a t i o n (CI) of the TMS derivatives and electron impact (EI) of the TMS and d-TMS derivatives indicated a MW of 248 for one of the unknown compounds and 310 for another unknown (Table V I I I ) . The absence of molecular ions (M ) i n TMS derivatives of hydroxy acids has been previously reported (43) . By comparing the EI spectra of TMS and d-TMS derivatives, the number of TMS groups was also deter mined. In the case of 3PLA, the difference i n mass between the TMS derivative (295) and the d-TMS (310) was 15. If the M+ was present i n the spectra, the mass difference (TMS vs. d-TMS) would have been 18 (Table VIII). Thus, the CI and EI spectra give the M+ and the number of s i l y l a t e d functions (Table V I I I ) . Once t h i s was determined, EI spectra of TMS derivatives were compared to standard spectra (44, 45) i n the l i t e r a t u r e . Standard spectra were then generated for 3ΡΙΆ and 3HBA. In so doing, t of the unknown compounds was also matched to standard compounds. AÏso, c o - i n j e c t i o n of TMS derivatives of standards and samples was used to further substantiate the i d e n t i fications. r

+

B i o l o g i c a l A c t i v i t y of Identified Compounds. Both 3HBA and 3PLA inhibited C. album hypocotyl length (HL) at 8 mM by 30 and 68%, respectively (Figure 3). It is probable that t o t a l seedling growth would be more s e n s i t i v e . Only 3P1A s i g n i f i c a n t l y decreased HL at lower concentrations (17% at 4 mM). Both compounds also inhibited C. album root length (RL) [Figure 4]. There was not a s i g n i f i c a n t difference i n effect on RL between the two compounds, although 3PLA caused s i g n i f i c a n t i n h i b i t i o n at a lower concentration (20% at 2 mM) as compared to the c o n t r o l . Hypocotyl length of A. retroflexus was more s e n s i t i v e to 3PLA i n h i b i t i o n than C. album (Figures 3 and 6). Hypocotyl length of C. album was inhibited by 17% at 4 mM. With A. r e t r o f l e x u s , O.8 mM caused a 17% i n h i b i t i o n of HL. Thus, 3PLA was 5X more inhibitory on hypocotyl growth of A. r e t r o f l e x u s . Also, 3PLA completely



a

^Compared to corresponding

—

—

—

a

a

+ M d-TMS

standards.

Not present i n mass spectra.

—

(2) 3-Phenyllactic acid

a

TMS

—

a

r

295

233

310

248

~T M -15 TMS d-TMS

311

249

M+l

339

277

351

289

CI (TMS) M+29 M+41

166

104

MW

b

l

,b 24.3

16.l

r

fc

Tabulation of Important Ions Present i n Mass Spectra, GC Retention Times (t ), Molecular Weights of Unknown and Standard Compounds and Number of T r i m e t h y l s i l y l (TMS) Groups per Molecule

(1) B-hydroxybutyric acid

Chemical

Table VIII.


2

2

No. of TMS groups

X

?

Ο τι > r r m r Ο

H ?

g

m

χ

η

m

Κ)

ON

17.

Rye and Wheat Mulch

SHILLING ET AL.


/5HBA

D

Concentration (mM) "95% confidence intervals. b

HL = 3.15-O.13 (0ΗΒΑ), F = 50.41 P>FO.0001R = O.84 2

°HL = 2.80-O.23 ( 0 P L A ) , F = 73.51 P>FO.0001R = O.85 2

Figure 3 .

The effect of DL-3-hydroxybutyric acid phenyllactic acid

(3PLA) on

(3HBA) and L - 3 -

album hypocotyl

length

(HL).

24

H

2.1 1.8 1.5 D) C Φ

Ο

ο oc

1.2 O.9 O.6 O.3 0

Concentration (m M) "95% mean confidence intervais. 2

°RL = 2.02-O.15 ( £HBA), F = 22.31 P>F O.0001 R = O.51 C

Figure 4 .

2

RL = 2.30-O.24 (£PLA), F = 179.75 P>F O.0001 R = O.95

The effect of DL-3-hydroxybutyric acid (3HBA) and L-3" phenyllactic acid (3PLA) on C. album root length (RL).



264


inhibited hypocotyl length of A. retroflexus at 8 mM (Figure 5). There was l i t t l e difference i n response between the two weed species to 3HBA (a 30% i n h i b i t i o n at 8 mM for C. album versus 27% f o r A. r e t r o f l e x u s ) . There was no s i g n i f i c a n t interaction effect on HL between the two compounds (F = O.97, Ρ > F O.50). The difference i n s e n s i t i v i t y of the weed species to the two compounds could have been due to the duration of the experiments (8 days for C^. album versus 60 hr for A. r e t r o f l e x u s ) . The longer duration may have resulted i n a lower concentration of the compound because of microbial degrada tion. Amaranthus retroflexus RL was s i g n i f i c a n t l y inhibited by both compounds (Figure 6). There was no s i g n i f i c a n t difference i n i n h i b i t i o n of C^. album root growth. This again indicates species s p e c i f i c i t y i n response to the two compounds. The effect on A. retroflexus RL indicated a s i g n i f i c a n t interaction between the two compounds. This interaction was antagonistic, as the combined i n h i b i t i o n by both compounds was less than the sum of the individual i n h i b i t i o n . At 2 mM 3PLA alone reduced RL by 59% and 3HBA alone reduced RL by 33%. However, i n combination the two compounds reduced RL by 62%, as opposed to an expected i n h i b i t i o n of 92% ( i . e . , 59 + 33 - 92%) had the interaction been a d d i t i v e . A l i p h a t i c acids such as butyric acid have been previously implicated as being a l l e l o p a t h i c compounds (46, 47, 23). Chou and Patrick (23) isolated butyric acid from soil amended with rye and showed that i t was phytotoxic. Hydroxy acids have also been shown to possess phytotoxic properties (48) but have not been implicated i n any a l l e l o p a t h i c associations. Since 3HBA is a stereo isomer, and the enantiomer was not i d e n t i f i e d because of impurity, a l l bioassays were run using a racemic mixture. The D-(-) stereo isomer of 3HBA has been isolated from both microorganisms and root nodules of legumes and is suspected to be a metabolic intermediate i n these systems (49). It is l i k e l y that only one enantiomer was present i n the extract; therefore, the true phytotoxic p o t e n t i a l of t h i s com pound awaits c l a r i f i c a t i o n of the phytotoxicity of the individual enantiomers. Most of the simple aromatic acids which have been implicated i n a l l e l o p a t h i c associations are derived from either cinnamic acid (in the case of phenolic acids) or benzoic acid (47). Therefore, 3PLA is unique among a l l e l o p a t h i c aromatic acids i n that i t is believed to be an intermediate (although of minor importance) i n the shikimic acid pathway and not an end product (5£, 51, 52^ 53). Although 3PLA is a stereo isomer and the exact enantiomer was not i d e n t i f i e d i n this study, Tamura and Chang (54) and Kimura and Tamura (55) isolated and i d e n t i f i e d L-3PLA from two fungal species and demonstrated plant growth regulator a c t i v i t y . These workers also showed that L-3PIA affected hypocotyl and root growth of lettuce, although i n both cases growth was stimulated i n this species. They also reported that D-3PLA did not a f f e c t growth as much as the L form. This could explain why DL-3PLA did not a f f e c t the growth of the bioassay species i n this study (data not shown). It is possible that both 3HBA and 3PLA were of microbial o r i g i n as most of the reports on these com pounds deal with plant microorganism associations. No d e f i n i t e conclusions can be drawn as to the potential use of these r e s u l t s i n explaining a l l e l o p a t h i c associations under f i e l d



17.

SHILLING ET A L .

265

Rye and Wheat Mulch

Concentration (mM) *95% mean confidence intervals.

Figure 5.

b

H L = 1.93-O.09 ( I H B A ) , F = 24.26 Ρ > F O.0003 R = O.65

C

HL = 1.82-O.39 ( β PLA) + O.02 (/?PLA) , F = 124.09 P > F O.0001

2

2

The e f f e c t of DL-3-hydroxybutyric acid (βΗΒΑ) and L - 3 ~ phenyllactic acid (3PLA) on A. retroflexus hypocotyl length (HL). 2.4 τ

τ 0

1 O.5

1 1.0

1 1.5

1 2.0

1 2.5

1 3.0

1 3.5

1 4.0

Concentration (mM) "Equal molar concentration of both compounds: RL • 1.99-O.34 ( 0 H B A ) - O . 1 4 (β PLA) -O.23 (β PLA) + O.16 (βΗΒΑ Χ β PLA), F = 47.78 P > F O.0001 R = O.82 2

2

b 9 5 % mean confidence intervals. °RL = 1.75-O.40 (0HBA)-O.29 (βΗΒΑ) , F = 19.36 P > F O.0001 R = O.68 2

2

d

Figure 6.

R L = 2.10-O.85 (#PLA) + O.11, F = 83.07 P > F O.0001 R = O.90. 2

The effect of DL-3-hydrοxybutyric acid (3HBA) and L - 3 phenyllactic acid (3PLA) i n d i v i d u a l l y and i n combination on A. retroflexus root length (RL).


266



conditions. However, both 3PLA and 3HBA are easily extractable by water and thus could be r e a d i l y leached from rye mulch to i n h i b i t weed growth. Wheat Mulch Studies. The effect of the aqueous wheat extract on morningglory and ragweed germination is shown i n Table IX. In the dark, the presence of the wheat extract reduced morningglory germi nation and root length 27 and 66%, respectively. The wheat extract did not, however, s i g n i f i c a n t l y reduce ragweed germination i n the dark, but did reduce ragweed root length 86%. Ragweed was chosen i n t h i s test because l e v e l s of this weed have not been observed to be reduced by n o - t i l l cropping practices. Greater phytotoxicity was observed when the bioassays were conducted i n the presence of l i g h t . The enhanced phytotoxicity due to l i g h t was p a r t i c u l a r l y apparent on ragweed. Ragweed seed wetted with the wheat extract and germinated i n the presence of l i g h t did not germinate, yet l i g h t alone had a s l i g h t stimulatory effect on ragweed germination. Morningglory germination and root length were reduced 65 and 62%, respectively, when seed wetted with the wheat extract were germinated i n the l i g h t . Table IX.

Effect of Light on Morningglory and Ragweed Germination with Aqueous Wheat Extract Morningglory Germination (%)

Treatment Dark + extract Dark check Light + extract Light check

53 73 30 85

b a c a

a

Ragweed

Root Length (cm) 1.5 4.4 1.0 2.6

c a c b

Germination (%) 29 34 0 45

b b c a

Root Length (cm) 1.1 7.6 O.0 3.0

c a d b

V a l u e s within columns sharing the same l e t t e r are not s i g n i f i cantly d i f f e r e n t according to the Waller-Duncan procedure, assuming a Κ r a t i o of 100. Reprinted with permission of Plenum Publishing Corp. (28). Isolation of Inhibitory Compound. The hydrolyzed extract of wheat straw yielded many compounds when subjected to TLC separation. When individual spots were removed and bioassayed for morningglory seed germination, only two of the spots s i g n i f i c a n t l y inhibited ger mination (Table X). Of the two i n h i b i t o r y compounds, the compound with R^ O.5 was the more i n h i b i t o r y . This compound reduced morningglory germination and root length 94 and 89%, respectively. Although the substance at Rf O.5 was found to be the most i n h i b i t o r y , concen t r a t i o n differences of the various components i n the ether extract may have been p a r t i a l l y responsible f o r the greater i n h i b i t i o n of the test solution containing the compound at R^ O.5. I d e n t i f i c a t i o n of Inhibitory Compound. Of the two compounds isolated by TLC that had a c t i v i t y on morningglory germination, only the one at R O.5 on the TLC plate was analyzed by GC-MS. From the f


17. SHILLING ET AL.

Rye and

267

Wheat Mulch

Table X. Effect of Compounds Isolated from Wheat by TLC on Morningglory Seedling Growth R.£ value

Germination


O.5 O.95 Check

(%)

Root length

6 c 67 b 94 a

(cm)

O.3 c 1.5 b 2.7 a

V a l u e s within columns sharing the same l e t t e r are not s i g n i f i cantly d i f f e r e n t according to the Waller-Duncan procedure assuming a Κ r a t i o of 100. Reprinted with permission of Plenum Publishing Corp. (28). mass spectral data, the i d e n t i t y of the compound at R determined to be f e r u l i c a c i d .

O.5

f

was

B i o l o g i c a l A c t i v i t y of Identified Compound, The effect of f e r u l i c acid on the germination and root length of morningglory, ragweed, p r i c k l y sida, and crabgrass is shown i n Table XI. Signifi cant reductions i n germination or root length of morningglory, crab grass, and p r i c k l y sida seed enclosed i n carpels were observed only at the highest concentration of f e r u l i c acid. F e r u l i c acid, however, had no effect on either ragweed or p r i c k l y sida seed with carpels removed. Of the four weed species bioassayed with f e r u l i c acid, crabgrass appeared to be the most sensitive. No crabgrass germina tion was observed at the high concentration of f e r u l i c acid. None of the concentrations of f e r u l i c acid used had any e f f e c t on the germination of corn or soybean (Table XII). F e r u l i c acid d i d , how ever, i n h i b i t the root growth of both corn and soybean at the high est concentration. Table XII. Influence of F e r u l i c Acid on Germination of Corn and Soybean Seed Corn

F e r u l i c acid 5 χ 10";? 1 χ 10_^ 5 χ 10 Check LSD (O.05)

(M)

Soybean

Germ. (%)

Root length (cm)

Germ. (%)

Root length (cm)

68 83 85 83 NS

O.9 3.7 3.3 2.9 1.2

63 65 70 82 NS

2.7 2.7 3.7 4.4 1.2

Reprinted with permission of Plenum Publishing Corp. (28). Of the many types of phytotoxic compounds released from decay ing plant material by microbial a c t i v i t y or leaching, the phenolic acids are probably the most common (47, 56). F e r u l i c acid, as well as other phenolic acids, is produced from intermediates of



75

15

Check

LSD (O.05)

O.6

1.7

1.8

NS

27

27

29

26

34

30

19

1.6

NS

29

0

O.3

1.1

1.0

O.9

0

Crabgrass Root Germ. Length (cm) (%)

1.6

1.6

1.5

Ragweed Root Length Germ. (cm) (%)

Reprinted with permission of Plenum Publishing Corp. (28).

65

1.4

68

3

1 χl(f

-4 5 χ 10

O.3

58

3

5 χ 10"

Ferulic Acid (M)

Morningglory Root Germ. Length (cm) (5)

NS

19

15

18

16

1.7

20

9

1.6

NS

O.3

2.1

1.7

O.3

21

13

3

1.8

1.6

1.4

P r i c k l y sida With Carpel Without Carpel Root Root Germ. Length Germ. Length (cm) (cm) (%) (%)

Table XI. Effect of F e r u l i c Acid on Morningglory, Ragweed, Crabgrass, and P r i c k l y Sida Seed Germination



17.

SHILLING ET A L .

Rye and

Wheat Mulch

269

respiratory metabolism v i a the shikimic acid pathway. F e r u l i c acid has been found i n a v a r i e t y of crop residues by a number of researchers (22, 57_, 58). The reported i n h i b i t o r y effects of f e r u l i c acid on germination and seedling growth have varied widely. Borner (59) found that a f e r u l i c acid concentration as low as 10 ppm inhibited the growth of wheat and rye roots. Guenzi and McCalla (22), however, reported that a 2500 ppm solution of f e r u l i c acid had no e f f e c t on the germination of wheat and reduced the growth of wheat roots by only about 50%. In work with soybean seedlings, Patterson (25) showed that soybean t o t a l dry weight, leaf area, plant height, and number of leaves were a l l s i g n i f i c a n t l y reduced when soybean plants were grown i n solution culture containing 194 ppm of f e r u l i c a c i d . Feru l i c acid had no effect on soybean growth when the f e r u l i c acid concentration was reduced to 19.4 ppm. Lodhi (57) reported that a f e r u l i c acid concentration of 194 ppm was very i n h i b i t o r y to the seed germination and r a d i c l e growth of radish (Raphanus sativus L . ) . With the extraction procedure we employed (22), f e r u l i c acid was isolated as the most i n h i b i t o r y component i n wheat straw. There could also be other unknown compounds i n the straw which would not be evident with t h i s procedure. In addition, we ignored the possible influence of toxin-producing microorganisms. Microorganisms may have influenced the phytotoxicity exhibited by the aqueous wheat extract in Table IX. Although the present study was not concerned with the phytotoxic e f f e c t s of m i c r o b i a l l y decomposed wheat straw, an i n f l u ence of microbial a c t i v i t y on f e r u l i c acid phytotoxicity was observed. From the results shown i n Table XI, i t appears that the presence of the p r i c k l y sida seed carpel enhanced the i n h i b i t o r y e f f e c t s of f e r u l i c acid. In addition to f e r u l i c acid i n test s o l u tions containing p r i c k l y sida seeds with carpels, a second compound, 4-hydroxy-3-methoxy styrene, was also found to be present. This compound is formed by the decarboxylation of f e r u l i c acid and was produced by a bacterium present on the carpel of p r i c k l y sida seed. The decarboxylation of f e r u l i c acid was detected i n aqueous solutions of f e r u l i c acid inoculated with the bacterium isolated from the carpels of p r i c k l y sida seed. No conversion occurred when the bacterium was not present. It seems most l i k e l y that the presence of the styrene compound was at least p a r t i a l l y responsible f o r the i n h i b i t i o n of p r i c k l y sida germination and root length, since f e r u l i c acid alone (prickly sida seed without carpels plus f e r u l i c acid) had no effect on p r i c k l y sida germination or root length (Table XI). The decarboxylation of phenolic acids to corresponding styrenes is known from studies on fungi and bacteria (60, 61). However, i n a number of studies d i r e c t l y concerned with the microbial decomposition of f e r u l i c a c i d , as well as other phenolic acids, no mention is made of any styrene compounds produced as a r e s u l t of phenolic acid decarboxylation (62, 63, 64, 65). It is u n l i k e l y that any one p a r t i c u l a r compound could be respons i b l e f o r reduced weed growth i n n o - t i l l . Higher plants and microorganisms produce a myriad of phytotoxic substances. If these substances are present i n the r i g h t combination and concentration, phytotoxic e f f e c t s may be observed. With the proper choice and management of various cover crops and plant residues, i t may be possible to supplement i f not reduce the number and amount of


270


herbicides used i n n o - t i l l cropping systems by u t i l i z i n g natural phytotoxic substances.


Acknowledgments "h?aper No. 9340 of the Journal Series of the North Carolina A g r i c u l t u r a l Research Service, Raleigh, NC 27695-7627. The work reported here was supported i n part from the Consortium of Integrated Pest Management Grant j o i n t l y funded by EPA (Agreement No. 806277-03) and USDA (Agreement No. 71-59-2481-1-2-039-1) and the North Carolina Tobacco Foundation, Inc. 2Current address: Monsanto A g r i c u l t u r a l Products Co., 800 N. Lindbergh Blvd., St. Louis, MO 63167. Literature Cited 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

Anderson, W.P. "Weed Science: P r i n c i p l e s " ; West Publishing Co.: New York; 1977; 598 pp. Edwards, W.M. Proc. Nat'l. No-tillage Systems Symposium, February, 1972, p. 30-41. Oschwald, W.R. (ed.). "Crop Residue Management Systems." Amer. Soc. of Agron., Crop S c i . Soc. Amer. and Soil S c i . Amer., Inc., 1979; Madison, Wisconsin, 248 pp. P h i l l i p s , R.E.; Blevins, R.L.; Thomas, G.W.; Frye, W.W.; P h i l l i p s , S.H. Science 1980, 208, 1109-13. Gallaher, R.N. Proc. South. Weed S c i . Soc., 1978, 31, 127-33. Putnam, A.R.; DeFrank, J. Abs. Weed S c i . Soc. Amer., 1980, p. 35. Guenzi, W.D.; McCalla, T.M.; Norstadt, F.A. Agr. J. 1967, 59, 163-4. Worsham, A.D. Proc. 35th Annu. Corn and Sorghum Res. Conf., 1980, 35, 146-63. L i e b l , R.A.; Worsham, A.D. Proc. South. Weed S c i . Soc., 1983, 36, 405-14. Forney, D.R.; Foy, C.L.; Wolf, D.C. Proc. South. Weed S c i . Soc., 1983, 36, 358. Overland, L. Amer. J. Bot. 1966, 53, 423-32. Putnam, A.R.; DeFrank, J. Crop Protection. 1983, 2(2), 173-81. Putnam, A.R.; DeFrank, J.; Barnes, J.P. J. Chem. Ecol. 1983, 9 (8), 1001-10. S h i l l i n g , D.G.; Worsham, A.D. Abs. Weed S c i . Soc. Amer. 1984, p. 56. S h i l l i n g , D.G.; Worsham, A.D. Proc. South. Weed S c i . Soc., 1983, 36, 404. Putnam, A.R.; DeFrank, J. Abs. Weed S c i . Soc. Amer. 1980, p. 35. Karssen, C.M. Acta Bot. Neerl. 1970, 19 (3), 297-312. Sauer, J. and Struik, G. Ecology 1982, 45, 884-86. Wesson, G.; Wareing, P.F. J. Exp. Bot. 1969, 20, 402-13. L a l , R. Plant and Soil. 1974, 40, 321-31. Guenzi, W.D.; McCalla, T.M. Soil S c i . Soc. Am. Proc., 1962, 26, 456-58. Guenzi, W.D.; McCalla, T.M. Agron. J. 1966, 58, 303-04. Chou, C-H.; Patrick, Z.A. J. Chem. Ecol. 1976, 2, 369-87. Guenzi, W.D.; McCalla, T.M. Soil S c i . Soc. Am. Proc., 1966, 30, 214-16. Patterson, D.T. Weed S c i . 1981, 29, 53-59.


17.


Rye and Wheat Mulch


26. 27. 28. 29. 30. 31.

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L i e b l , R.A.; Worsham, A.D. J. Chem. Ecol. 1983, 9 (8), 1027-43. Shettel, N.L.; Balke, N.E. Weed S c i . 1983, 31, 293-98. Putnam, A.R.; Duke, W.B. Science 1974, 185, 370-72. Leather, G.R. Weed S c i . 1983, 31, 37-42. Fay, P.K.; Duke, W.B. Weed Sci. 1977, 25, 224-28. Norstadt, F.A.; McCalla, T.M. Soil S c i . Soc. Am. Proc., 1968, 32, 241-45. 32. Cockran, V.L.; Elliot, L.F.; Papendic, R.I. J. Soil S c i . Soc. Am. 1977, 41, 903-08. 33. Kimber, R.W.L. Plant Soil 1973, 38, 347-61. 34. Toussoun, T.A.; Weinhold, A.R.; Linderman, R.G.; Patrick, Z.A. Phytopathology 1968, 58, 41-54. 35. Norstadt, F.Α.; McCalla, T.M. Science 1963, 140, 410-11. 36. McCalla, T.M.; Haskins, F.A. B a c t e r i o l . Rev. 1964, 28, 181-207. 37. Patrick, Z.A.; Koch, L.W. Can. J. Bot. 1985, 36, 621-47. 38. Patrick, Z.A. Soil S c i . 1971, 111, 13-18. 39. Barnes, J.P.; Putnam, A.R. J. Chem. Ecol. 1983, 9, 1045-57. 40. Helwig, J.T.; Council, K.A. (ed.) "SAS User's Guide"; SAS Institute, Inc.: Cary, N.C., 1979, p. 1-101. 41. Van Sumere, C.F.; Wolf, G.; Tenchy, H.; Kint, J.J. Chromatog. 1965, 20, 48-60. 42. Pierce, A.E. " S i l y l a t i o n of Organic Compounds." Pierce Chem ical Company: Rockford, IL, 1983. 43. McLafferty, F.W. "Interpretation of Mass Spectra"; University Science Books: M i l l Valley, CA, 1980. 44. Stenhagen, E.; Abrahamsson, S.; McLafferty, F.W. "Registry of Mass Spectral Data"; John Wiley and Sons: New York, 1974. 45. Markey, S.P.; Urban, W.G.; Levine, S.P. (eds.). "Mass Spectra of Compounds of B i o l o g i c a l Interest"; Technical Information D i v i s i o n , U.S. Atomic Energy Commission (26553 P1-P3), 1975, V o l . I-II, Oak Ridge, TN. 46. Rao, O.N.; Mikkelsen, D.S. Agr. J. 1977, 69, 923-28. 47. Rice, E.L. "Allelopathy." Academic Press: New York, 1974. 48. Gross, D. Phytochem. 1975, 14, 2105-12. 49. Wong, P.P.; Evans, H.J. Plant Physiol. 1971, 47, 750-55. 50. Pridham, J.B. Ann. Rev. Plant Physiol. 1965, 16, 13-36. 51. Gamborg, O.L. Can. J. Biochem. 1966, 44, 791-99. 52. Robinson, T. "The Organic Constituents of Higher Plants"; Cordus Press: North Amherst, MA, 1980. 53. Conn, E.E. (ed.). "The Biochemistry of Plants: Secondary Plant Products"; Academic Press: New York, 1981. 54. Tamura, S.; Chang, C-F. Agr. B i o l . Chem. 1965, 29, 1061-62. 55. Kimura, S.; Tanura, S. Agr. B i o l . Chem. 1973, 37, 2925. 56. Whittaker, R.H. In "Chemical Ecology"; Sondheimer, E.; Simone, J.B., Eds.; Academic Press: New York, 1970; pp. 43-70. 57. Lodhi, M.A.K. J. Chem. Ecol. 1979, 5, 429-37. 58. Wang, T.S.C.; Yang, T.K.; Chuang, T.T. Soil S c i . 1967, 103, 239-46. 59. Borner, H. Bot. Rev. 1960, 26, 393-424. 60. Finkle, B.J.; Lewis, J.C.; Corse, J.W.; Lundin, R.E. J. B i o l . Chem. 1962, 237, 2926-31. 61. Indahl, S.R.; Scheline, R.R. Appl. M i c r o b i o l . 1968, 16, 667. 62. DiMenna, M.E. Gen. Microbiol. 1959, 20, 13-20. 63. Henderson, M.E.K. Pure Appl. Chem. 1963, 7, 589-602. 64. Henderson, M.E.K.; Farmer, V.C. J. Gen. M i c r o b i o l . 1955, 12, 37-46 65. Turner, J.A.; Rice, E.L. J. Chem. Ecol. 1975, 1, 41-58. RECEIVED August 21, 1984 In The Chemistry of Allelopathy; Thompson, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

Rye (Secale cereale L.) and Wheat (Triticum aestivum L.) Mulch: The

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