Allelopathic effects of phenolic acids detected in buffalograss (Buchloe dactyloides) clippings on growth of annual bluegrass (Poa annua) and buffalograss seedlings

Environmental and Experimental Botany 39 (1998) 159 – 167

Allelopathic effects of phenolic acids detected in buffalograss (Buchloe dactyloides) clippings on growth of annual bluegrass (Poa annua) and buffalograss seedlings Lin Wu *, Xun Guo, M. Ali Harivandi Department of En6ironmental Horticulture, Department of Vegetable Crops, and Cooperati6e Extension, Uni6ersity of California, Da6is, CA 95616, USA Received 20 June 1997; accepted 31 October 1997

Abstract Fourteen different phenolic acids were detected in water extracts of buffalograss clippings. Six of the 14 phenolic acids, including p-coumaric acid, ferulic acid, gentisic acid, homoveratric acid, p-hydroxybenzoic acid, and vanillic acid, were examined for water and base extractable tissue concentration, and their effects on growth of seedlings of annual bluegrass (Poa annua) and buffalograss (Buchloe dactyloides). The tissue phenolic acid concentrations were found to be significantly different among the three buffalograss varieties examined. The variety ‘Prairie’ had a higher total tissue phenolic acid concentration than the concentrations detected in the varieties ‘UCHL-1’ and ‘NE609’. Seed germination was not affected by the six phenolic acids, but root growth of seedlings was severely inhibited. Seedling establishment was tested in buffalograss turf plots. No annual bluegrass became established in the buffalograss turf, and only 1% of buffalograss seedlings became established. The allelopathic effects of the phenolic acids may be, at least partly, responsible for prevention of establishment of new plants in the sward. The allelopathic effects of these phenolic acids seem not to be species specific, but they act like a broad-spectrum preemergence herbicide that affects seedling establishment while not affecting established turfgrass. This character may exist in other turfgrass species, and it is a potentially useful trait for turfgrass breeding. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Allelopathy; Buchloe dactyloides; Growth inhibition; Phenolic acid; Poa annua; Seedling

1. Introduction In the last several decades, a great number of research reports of secondary metabolic organic compounds produced by plants (allelopathy) emphasized the interpretations of plant growth response to allelopathic compounds and their * Corresponding author.

inhibitory effects on various crop plants (Tinnin and Muller, 1971; Chou and Chung, 1974; Newman and Rovira, 1975; Wu et al., 1976; Miller, 1996). Only a few studies employed the allelopathic concept as a means of biological control of weeds. This concept was briefly discussed by Rice (1984). Recently, greater interests in the application of allelopathic compounds as biological control agents was reviewed by Einhellig (1995) and

S0098-8472/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 9 8 - 8 4 7 2 ( 9 7 ) 0 0 0 4 0 - 3

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Inderjit (1996). Putnam and Duke (1978) examined several different species and strains of Cucumis and found eight species in the genus Cucumis that exhibited strong germination inhibition to certain weed species. Naqvi and Muller (1975) identified four allelopathic compounds in the leaves of Lolium multiflorum. Tukey (1971), Duke (1986) and Weston et al. (1989) reported that the metabolites leached from plants consisted of a variety of substances, such as mineral nutrients, carbohydrates, amino acids, and other organic compounds. These substances may inhibit or sometimes stimulate plant growth, depending on the concentration, the leachability, the season, and the age of the plants. They mostly are natural metabolites and degrade rapidly in the environment. Chou et al. (1989) reported that allelopathic compounds produced by Kikuyugrass (Pennisetum clandestinum) inhibited weed invasion but had no negative effect on seedling establishment and growth of forest trees. The above discoveries initiated research to determine if certain grass species may be used as cover or companian crops for orchards, vineyards, and forest tree farms. In buffalograss, under minimal field management conditions, it is able to form a solid turf stand without any weed control program. In addition, natural stands of single sex buffalograss several meters in diameter are commonly observed in the field (Quinn, 1991). Our hypothesis was that buffalograss may release allelopathic compounds from its plant tissue, and these chemical compounds may be responsible for the prevention of establishment of new plants in the sward. It is the purpose of this paper to report the discovery of phenolic acids in buffalograss clippings and their suppression effects on growth of seedlings of annual bluegrass (Poa annua) and buffalograss (Buchloe dactyloides).

2. Materials and methods

2.1. Buffalograss field turf plot establishment Buffalograss (Buchloe dactyloides (Nutt.) Engelm.) field plots (3×3 m) were established 6 years ago in the experimental field of the Depart-

ment of Environmental Horticulture, University of California, Davis campus. The buffalograss variety ‘UCHL-1’ was seeded with a seeding rate of 20 g m − 2. The vegetatively propagated varieties ‘Prairie’ and ‘NE609’ were established by 2.5-cm diameter plugs and planted at 30-cm intervals. Four replicate plots of the three buffalograss varieties were randomly arranged in the field along with five other buffalograss varieties (these varieties are not discussed in this study). The soil was loam clay with a pH of 6.5. The buffalograss turf plots became well established in 12 months after seeding and plugging. The turf plots were fertilized with 16–6–6 (N–P–K) fertilizer at a rate of 30.5 g m − 2 once in May, once in July, and once in September. During the dry season, from May to October, the turf plots were irrigated once a week and mowed at 5 cm high. No herbicide was used during the experiment, yet no apparent weed invasion was noticed in the buffalograss turf plots over the 6 years.

2.2. Preparation of buffalograss clipping extracts Buffalograss clippings were collected in September 1995 using a rotary mower and mowing at 5 cm high. The clippings were oven dried for 72 h at 60°C. The clippings were extracted for phenolic acids using either deionized water (pH 6.8, EC= 0.05) or 0.2% (w/v) of NaHCO3 (pH 8.0, EC= 1.7). The reason for using a base solution for phenolic acid extraction was that under field management conditions in arid and semi-arid regions of the western United States and California, high salinity and high pH irrigation waters are common. Base extraction of the phenolic acids may provide additional information for extractable concentrations of phenolic acids under high pH conditions. Fifty grams of dry turf clippings were immersed in 200 ml of the extraction solution in a 5-cm deep, 15× 20-cm plastic tray, and the clippings were pressed down with a 2-cm thick glass plate and kept at 5°C in a incubator. The clippings were turned over and squeezed by hand once a day for a period of 5 days. At the end of the fifth day, the extracts were separated from the plant materials for chemical analysis.

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2.3. Phenolic acid extraction For separation of phenolic acids from the crude clipping extract, the extract was acidified by adding 15 ml of concentrated sulfuric acid to 60 ml of the extract. After cooling down to room temperature, the solution was filtered through Whatman No. 1 paper and centrifuged at 3500× g for 5 min, and the clear extract was collected. Ten ml of ethyl acetate was added to the clear extract and shaken manually for 30 s. The organic (ethyl acetate) phase was carefully collected using a 150-ml separating funnel, and the aqueous phase was repeatedly extracted three times using 4 ml ethyl acetate. The ethyl acetate extract was used for the following chemical analysis.

2.4. Esterification and gas chromatographic analysis Before the analysis of phenolic acids using capillary gas chromatography (GC), esterification was conducted to increase the volatility of the compounds. Standard chemical compounds were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The method described by Husek (1991) was used for the present study. Heptafluorobutyric-isobutanol amino acid (HFB-IBA) was used as an esterification reagent. The organic phase extract (about 70 ml in volume) was concentrated and the volume was reduced to about 200 ml by a stream of dry N2 in an esterification tube. A reaction solution (1200 ml; dichloromethane and acetyl chloride 10:3, v/v) was added into the esterification vessel, capped, and heated at 135°C for 50 min. After heating, it was vaporized to dryness under a dry nitrogen stream. After cooling, 1200 ml of dichloromethane and 400 ml of heptafluorobutyric anhydride were added to the vessel, the cap was sealed and the vessel was heated at 130°C for at least 15 min. After the heating process, the vessel was removed and placed in an ice-bath for 2 min. The solvent was vaporized to dryness under a stream of dry nitrogen. The residue was redissolved with 500 ml of ethyl acetate for GC analysis. A Hewlett-Packard 5890 gas chromatograph equipped with a split/splitless injection port, flame

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ionization detector (FID), and a DB-1HT 30 m × 0.32 mm× 0.1 mm column was used for this study. The oven temperature ramps were at 75°C for 1 min, then increased at 25°C min − 1 up to 280°C and held there for 1 min. The split injector was set at a rate of 46:1 and the temperature was 250°C. The FID detector temperature was set at 320°C. Helium was used as the carrier gas at a flow rate of 1.5 ml min − 1, with make up gas at 25 ml min − 1. Based on the retention time of each standard phenolic acid, the phenolic acids separated by GC chromatograms can be readily identified. For further confirmation, the method of direct standard addition was used. One mmol of each standard phenolic acid was individually added into the sample extract before esterification. The direct standard phenolic acid addition response was detected by the GC chromatograms. Recovery rates for the six phenolic acids (pcoumaric acid, ferulic acid, gentisic acid, homoveratric acid, p-hydroxybenzoic acid, vanillic acid) ranged from 83 to 104%. A standard analytical curve method was used for quantitative analysis. Detection limits for the six phenolic acids were from 1 to 3 nmol.

2.5. Seed germination test The p-coumaric acid, ferulic acid, gentisic acid, homoveratric acid, p-hydroxybenzoic acid, and vanillic acid were chosen for the study, because these six phenolic acids showed a relatively consistent tissue concentration among sample preparations. They were tested for water and base extractability and seedling growth effects of annual bluegrass and buffalograss. Seeds of Annual Bluegrass (Poa annua L.), a golf green perennial biotype that came from golf greens of the Davis municipal golf course, were produced in the greenhouse in 1993. The seeds of the UCHL-1 buffalograss variety were harvested from the experimental field of the Department of Environmental Horticulture at UC Davis in the fall of 1993 and dehulled. The germination rates of both species were greater than 85%. For the seed germination test, 5-cm diameter by 10-cm tall glass jars were used. The bottom 4 cm of the jar was

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filled with facial tissue and saturated with the phenolic acid treatment solution. A piece of Whatman c1 filter paper was placed on top of the saturated facial tissue. The six phenolic acids used for this experiment were p-coumaric acid, ferulic acid, gentisic acid, homoveratric acid, phydroxybenzoic acid, and vanillic acid. The treatment solutions were prepared using 100 mg l − 1 of the phenolic acids dissolved either in distilled water or in 0.2% NaHCO3. Crude buffalograss clipping extract was used for the seedling growth test comparisons. A treatment of distilled water without phenolic acid was used as the control. One hundred seeds of either species were placed on the filter paper in the jar, and the jar was covered with a 6-cm diameter petri dish lid. Four replications were used for each treatment; the jars were completely randomized and kept at 20°C under incandescent light, with a photon flux density of 200 mmol m − 2 s − 1. After 14 days, the rate of germination, root length, and shoot length of 20 seedlings from each treatment replication were recorded.

2.6. Field seedling establishment test For testing the established buffalograss turf on seedling establishment of annual bluegrass and buffalograss, 30 × 30-cm quadrats were marked in the buffalograss plots (described previously) by punching 2.5-cm diameter wooden sticks into the ground at the four corners of each quadrat. The quadrats designated in the bare ground adjacent to the turf plots were used as control plots. Annual bluegrass is a cool-season winter weed. Therefore, the seedling establishment of annual bluegrass was conducted in a dormant buffalograss turf in November of 1994. Buffalograss is a warm-season species and the seedling establishment experiment was conducted in June of 1995 on an actively growing buffalograss turf. Three hundred seeds of annual bluegrass or buffalograss were sown into each quadrat and the seeds were pressed down to the ground surface by hand. Irrigation was applied to ensure sufficient moisture for germination. Three weeks after seeding, the number of seedlings found in each quadrat was recorded. After an additional 4 weeks, the

number of established seedlings (those having produced three or more tillers) was recorded.

2.7. Data analysis One-way and two-way analyses of variance were performed on differences of phenolic acid concentrations detected between water and base solution extracts, on seedling growth under different phenolic acid compound treatments, and on the effects of buffalograss turf on seedling establishment. The Statistical Analysis System (SAS) version 6.03 was used for the analyses (SAS Institute, Inc., 1988).

3. Results The preliminary study showed that 5 days of soaking of the clippings in the extraction solution attained a maximum extractable phenolic acid concentration. Fig. 1 shows that at least 14 different phenolic acid compounds were separated and identified from the buffalograss clipping extracts, including benzoic acid, salicylic acid, gallic acid, gentisic acid, p-hydroxybenzoic acid, trans-cinnamic acid, vanillic acid, homoveratric acid, 3-(phydroxyphenyl) propionic acid, 3,4,5-tri-methoxybenzoic acid, p-coumaric acid, syringic acid, caffeic acid, and ferulic acid. Tissue concentrations of the six phenolic acids detected in the water and base extracts of the three buffalograss varieties are presented in Table 1. The results revealed several patterns regarding the distribution of these phenolic acids in the three buffalograss varieties: (1) higher concentrations were found in the base extracts than in water extracts for p-coumaric acid and gentisic acid in all three buffalograss varieties; (2) higher ferulic acid concentrations were found in the base extracts than in the water extracts for UCHL-1 and NE609, but there was no difference for Prairie; (3) homoveratric acid was found in lower concentrations in base extracts than in water extracts for all three varieties; (4) the p-hydroxybenzoic acid concentration was higher in the water extract than in the base extract for UCHL-1, but the higher concentration was found in the base extract for Prairie,

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Fig. 1. Phenolic acids detected in water extract of UCHL-1 buffalograss clippings separated by gas chromatography.

while there was very little difference between the water and base extracts for NE609; and (5) the vanillic acid concentrations were considerably higher in the water extracts than in the base extracts for UCHL-1 and NE609, while the opposite result was found for Prairie. The extractable phenolic acid concentrations, expressed on the basis of tissue dry weight (Table 1), were found to be quite different among the three buffalograss varieties. The average p-coumaric acid concentration detected in Prairie were 299 mg g − 1 dry tissue for the water extract and 399 mg g − 1 for the base extract. UCHL-1 and NE609 only had about one-tenth of the concentrations found in Prairie. Ferulic acid generally was low, and only found in the base extracts of UCHL-1 and NE609. The highest concentrations of gentisic acid were detected in the base, 337 mg g − 1, and water, 79 mg

g − 1, extracts of the Prairie variety. For UCHL-1 and NE609, gentisic acid only ranged from about 16–34 mg g − 1. Homoveratric acid was highest, 63 mg g − 1, in the water extract and lowest in the base extract, 0.01 mg g − 1, of Prairie. UCHL-1 and NE609 had homoveratric acid concentrations ranging from 7 to 22 mg g − 1 dry weight. p-Hydroxybenzoic acid concentration was lower in NE609 than in UCHL-1 and Prairie. High concentrations of vanillic acid were found in both the water, 329 mg g − 1, and base, 424 mg g − 1, extracts of the Prairie variety. Vanillic acid concentrations in the base extracts of UCHL-1 and NE609 were extremely low, but its concentrations in the water extracts were found to be 50 mg g − 1 in NE609 and 101 mg g − 1 in UCHL-1. Overall, Prairie had the highest tissue phenolic acid concentrations for both the base extract and the water extract.

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Table 1 Concentrations of the six phenolic acids detected in distilled water or sodium bicarbonate extracts of clippings of the three buffalograss varieties Phenolic acids

p-Coumaric acid Ferulic acid Gentisic acid Homoveratric acid p-Hydroxybenzoic acid Vanillic acid Total ANOVA Between extracts Between varieties Variety×Extract interaction a

UCHL-1 (mg g−1 clipping dry weight)

Prairie (mg g−1 clipping dry weight)

NE609 (mg g−1 clipping dry weight)

Water extract

Base extract

Water extract

Water extract

359 1 89 0 279 8 69 0 39 0

299 940 0 90 79 97 63 93 32 91

399 95 0 90 337 94 090 48 91

25 97 1 910 33 914 14 9 3 991

38 92 490 34 9 1 690 692

09 0 82

329 9 17 804

424 9 27 1208

50 912 135

090 89

22 9 4a 090 169 1 219 5 349 4 1019 5 202

F = 4.91, PB0.05 F= 26.04, PB0.001 F = 2.10, P\0.05

Base extract

F= 1.44, P\0.05

Base extract

F=5.50, PB0.05

Mean and standard deviation of three samples.

Analysis of variance indicates (Table 1) that, over all, within variety, phenolic acid concentrations detected between water and base extracts were significantly different (at 5%) for UCHL-1 and NE609, but for Prairie concentrations of the phenolic acids were not significantly different between the water and base extracts. Phenolic acid concentrations between buffalograss varieties were significantly different at 1%, and Prairie had the highest total phenolic acid concentration. The plant variety× phenolic acid concentration interaction was not significantly different. The results of the laboratory tests of the effects of phenolic acids on seed germination and seedling growth are presented in Table 2. Except for seed germination, the six phenolic acids produced various degrees of shoot and root growth inhibition on annual bluegrass seedlings. In distilled water (control), after 14 days growth, the mean shoot length of annual bluegrass was about 32 mm. Under the treatment using the buffalograss extract, the mean shoot length was 66% of the control. For seedlings in 100 mg l − 1 phenolic acid, the shoot length ranged between 20 mm (63% of control) for those treated with p-coumaric acid and 29 mm (90% of control) for those treated with p-hydroxybenzoic acid. The root growth of annual bluegrass was

more severely inhibited by the buffalograss extract and the phenolic acids than the shoot growth. The root length in the control treatment was about 25 mm, but the root length was reduced to a range from 14 mm (56% of the control) in the p-hydroxybenzoic acid treatment to 1 mm (4% of control) in the p-coumaric acid treatment. The buffalograss shoot growth was not inhibited by the buffalograss extract or the phenolic acids. The shoot length of the buffalograss seedlings in distilled water was about 18 mm, and its mean shoot length ranged from 16 mm in p-coumaric acid to 21 mm in the p-hydroxybenzoic acid and vanillic acid treatments. However, the root growth of the buffalograss seedlings was inhibited to various degrees. The mean root length in the control treatment was 32 mm. The root length declined to 56% of the control treated with buffalograss clipping extract and reduced to 59% treated with vanillic acid. Root length was reduced to 75% by the homoveratric acid and gentisic acid treatments and to 16 and 13% by the ferulic acid and p-coumaric acid treatments, respectively. Root length of the buffalograss seedlings was only slightly affected by p-hydroxybenzoic acid treatment. Analysis of variance indicates that between-treatment difference was significant at 1% for both the shoot

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Table 2 Seed germination, shoot length, and root length of seedlings of Annual Bluegrass (Poa annua) and buffalograss (Buchloe dactyloides) after a 2-week incubation in extracts of buffalograss clippings and in solutions of six phenolic acids (100 mg l−1) Treatment

Distilled water Buffalograss extract p-Coumaric acid Ferulic acid Gentisic acid Homoveratric acid p-Hydroxybenzoic acid Vanillic acid ANOVA Between treatments Between species (Shoot) Between species (Root) a b

Annual Bluegrass

Buffalograss

Germination (%)

Shoot length (mm)

Root length (mm)

Germination (%)

Shoot length (mm)

Root length (mm)

899 2 9091 87 9 3 899 2 859 4 82 95 909 3

329 4a 21 9 2 (65)b 20 9 4 (62) 2495 (75) 28 9 6 (88) 25 94 (78) 29 95 (90)

25 9 6 8 92 190 59 2 592 89 2 14 9 3

(25) (4) (20) (20) (32) (56)

81 93 82 9 5 85 93 87 95 86 9 4 80 9 4 82 9 3

18 98 20 9 9 16 9 8 17 93 20 96 19 9 6 21 9 4

(111) (80) (85) (111) (105) (116)

32 9 11 18 9 6 (56) 5 9 2 (16) 4 92 (13) 9 92 (75) 1194 (34) 25 95 (71)

86 9 6

249 3 (75)

7 9 2 (28)

87 9 4

21 9 8 (116)

199 7 (59)

F = 1.2, P\0.05 F= 103.2, PB0.001 F= 32.4, PB0.001

F =15.0, PB0.001

F =1.4, P\0.05

F= 1.4, P\0.05

F= 58.5, PB0.001

F=111.5, PB0.001

Mean and standard deviation of 20 measurements. Values in the parentheses are means of % of growth to the control treatment.

and the root length of annual bluegrass and for root length of buffalograss. Between-treatment difference in shoot length of the buffalograss was not significant (Table 2). The effects of the phenolic acids on both shoot growth and root growth between the two grass species were significantly different at the 1% level (Table 2). Buffalograss turf reduced seedling (in November) establishment of annual bluegrass and buffalograss (Table 3). Seven weeks after seeding, no annual bluegrass became established in the buffalograss turf, but 62% of seedlings became established and produced more than three tillers in the bare soil plots. Only 0.9% of buffalograss seedlings became established (in June) in the Buffalograss turf, but 50.3% seedling establishment was found in the bare soil plots.

4. Discussion Although buffalograss is still mainly used for forage on western rangelands, it is emphasized for

use as a low-maintenance turfgrass and for soil stabilization because of its drought tolerance, low nutritional requirements, and short growth stature (Rizvi et al., 1980; Engelke and Hickey, 1983; Wu et al., 1984; Wu and Lin, 1994). This species is dioecious; unisex clones of buffalograss several meters in diameter are commonly observed in the field (Quinn, 1991). In predominantly vegetatively propagated plant populations, recruitment of sexually reproduced individuals has been found to be rare or absent (Turkington and Harper, 1979; Doust, 1981; Ellstrand and Roose, 1987). In ecology and population biology, the inability to establish seedlings in well-established perennial swards is interpreted as being due to competition of the aggressiveness of the established plants, but the mechanisms of aggressiveness have not been well defined. Viles and Reese (1996) reported that water extracts from plant tissue of Echinacea angustifolia have the potential to act allelopathically and inhibit seed germination and seedling growth of lettuce (Lactuca sati6a), dropseed (Sporobus helerolopis), and switchgrass (Panicum 6irgatum).

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Table 3 Seedling establishment of Annual Bluegrass (Poa annua L.) and buffalograss (Buchloe dactyloids (Nutt.) Emgelm.) in established buffalograss turf Seeds of plant species

Field

No. of seeds sown in the field

Seedlings found in turf

Annual Bluegrass

Buffalograss turf

300

142 918

Bare field Buffalograss turf Bare field

300 300 300

203 925 165 9 15 191 9 16

Buffalograss

No. of seedlings became established 0 128 9 18 1.390.5 97 913

Rate of establishment (%) 0 63 9 6 1 90 50 912

Annual Bluegrass establishment (produced three or more tillers) was tested in December in dormant buffalograss turf, and buffalograss establishment was tested in June in actively growing buffalograss turf.

The phenolic acids found in extracts of buffalograss clippings showed various degrees of seedling growth inhibition, especially on root growth. Therefore, the allelopathic effects of these phenolic acids may be, at least partly, responsible for the exclusion of the establishment of new plants in well-established buffalograss stands. Most allelopathic compounds have been termed secondary substances and do not appear to play a role in the basic metabolism of organisms. There are many thousands of such secondary compounds and they could be classified into five major categories: phenylpropanes, acetogenins, terpenoids, steroids, and alkaloids (Whittaker and Feeney, 1971; Inderjit, 1996), but only a limited number of them have been identified as toxins involved in allelopathy (Rice, 1984). Rizvi et al. (1980) stated that pesticides from plant sources are more systemic and more easily biodegradable than synthetic pesticides. For example, the active phytotoxic compound (caffeine) isolated from extracts of leaves and seeds of coffee (Coffea arabica) completely inhibited seed germination of Amaranthus spinosus at a concentration of 200 mg l − 1, but it did not affect seed germination and subsequent growth of black gram (Phaseolus mungo). Thus, this compound appears to be a promising selective herbicide, at least for some crops (Rizvi et al., 1981). Phenolic acids such as ferulic acid, vanillic acid, and p-hydroxybenzoic acid produced by chaparral shrub (Adenostoma fasciculatum) were found to inhibit the growth of lettuce seedlings at a concentration of 200 mg l − 1 (McPherson and Muller, 1969). The present study

showed that the root growth of seedlings of Annual Bluegrass was severely inhibited by 100 mg l − 1 of these compounds. Buffalograss seedlings were less severely inhibited, however the growth inhibition was significant. Therefore, the allelopathic effects of these phenolic compounds seem not to be species specific, but act like a broad spectrum preemergence herbicide. A seedling with impaired root growth is unlikely to be able to survive in a well-established sward. This is especially true where there is infrequent irrigation and drying of the field, which is common for buffalograss. Concentrations of the phenolic acids detected from the clippings of the buffalograss varieties were different. The pattern of distribution of concentrations of the six phenolic compounds was similar between the diploid variety ‘UCHL-1’ and the hexaploid variety ‘NE609’, but the tetraploid variety ‘Prairie’ had much higher concentrations of p-coumaric, gentisic, and vanillic acids. The differences seem not to relate to the ploidy levels, but it is a potentially genetically manipulatable character. The concentration of allelopathic compounds was found to be affected by environmental factors and the life history of the plants, such as water stress (Del Moral, 1972), temperature (Koeppe et al., 1970), and maturity of the plants (Tukey, 1971). In addition, the chemical conditions of soil and water in the field may affect the potential of allelopathy greatly. For example, the application of lime and ammonium sulfate was able to reduce phytotoxicity of rice residues in paddy fields (Chandrasekaran and Oshid, 1973). In arid and semi-arid regions of the western

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United States, soils are usually alkaline and irrigation water salinity is high. Therefore, under field conditions the effectiveness of weed inhibition by buffalograss is expected to be less predictable than the results of the laboratory test. However, it is a potentially useful trait for the buffalograss breeding, and future research should emphasize testing for allelopathic potential among genotypes in this species and identifying environmental and biological factors that may affect the amounts and effectiveness of allelopathic compounds produced by the plants.

Acknowledgements This work was supported by the Agricultural Experiment Station Project, UC Davis, Ca-D*EHT 4063-H.

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