Allelopathy in the Search for Natural Herbicide Models - ACS

Chapter 23

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Allelopathy in the Search for Natural Herbicide Models Francisco A. Macias Departamento de Quimica Orgánica, Universidad de Cádiz, Facultad de Ciencias, Apartado 40, 11510 Puerto Real, Cádiz, Spain

The existence of allelopathy has been well documented during the past few decades on natural as well as in agroecosystems. The potentiality that allelopathy can offer useful clues in the search of new natural herbicide models, more specific and less harmful than those synthetic, at present used in agriculture is discussed. Three different strategies are presented considering allelopathic studies on natural ecosystems, agroecosystems and natural product models as natural herbicides with applications in agronomical important crops such as barley, oat, wheat, grapes, etc. The bioactivity levels of the present synthetic herbicides and those of several allelochemicals reported in the literature are compared and discussed.

The weed problem supports an important part of the agriculture research. As a consequence of this research, many chemicals have been developed since the 50 's, and their utilization are widely extended (7). There are about 250 plant species sufficiently troublesome in agriculture to termed weeds (2). In spite of modern control methods, even in developed countries that rely heavily on chemical herbicides for control, losses due to weeds, including efforts to control them plus losses in yield and quality, are relatively high. Herbicides will continue to be a key component in most integrated weed management systems in the future. Nevertheless, the increasing of the chemical control has become overwhelming economical border, and more important, it could pose a serious threat of the public health and of the environment as it has been proved in recent studies (3,4)fromwhich the following facts are concluded: a) a considerable decreasing of the crop yields; b) the appearance of highly resistant species to commercial innocuous products traditionally used; c) a clear and evergrowing pollution on the phreathical layer. During last few decades the extensive use of synthetic herbicides and pesticides has been the cause of concern from both environnmental and health considerations. Most of the synthetic chemicals are more hazardous due to their long

0097-6156/95/0582-0310$08.00/0 © 1995 American Chemical Society In Allelopathy; Dakshini, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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Search for Natural Herbicide Models

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persistence, non-target toxicity, pollutive, carcinogenic and mutagenic activity (5-8). In addition some problems as crop injury, increased cost of discovering and developing new herbicides, enhanced soil biodegradation, and container disposal, are receiving increasing attention and concern. Because of these problems much attention is being focused on alternative ways for weed control. Allelopathy, which studies biochemical plant-plant interactions including positive and negative effects (9,10), has been proposed as a possible alternative weed management strategy (11-13). The multidisciplinary character of this science, where biologists, plant physiologists, ecologists, edaphologists, agricultural chemists, agronomical engineers, natural product chemists, etc. play an important role, can offer additional routes for weed control (12-16) by developing new techniques involving allelopathy for weed suppression, such as: 1. The use of natural or modified allelochemicals as herbicides. 2. Genetic transfer of allelopathic traits into commercial crop cultivars. 3. The use of allelopathic plants in crop rotation, companion plantings and smother crops. 4. The use of phytotoxic mulches and cover crop management for weed suppression, especially in conservation and no-tillage crop production. The potentiality that allelopathy can offer helping in the search of natural herbicide models, more specific and less harmful than those synthetic at present used in agriculture, will be discussed in this chapter. Developments in Weed Control Developments in weed control can be divided into three periods. The first, before 1945, was marked by inorganic and organic herbicides having very low activity and no selectivity i.e. copper sulphate and dinitro-ortho-cresol "DN". The basic idea behind the research of the early herbicides was to spray a group of plants with a compound in order to kill the weeds leaving the crop unharmed. This type of compound was called postemergence herbicide. The modern era began in the mid 1940's with the discovery of the phenoxy herbicides, followed during the next 30 years by substituted phenylureas, triazines, glyphosate, and others. They allowed for the first time selective pre- and postemergence weed control in seeded crops. With this second generation compounds came the finding that many different steps in the plant's biochemistry are susceptible to chemical exploitation (7 7). Those pathways that are different from other forms of life are primary targets for attack in the design of new agrochemicals (18). The discovery of the sulfonylurea herbicides in the mid 1970's by Levitt (19-21) signed the start of the present low dose era of chemical herbicide, which is characterized by selective weed control on crop at very low use rate. Strategies in the Search of New Herbicides Strategies to search novel herbicides can be divided into four basic approaches:

In Allelopathy; Dakshini, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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ALLELOPATHY: ORGANISMS, PROCESSES, AND APPLICATIONS

a) New Structural Types: Synthesis of new compounds for broad biological screening motivated purely by structural novelty. It is not possible to establish any direct relationship between structural complexity and activity. Many more "simple" molecules of high activity can be found, considering the almost infinite ways in which the elements can be combined. b) New Ideas in Known Areas: It depends on other approaches for its starting point but in the hands of creative chemists adds significant new value to existing classes of chemicals. Seldom is the initial product the last word for any area. c) Biochemically Directed Synthesis: "Biorational herbicides". The common problem of poor correlation between "in vitro" enzyme inhibition and whole-organism activity should begin yielding to better understanding of xenobiotic penetration movement on metabolism. This strategy can be supported from the outset by biochemistry and biotechnology. Specific new modes of action can be sought intentionally. d) Natural Products: As traditional methods of discovering new herbicides become more difficult and expensive, interest in natural products as sources of new herbicide chemistry increases. Natural products are an attractive source of potential leads to new natural herbicides, not only for the diversity and novelty of chemical structures produced by living organisms, but also for the potential specificity of biological action and the greatly reduced likelihood of harmful bioaccumulation and/or soil and ground water residues. Indeed, natural products can be used in any of the other three strategies mentioned above. Allelopathy, which studies biochemical plant-plant interactions, can offer an excellent opportunity to help in the search of new natural herbicide models. Knowledge of chemistry and biology of allelochemicals is necessary to their exploitation in biocontrol programmes. The knowledge of the allelochemicals involved in one specific interaction, their mechanisms of action and the receptors can allow us to develop new strategies in the search of natural herbicides models. Learning from nature how a specific plant can biochemically interact with another we can focus our attention to the natural products isolation based on the corresponding bioassay in order to found new structural types of herbicides more specific and less harmful than those synthetic at present in use in agriculture. The sources for allelopathic agents can be classified into three groups: 1. - Secondary metabolites from species belonging to the own studied ecosystem (natural or agroecosystem). 2. - Secondary metabolites from other ecosystems, not necessarily related with the studied one. (i.e. from marine organisms). 3. - Synthetic analogues of the allelochemicals above mentioned. Consequently three different strategies can be formulated depending on the origin of the allelopathic compound:



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313

- The search of natural herbicide models from a particular ecosystem (natural or agroecosystem) with application on the own ecosystem. - The search of natural herbicide models from a particular ecosystem with application on a different one. - The synthesis of analogues of the previous allelopathic compounds in order to establish the structural requirements needed for a specific bioactivity. With these concepts in mind and with the notion that allelopathic compounds have a wide diversity of skeleton type we have initiated several years ago two different and complimentary research projects: "Natural Product Models as Allelochemicals" and "Allelopathic Studies on Cultivar Species". We have initiated a systematic allelopathic activity studies on natural and agroecosystems as well as with synthetic bioactive natural product models in order to evaluate their potentiality as allelopathic agents and consequently as natural herbicides models. The plant material selection is based on field observations and on preliminary bioassay of the crude water extract using 1:10, 1:20 and 1:40 (v:v) dilutions. After the first chromatographic separation a second bioassay is performed and the fractions are selected on the basis of their bioactivity. Each pure compound resulting from the separation is tested using a series of aqueous solutions at 10" -10" M in order to establish a structure-allelopathic activity relationship. Finally the synthesis of analogues is carried out in order to establish the specific structural requirements needed for this bioactivity. In this chapter two examples related with the two first proposed strategies using allelopathic studies are presented. The first one belonging to a natural ecosystem: Melilotus messanensis (L.) A l l . and the second one from an agroecosystem: cultivar sunflowers (Helianthus annuus L.) var. SH-222. 4

9

Allelopathic Studies on Natural Ecosystems: Melilotus messanensis (L.) All. Melilotus messanensis (L.) A l l . is a small shrub (less than 50 cm tall) endemic to the Mediterranean Basin (22), it is one of the 23 species that belonging to the genus Melilotus (Fabaceae). Recently, the potentiality of different Melilotus species such as M. segetalis or M. messanensis (L.) All. ecotypes from SW Spain, able to grow in saline soils, has been evaluated as forage resources, green manure and as source of biocide compounds (23,24). It must be pointed out that about 15 million ha in the Mediterranean Basin are affected by salinity and a diversified use of these "marginal land" must include extensive grazing. Following the general proposed strategy a preliminary bioassay of M. messanensis (L.) A l l . crude extract was performed. It shows to be active on germination (stimulation) and radicle length (inhibition) of Lactuca sativa L . Results on the germination, root and shoot length (Figure 1) are expressed on % units over the control, consequently 0 means that the observed value was identical to the control and obviously no effect, a positive value means stimulation and a negative value represent inhibition. Extraction of the fresh M. messanensis aqueous extract with Dichloromethane (DCM) afforded, after chromatography following the levels of bioactivity exhibited by the fractions over Lactuca sativa, on the second bioassay (Figure 1), six lupane-type




314

Figure 1.

Strategy for isolation of allelopathic agents from Melilotus messanensis (L.) A l l .


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315


triterpenes of increasing polarity, the known lupeol (1) (25,26), betulin (2) (27), betulinaldehyde (3) (28,29), betulinic acid (4) (30) and 6 (57) and a new nor-lupane messagenin (5).They were identified and characterized by spectroscopic techniques (IR, MS and ID, 2D N M R experiments) and chemical correlation (32) (Figure 2).

1

R = CH

R = H

2

R = CH OH

R = H

3

R = CHO

R = H

4

R = COOH

R = H

6

R = CH OH

R = OH

3

1

1

2

Figure 2.

2

5

1

1

1

Allelopathic lupanic triterpenes isolated from bioactive fractions of Melilotus messanensis (L.) All.

In order to evaluate their potential allelopathic activity and to obtain information about the specific structural requirements needed for their biological activity, we have studied the effect of a series of aqueous solutions at 10" -10" M of six natural lupanic triterpenes 1-6 on germination, root and shoot lengths of Lactuca sativa seedlings (dicotyledons) (Figure 2) and Hordeum vulgare and Triticum aestivum seedlings (monocotyledons) (32). In the literature, only oleanic-type triterpenes as ursolic acid (33), medicagenic acid and their glycoside derivatives (34) or soyasapogenol B (35) have been reported to influence the growth of surrounding plants where the effect was inhibition for the acid derivatives and stimulation for soyasapogenol B. As observed with the water extract and the fractions C,D,G and K (Figure 2) from which they were isolated, 1-5 showed a high stimulatory activity on the germination of Lactuca sativa seeds in high and low concentration, pointing out 2, 5 and 6 (10- ,10- ,10" M , 2: +38%; 10" M, 5: +73%; 10" M, 6: +75%) (Figure 2). The effects on the radicle and shoot length are, in general, of little or no significance. These compounds have low effects on the germination and seedlings growth of Hordeum vulgare L . and Triticum aestivum L. (32), except for 3 over H. vulgare L . Compound 3 has an inhibitory effect on the shoot length (10~ M, -42%; 10" M, -44%) and there are stimulatory effects on germination promoted by 3 (10" M, +30%). These data suggest that the bioactivity of these compounds can be related with the presence of a free hydroxyl group at C-3, a - C H O H at C-17 as shown by 2, 5 and 6 and this is increased when a methyl and ketone groups or C H O H and methylene is attached at C-20. 4

4

7

9

6

9

6

6

7

9

2

2



316


The concentrations of compounds 1-5 in the 1:10 aqueous extract are in the same range as those that were active in the bioassay (32). The above findings suggest that the lupane triterpenes are very likely responsible for the allelopathic activity of M. messanensis aqueous extract with a certain specificity over some dicotyledons species. This is a very interesting example where the profile of activity shown by the original crude aqueous extract and the fractions C,D,G and K from which triterpenes 1-6 were isolated is perfectly correlated with the corresponding of pure compounds. The stimulatory effects shown over germination of some dicotyledons species suggest that these lupane triterpenes are excellent candidates to be used as a pre-emergence herbicides at very low doses (10" -10" M). 4

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Allelopathic Studies on Agroecosystems: Cultivar Sunflowers (Helianthus annuus L.)varSH-222 Cultivation of sunflowers is predominantly performed to produce oil and plays an important role in southern parts of Europe. Biochemical investigations on sunflower reveal that this species (Helianthus annuus L.) is a rich source of sesquiterpenoids (36,37) and other plant metabolites with a wide spectrum in biological activities (38), nevertheless little is known about the function of its compounds. Recent investigations have shown that sunflowers can actively influence the growth of surrounding plants (39,40), but the mechanism of these allelopathic effects is unresolved. In Andalusia region (Spain), 26 different varieties of sunflowers for crop production are used. Following the proposed strategy, we perform a preliminary bioassay with those varieties during four different plant development stages in order to establish which variety shows a better significative profile of activity and when is the best stage for use the plant material (fresh leaves) without injury the plant for the main crop production. As result of this previous bioassay H. annuus var. SH-222 during the third plant development stage (plants 1.2 m tall with flowers, 1 month before harvest) was one of the selected varieties (Figure 3). The subsequent bioassays withfractionsobtained from the first chromatographic separation, where fraction G (Figure 3) shows a good correlation respect to the crude extract (inhibition of the germination of lettuce seeds, -37%), guided to the isolation of the active principles. From fraction G five new guaianolides named annuolides A-E (7-11) were isolated (41) as well as a sesquiterpene heliannuol A (12) the first member of a novel class of sesquiterpene (37) (Figure 4). In order to evaluate their potential allelopathic activity, we have studied the effect of a series of aqueous solutions at 10' -10" M of compounds 7-12 on root and shoot lengths of Lactuca sativa L. and Hordeum vulgare L. seedlings. There are several contributions about the regulatory activity on the germination and plant growth of sesquiterpene lactones (42,70,72) where it is reported that the activity is clearly affected by the conformation of the molecules and the accessibility of groups which can be alkylated as an a-methylene-y-lactone moiety. 4

9




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Figure 3.

Strategy for isolation of allelopathic agents from Helianthus annuus L . var. SH-222.


317


318


11

Figure 4.

R^CHa R = H 2

Allelopathic agents isolated from bioactive fractions of Helianthus annuus L. var. SH-222.

As observed with the fraction from which they were isolated, 8 and 11 showed (Figure 3) a high inhibitory activity on the germination of lettuce seeds in high and low concentrations (10 M 8: -71%; 10 M 11: -62%). The effects on the radicle and shoot length are, in general, of little or no significance, as well as with germination and growth of barley seeds (41), except for 7 and 10. There are stimulatory effects on germination promoted by 7 (1(T M , 27%) and 10 (10" M , 17%; 10" M , 23%). The novel sesquiterpene 12 shows a homogeneous inhibitory profile of activity with an average of 40% of inhibition on the germination of lettuce from 10" -10* M and small effect on the germination and growth of barley seeds. The above findings suggest that the guaianolides 7-11 and the heliannuol 12 are likely to be significantly involved in the allelopathic action of cultivar sunflowers with certain specificity over some dicotyledons species. The inhibitory effects shown over germination of some dicotyledons species as well as the stimulatory effects shown over germination of some monocotyledons species suggest that these guaianolides and the heliannuol are excellent candidates to be used as a pre-emergent herbicides on a monocotyledons crop at very low doses (10" -10- M) where even some stimulation over the germination of the monocotyledons can be expected. This suggestions are in agreement with field observations in southern parts of Mexico (unpublished data) where the corn field present an important number of Helianthus maximiliani (43) and H microcephalus (44) plants with positive effects over the crop, from which guaianolides similar to annuolides A (7) and D (10) has been isolated. -5

-6

5

5

6

4

4

9

9

Experiments for Bioassays Seed germination bioassay. Seeds of Lactuca sativa L var. nigra., Hordeum vulgare L. and Triticum aestivum L. 1991 crop, were obtained from Rancho La Merced, Junta de Andalucia, Jerez, Cadiz, Spain. Seeds were selected for uniformity of size and damaged seeds were discarded.


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Germination bioassays consisted of germinating 25 lettuce seeds for 5 days (3 for germination and 2 for root and shoot growth) and 5 Triticum aestivum L . and Hordeum vulgare L. seeds for 3 days, in the dark at 25°C in 9-cm plastic Petri dishes containing a 10-cm sheet of Whatman no. 1 filter paper and 10 ml of a test or control solution for lettuce and 5 ml for barley and wheat. Test solutions of water extract were prepared by diluting the original extract to 1:10, 1:20 and 1:40 (V. extract: V. H 0 ) using deionized H 0 and for the fractions by diluting the appropriate amount of each fractions to obtain a similar concentration to 1:10, 1:20 and 1:40 aqueous extract. Test solutions (10~ M) were prepared using deionized water and test solutions 10' -10' M were obtained by diluting the previous solution. There were 3 replicates for L. sativa L. and 19 for H. vulgare L . and T. aestivum L . of each treatment and of parallel controls. The number of seeds per replicate, time and temperature of germination were chosen in agreement with a number of preliminary experiments, varying the number of seeds, volume of test solution per dish and the incubation period. A l l the pH values were adjusted to 6.0 before the bioassay using MES (2-[NMorpholino]ethanesulfonic acid, 10 mM). The osmotic pressure values were measured on a Vapor Pressure Osmometer WESCOR 5500 and are on a range between 30-38 m osmolar. The germination, root and shoot length values were tested by the Student's ttest; the differences between the experiment and the control were significant at value of P=0.01. 2


319


2

4

5

9

Natural or Analogs Allelopathic Compounds as Herbicides There are excellent reviews about the potential use of allelochemicals as herbicides where allelochemicals from higher plants and microbes (12,45,46) from microbes (47,48) or plants (49,50) with a wide diversity of structural types are proposed. Their potential use is discussed on the basis of their stability in the soil (some of the degradation products are more active than their precursors, particularly phototoxins), the environmental safety (biodegradability), the site of action (it has only begun to tap potential sites of herbicide action that can provide a start for biorational herbicides design around these sites), the accessibility (knowledge of the location of active principles within the plant might be crucial to determine the real possibilities of practical applications on agriculture) and the level of activity (to be successful, natural phytotoxins might be active at lower concentrations in comparison with synthetic herbicides). In this section only allelochemicals from higher plants and particularly their level of activity are discussed. The focus here is to compare the level of activity between the traditional candidates, the more recent allelochemical discovered, which may impact weed science, and the synthetic herbicides. Specifically these are compounds released either by crops, weeds, their residues or synthetic analogues. The dose of synthetic herbicides used for weed control has been changed along the different periods mentioned above. Trichloroacetic acid, corresponding to the first period, was used for non-selective weed control at rates of 55-225 kg/ha, ca. 5.5x10 2.25x10 ppb (soil weight basis, distributed 10 cm deep). 4

5


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Table I. Selected Allelopathic Compounds with Potential Use as Natural Herbicides


Name

Activity range (ppb)

Target species

3

Type of activity

Simple acids and esters L- p-Hydroxybutyric ac. (57) (13) Ethyl propionate (52,55) (14)

2.2xl0 -8.3xl0

Ethyl 2methylbutyrate (52) (15)

8.9xl0 -4.4xl0

3

4

2

3

7xl0 -3.4xl0

2

3

Growth(-) Chenopodium album L. Amaranthus retroflexus L.Growth(-) Allium cepa L. Germination(ret) Daucus car ota L. Germination(-) Lycopersicon esculentum Germination(ret) Miller Allium cepa L. Germination(-) Germination(ret) Daucus carota L. Lycopersicon esculentum Germination(ret) Miller

Polyacetylenes ta-DME

(54) (16)

Cw-dihydro-ME (54) (17)

3

10

3

4

5xl0 -5xl0

Echimochloa crus-galli (L.) Growth(-) Beauv. Growth(-) Oryza sativa L.

Long chain fatty acids Arachidic ac. (55) (18) Behenic ac. (55) (19) Myristic ac. (55)

(20)

3

Cynodon dactylon (L.) Pers. Germination(-)

3


3


5xl0 5xl0

5xl0

Alkaloids b

BOA (56) (21)

b

AZOB (57)

(22)

Caffeine (58) (23)

5

10

5xl0

5

4

5

10 -4xl0

Echinochloa crus-galli (L.) Beauv. Lepidium sativum L. Cucumis sativus L. Phaseolus vulgaris L. Echinochloa crus-galli (L.) Beauv. Lepidium sativum L. Cucumis sativus L. Phaseolus vulgaris L. Lactuca sativa L.

NA Growth(-) Growth(-) Growth(-) Growth(-) Growth(-) Growth(-) Growth(-) Growth(-)

a

Results denoted by (+) = stimulation; (-) = inhibition; (ret.) = retarding; NA = Non Active; (+,-) = stimulation or inhibition pending on the concentration; ac = acid.

b

B O A = 2(3H)-Benzoxazolinone; AZOB= 2,2-Oxo-1,1-azobenzene.


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Table I. continued Name

Activity range

Target species

Type of activity

3

(ppb)


Benzoic acid derivatives

/?-Hydroxybenzoic ac. (59-61) (24)

Allelopathy in the Search for Natural Herbicide Models - ACS

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