BIOLOGICALLY ACTIVE NATURAL PRODUCTS: AGROCHEMICALS - CHAPTER 5 ppt

In one field trial, ailanthone was sprayed postemer-gence onto seedlings of 17 species of weeds and crops.. Ailanthone exhibits a number of the same problems that have pre-vented other na

Development of an Allelopathic Compound

From Tree-of-Heaven (Ailanthus altissima)

as a Natural Product Herbicide

Rod M Heisey

CONTENTS

5.1 Introduction

5.2 Tree-of-Heaven (Ailanthus altissima)

5.3 Ailanthone

5.4 Pre- and Postemergence Effects of Ailanthone

5.5 Effects of Ailanthone on Weeds and Crops

5.6 Conclusions

References

ABSTRACT Since 1959, it has been known that Ailanthus altissima produces one or more

phytotoxic compounds The major toxin was recently identified as ailanthone, a member

of the quassinoid group In greenhouse trials, purified ailanthone exhibited moderate preemergence herbicidal activity and strong postemergence activity It also had potent her-bicidal activity under field conditions In one field trial, ailanthone was sprayed postemer-gence onto seedlings of 17 species of weeds and crops The lowest application rate (0.3 kg/ha) reduced shoot biomass of six species to ≤ 10% of the control and 10 species to

≤ 50% of the control Ailanthone is relatively nonselective, but cotton, yellow nutsedge, and

A altissima seedlings show a high level of tolerance Ailanthone also was tested in the field

for its ability to control weeds in several vegetable crops (beans, tomato, cauliflower, corn)

A marked reduction in weed population occurred for a few weeks after application, but the herbicidal activity was short-lived Nine weeks after spraying, weed biomass in the most effective treatment (0.6 kg/ha) was 60% of the weed biomass in the untreated control Ail-anthone is rapidly degraded by soil microorganisms It killed many of the weed seedlings present at the time of application, but its herbicidal activity was rapidly degraded As a result, weeds that were not killed were able to recover and a new crop of weeds was able

to emerge and grow Ailanthone exhibits a number of the same problems that have pre-vented other natural products from being developed as commercial herbicides; however, its striking herbicidal activity under field conditions justifies further investigations

5.1 Introduction

Much interest exists in using natural products to control weeds in agroecosystems, but few natural products have actually been developed into commercial herbicides.1-5 Bialaphos and glufosinate (= phosphinothricin) are the most successful natural product herbicides to date.6-9 Both are metabolites of bacteria in the genus Streptomyces, although glufosinate is

currently produced synthetically These compounds are now available as commercial her-bicides and are the only two agriculturally important herher-bicides to have resulted from phy-totoxins produced by microorganisms.5

From plants, no single compound in its naturally occurring form has yet been developed into a commercial herbicide, to the best of my knowledge.5 Many plants reportedly cause allelopathic effects, and numerous metabolites from plants are phytotoxic in laboratory bioassays,10 but few possess the level of activity necessary for development as herbicides Corn gluten meal, which was recently patented for use as a preemergence herbicide, may

be somewhat of an exception.11 The most active herbicidal components of this product are dipeptides released during hydrolysis of the corn meal,12 suggesting the involvement of microorganisms in producing the phytotoxic effects The hydrolysate is a potent inhibitor

of root system development in germinating grass seedlings.13

A number of synthetically derivatized metabolites from plants have been developed, or have potential for development, as commercial herbicides Benzoic acid, which is produced

by many plant species, has been derivatized to yield the herbicides dicamba (3,6-dichloro-2-methoxybenzoic acid) and chloramben (3-amino-2,5-dichlorobenzoic acid) The allelo-pathic terpenoid 1,8-cineole has served as a model for the development of cinmethylin, an experimental herbicide that has undergone field trials, but has not yet been released.14-16

5.2 Tree-of-Heaven (Ailanthus altissima)

Tree-of-heaven (Ailanthus altissima, Simaroubaceae) has been known since the work of

Mer-gen in 1959 to produce one or more phytotoxic and potentially allelopathic compounds.17

A altissima was introduced into the U.S from China about 300 years ago.18 It has since become naturalized in many parts of the U.S., and is especially abundant in the milder regions of the Northeast.19,20A altissima is considered an undesirable woody weed having

almost no value for timber, paper pulp, or fuelwood

In the southern parts of Pennsylvania, New York, and Connecticut, A altissima often

col-onizes disturbed sites such as abandoned fields or vacant lots20,21 and seems to inhibit the invasion of other tree species, suggesting an allelopathic effect.22 It commonly forms dense, nearly monospecific thickets, which may persist for decades.19A altissima is a vigorous

com-petitor It grows rapidly and reproduces prolifically from root sprouts, stump sprouts, and abundantly produced samaras.23-25 Aqueous extracts of A altissima tissues are strongly

phy-totoxic in laboratory bioassays.26-28 The question of whether the invasiveness of A altissima

is due to allelopathy or to its superior competitive ability, or both, remains unanswered.29

5.3 Ailanthone

The major phytotoxin produced by A altissima was recently identified as ailanthone

(Figure 5.1) This compound was first isolated and identified from A altissima in the

1960s,30-32 but it was not recognized to be the major phytotoxic component until the 1990s.33-35 Ailanthone belongs to the class of bitter-tasting compounds called quassinoids that are commonly found in members of the Simaroubaceae Quassinoids are heavily oxygen-ated lactones The majority have a C-20 basic skeleton, but C-18, C-19, and C-25 quassinoids also exist.36,37 Quassinoids are degraded triterpenoids and are believed to be synthesized in plants from ∆ 7-euphol or ∆ 7-tirucallol Over 75 quassinoids are known.38

Quassinoids exhibit a wide range of biological activity Soon after its discovery, ailanthone

was reported to have activity against the amoeba Entamoeba histolytica.39 Interest in the quassinoids was greatly stimulated by the discovery in the 1970s of antileukemic effects and other forms of anticancer activity.38,40 Additional effects have since been discovered,

includ-ing activity against the malarial parasite Plasmodium falciparum,41 antifeedant and growth inhibitory activity against insects,42-44 antiviral activity against the Rous sarcoma virus,45 and

antifungal activity against Plasmopara viticola, the cause of grape downy mildew.46

The phytotoxic effects of quassinoids were not reported until recently In 1993, holocan-thone was mentioned to have pronounced phytotoxicity at 100 ppm on grape plants.46 The herbicidal effects of purified ailanthone were first reported in 199333 and were described more extensively a few years later.34,35

In mature A altissima trees, ailanthone is most abundant in the bark of roots, bark of

branches, and the inner bark of trunks.28 Concentrations are intermediate in leaves and low

in the wood and the outer bark of the trunk Ailanthone is soluble in polar solvents such as water and methanol, and it can be easily extracted from roots and bark with pure methanol

or aqueous methanol To purify ailanthone for use in greenhouse bioassays of herbicidal activity, a crude extract of macerated roots and root bark was made with aqueous metha-nol The extract was concentrated under vacuum to remove the solvents, and the concen-trate was redissolved in water and sequentially partitioned against hexane, diethyl ether, dichloromethane, and ethyl acetate The phytotoxicity of the resulting fractions was

mon-itored with a bioassay using seeds of garden cress (Lepidium sativum) The dichloromethane

and ethyl acetate fractions were the most phytotoxic These two fractions were combined and purified by low-pressure column chromatography on silica gel and C18 packing to yield purified ailanthone.35

5.4 Pre- and Postemergence Effects of Ailanthone

Ailanthone exhibits potent herbicidal activity and appears to be a promising candidate for development as a natural product herbicide The purified compound was tested for pre-and postemergence activity in the greenhouse using seeds pre-and seedlings of weeds pre-and crops planted in field soil in small flats.35 For preemergence tests, purified ailanthone was sprayed onto the soil surface after the seeds had been planted The soil was immediately watered to carry the ailanthone down into the seed zone For postemergence tests, the

FIGURE 5.1

Structure of ailanthone (molecular mass = 376),

the major phytotoxic compound produced by

A altissima (From Heisey, R M., Am J Bot., 83,

194, 1996 With permission.)

seeds of weeds and crops were sown in flats The resulting seedlings were sprayed with ail-anthone 4 to 6 days after emergence The purified ailail-anthone was dissolved in distilled water containing 0.36% X-77 surfactant and sprayed at rates ranging from 0 (control) to 8 kg/ha

5.5 Effects of Ailanthone on Weeds and Crops

Even the lowest application of ailanthone (0.5 kg/ha) slowed or reduced seedling emer-gence Emergence was completely inhibited for at least 6 days after spraying in treatments receiving 4 and 8 kg/ha (Figure 5.2) Eventually some seedlings emerged, even in the

8 kg/ha treatment, but the number was greatly reduced relative to the control

The postemergence effects of ailanthone were more striking (Figure 5.3) Even the lowest

dose (0.5 kg/ha) killed all seedlings except those of velvetleaf and A altissima Velvetleaf

was somewhat resistant to ailanthone, but at the higher rates it was severely injured or

killed A altissima seedlings were remarkably tolerant to ailanthone and showed no visible injury even at the highest application (8 kg/ha) This result indicates A altissima possesses

one or more mechanisms to avoid autotoxicity

The strong postemergence activity of ailanthone suggested its herbicidal effects should

be tested under actual field conditions Much larger quantities of ailanthone were needed for field trials Because of the difficulties associated with purifying such a large amount of ailanthone, a crude extract prepared from the inner bark of the trunk was used as the spray material The amount of ailanthone in the crude extract was first quantified with a bioassay Appropriate quantities of the crude extract were then applied to the field plots to deliver the desired amounts of ailanthone

The first field trial was designed to examine the herbicidal effects and selectivity of ail-anthone on 17 species of weeds and crops under actual field conditions A randomized block design containing four replicates (only two in 4.5 kg/ha treatment) was used Each

of the 17 plant species was sown in two adjacent rows running the length of the blocks Each block was randomly subdivided into six treatments (0.4 × 7.8 m) containing two adja-cent rows of all 17 species

FIGURE 5.2

Preemergence effects of purified ailanthone shown 6 days after application to seeds planted in soil Application rates (left to right) are 0 (control), 0.5, 1, 2, 4, and 8 kg of ailanthone per hectare The plant species, as shown by the control, are (front to rear of flats) redroot pigweed, garden cress, velvetleaf, foxtail, barnyard grass, and corn

(cv Silver Queen) (From Heisey, R M., Am J Bot., 83, 196, 1996 With permission.)

The crude extract of A altissima bark was sprayed onto the seedlings 3 weeks after

plant-ing Application rates were adjusted to provide 4.5, 2.2, 1.1, 0.6, 0.3, and 0.0 (control) kg/ha

of ailanthone, based on the 1.2% content of ailanthone in the crude extract For the treat-ments of 2.2, 1.1, 0.6, and 0.3 kg/ha, the extract was dissolved or suspended in distilled water containing 0.36% X-77 surfactant These solutions were applied with a double-nozzle spray boom on a knapsack sprayer adjusted to deliver 935 L of solution per hectare at

30 psi The control received a similar application of distilled water containing 0.36% X-77 Only two replications of the 4.5 kg/ha treatment were possible because of the limited amount of extract available Because of difficulties in dissolving and spraying such a high concentration of extract, the 4.5 kg/ha treatment was sprayed twice with a solution having the same concentration of extract as the 2.2 kg/ha treatment (but half the concentration of X-77) The effects of the treatments were evaluated 19 days after spraying At this time, all plants were cut at soil level, dried at 55 to 60°C, and weighed for biomass determination The results of this investigation showed that the potent postemergence effects of ailan-thone previously observed in the greenhouse also occurred under field conditions (Table 5.1) Symptoms of phytotoxicity were evident on certain species in less than 24 h after spraying Even the lowest application rate tested (0.3 kg/ha) reduced shoot biomass

in 10 of the 17 species to ≤50% of the control Shoot biomass of the six most sensitive species (redroot pigweed, common lambsquarters, bindweed, sunflower, tomato, and radish) was reduced to ≤10% of the control by 0.3 kg/ha of ailanthone

A wide range in sensitivity to ailanthone occurred (Table 5.1) Yellow nutsedge and cotton were the most resistant and showed no reduction in shoot biomass, even at the highest appli-cation (4.5 kg/ha) Velvetleaf, large crabgrass, and Johnson grass also exhibited a high level

of resistance Broadleafed species (dicots) generally were more sensitive to ailanthone than grasses and yellow nutsedge (monocots) The resistance of cotton and velvetleaf suggested species in the Malvaceae may be tolerant to ailanthone and indicated additional members of this family should be examined in future tests All crop species other than cotton (oats, soy-bean, sunflower, tomato, squash, radish, corn) exhibited high-to-moderate sensitivity

FIGURE 5.3

Postemergence effects of purified ailanthone shown 5 days after spraying on emerged seedlings The application rates and species are identical to those in Figure 5.2 , except an additional row at the rear of the flats contains

A altissima seedlings (From Heisey, R M., Am J Bot., 83, 197, 1996 With permission.)

These results provided strong evidence that ailanthone has significant herbicidal activity under field conditions Therefore, a second field trial was conducted to test the effective-ness of ailanthone in controlling weeds in an actual cropping system This trial was based

on the following rationale Ailanthone is very expensive to produce, even in the form of a crude extract If it is going to be developed as a herbicide, its most likely market would be

as a specialty product for organic growers or home gardeners who want to control weeds chemically but without synthetic pesticides This field trial had two goals: (1) to evaluate the ability of ailanthone to control weeds in an actual agricultural system, and (2) to deter-mine what effects ailanthone would have on certain vegetable crops that might be planted

by a home gardener or organic grower

The field trial was set up in a randomized block design with four replicates Four

vege-table crops (green bean, Phaseolus vulgaris cv Tendergreen; tomato cv Roma; cauliflower,

Brassica oleracea cv Snow Crown Hybrid; and sweet corn cv Silverado) were planted in

rows running the length of the blocks The tomatoes and cauliflower were planted as trans-plants and the beans and corn were direct seeded The plots were sprayed about 1 week after planting At this time a high population of recently-emerged weed seedlings ranging from 0 to 5 cm tall was present The crude extract was dissolved or suspended in distilled water containing 0.36% X-77 surfactant and sprayed directly onto the weeds and crops at rates calculated to deliver 1.1, 0.6, 0.3, 0.15, and 0 (control) kg/ha of ailanthone Weed den-sity and crop injury were monitored for several weeks after spraying Crop yield and weed biomass were determined 6 to 9 weeks after spraying, when the investigation was ended The initial results demonstrated ailanthone did have very impressive herbicidal effects

The major weeds in the plots were galinsoga (Galinsoga ciliata), black nightshade (Solanum

nigrum), common lambsquarters, redroot pigweed, carpet weed (Mullugo verticillata), and

several grasses (Figure 5.4a) Even at the lowest application (0.15 kg/ha), a reduction in the weed population was noticeable and the crops were not seriously harmed A greater reduc-tion in weed populareduc-tion occurred at 0.3 kg/ha, but damage to cauliflower was evident (Figure 5.4b) Weed control increased in the 0.6 kg/ha treatment, but crop damage became

TABLE 5.1

Applications of Ailanthone (kg/ha) Needed to Reduce Shoot Biomass

of Weeds and Crops to 50% (ID50) and 10% (ID10) of Control Biomass

Velvetleaf (Abutilon theophrasti) 1.1 >4.5

Redroot pigweed (Amaranthus retroflexus) <0.3 <0.3

Oats (Avena sativa cv Ogle) <0.3 0.6

Common lambsquarters (Chenopodium album) <0.3 <0.3

Field bindweed (Convolvulus arvensis) <0.3 <0.3

Yellow nutsedge (Cyperus esculentus) >4.5 >4.5

Large crabgrass (Digitaria sanguinalis) 1.1 >4.5

Barnyard grass (Echinochloa crus-galli) <0.3 2.2

Soybean (Glycine max cv Agripro 4420) <0.3 4.5

Cotton (Gossypium hirsutum cv Koker 315) >4.5 >4.5

Sunflower (Helianthus annuus cv Black Oil Hybrid) <0.3 0.3

Tomato (Lycopersicon esculentum cv Roma) <0.3 <0.3

Squash (Cucurbita pepo cv Black Beauty zucchini) <0.6 4.5

Radish (Raphanus sativus cv Cherry Belle) <0.3 <0.3

Johnson grass (Sorghum halepense) 0.6 >4.5

Corn (Zea mays cv Iochief) <0.3 <0.6

Note: Applications used in this investigation were 4.5, 2.2, 1.1, 0.6, and 0 (control)

kg/ha Data were collected 19 days after ailanthone application.

(b) FIGURES 5.4a-d

Effects of ailanthone on weeds and crops shown 21 days after spraying (a) Control (0 kg/ha ailanthone), (b) treatment receiving 0.3 kg/ha ailanthone (=0.25 lb/acre), (c) treatment receiving 0.6 kg/ha ailanthone (=0.5 lb/acre), and (d) treatment receiving 1.1 kg/ha ailanthone (=1.0 lb/acre) The four crops present in rows running the width of the plots are (front to rear) corn, cauliflower, tomato, and green beans.

(d) FIGURE 5.4 (continued)

more serious (Figure 5.4c) Very strong suppression of weeds occurred at 1.1 kg/ha, but moderate to severe injury was present on all crops except beans (Figure 5.4d) Cauliflower was the most sensitive of the four crops tested and some of the plants were killed at the higher applications of ailanthone Corn and tomato were intermediate in sensitivity, and beans were the most tolerant

Although the weed suppression caused by ailanthone initially was impressive, it even-tually became apparent that the herbicidal effects were short-lived Weed biomass in the treated plots was reduced throughout the duration of the investigation, but the results at the end of the investigation (9 weeks after spraying) were not as striking as those observed initially (Table 5.2) The reduction in weed biomass for the 0.3, 0.6, and 1.2 kg/ha treat-ments was statistically significant (P = 0.05) compared to the 0 kg/ha control The greatest reduction was in the 0.6 kg/ha treatment, where biomass was reduced to 60% of that in the control The reason why 0.6 kg/ha reduced weed biomass more than 1.1 kg/ha was not readily apparent

The reason the suppression in weed biomass at the end of the investigation was less impressive than the reduction in weed population immediately after spraying is because ailanthone degrades rapidly in the field Ailanthone killed many of the weeds that were present at the time of spraying, but its toxicity disappeared quickly As a result, weeds that were not killed were able to recover and a new crop of weeds was able to emerge and grow normally This result was not surprising because previous work had shown ailanthone is degraded by microorganisms within several days of application to soil.28,35 The microbial transformation of other herbicidal natural products also has been reported for juglone from black walnut,47 2(3H)-benzoxazoline from rye,48 and phenolic compounds.49

It is difficult to compare the herbicidal effects of ailanthone with those of other natural products from plants because few of these compounds have been tested as herbicides under actual field conditions Shettel and Balke investigated the pre- and postemergence herbicidal effects of salicylic acid, coumarin, and caffeine.50 Their postemergence effects were stronger than the preemergence effects, but even at 11.2 kg/ha these compounds showed only mild herbicidal action Ailanthone appears to be considerably more active under field conditions The comparatively high phytotoxicity of ailanthone under field conditions justifies further investigations of its potential for development as a herbicide Despite its potential, ailanthone exhibits several of the problems that have traditionally inhibited the development of natural product herbicides Ailanthone and many other nat-ural products are degraded rapidly in the environment by microbial or physicochemical processes Short persistence may be advantageous from the standpoint of safety to humans and the environment, but it can limit herbicidal efficacy Ailanthone, like many other sec-ondary plant products, is produced only in small amounts and has a relatively complex molecular structure The ailanthone content of the bark used to produce the crude extracts

TABLE 5.2

Shoot Biomass of All Weed Species Present in Plots

9 Weeks after Spraying with Ailanthone

Ailanthone Application Rate (kg/ha)

0 0.15 0.3 0.6 1.1 Weed biomass 100a 84ab 74b 60b 76b

Note: Data are expressed as percentage of biomass in

control plots (0 kg/ha) Data followed by different letters differ at 0.05 probability level in Duncan’s multiple range test.

in these investigations was about 0.06% of bark fresh weight These characteristics limit yields of the compound from natural sources and hinder commercial synthesis at a price competitive with artificial pesticides

The question of how to produce ailanthone in large quantities at a competitive price must

be answered if ailanthone is to be developed as a commercial herbicide Ailanthone has not yet been synthesized artificially in the laboratory, to the best of my knowledge Numerous attempts have been made to synthesize related quassinoids Only a few have been success-ful, the procedures are complex, and the yields are low.37,51-53

Extraction from trees currently is the best way to obtain ailanthone, but doing so would

be expensive on a large scale For example, a typical 15 year-old tree will have a trunk diameter of about 25 cm and will yield about 25 kg (fresh-weight) of easily harvestable bark with an ailanthone content of about 0.06% A tree this size, therefore, would yield 14 g

of pure ailanthone which is enough to treat 100 m2 (0.01 ha) at 1.2 kg/ha If one were to

grow A altissima trees 4 m apart in plantations, giving a population of 625 trees per hectare,

the yield of ailanthone would be 8.75 kg in 15 years This would be enough to treat about

7 ha at 1.2 kg/ha Obviously, producing ailanthone this way would be expensive and not economically competitive with synthetic herbicides This is a good example of why the pes-ticide industry currently favors artificial synthesis over biosynthesis.3

Cell culture is another possibility for producing ailanthone A altissima cells have already

been cultured by other investigators Ailanthone production has been demonstrated, but the concentrations have been below those in the bark of intact trees.54,55 Therefore, if ailan-thone is to be developed as a herbicide, large-scale production is an issue that must be addressed

5.6 Conclusions

Ailanthone exhibits strong postemergence herbicidal activity, not only in the greenhouse, but also under actual agricultural conditions in the field Its herbicidal activity, however, appears to be degraded within several days under field conditions Weed seedlings present

in the field at the time of spraying are killed or injured, but after several days the ailanthone

is degraded and a new cohort of weeds is able to emerge More research is needed to develop ways to extend the period of activity Multiple applications over the growing sea-son might alleviate the problem, but also would increase costs The phytotoxicity of ailan-thone to certain crops is another issue One solution would be to develop ways to apply ailanthone without getting it on the crops Methods to produce ailanthone in large quanti-ties at a competitive price also must be investigated The fact that ailanthone is a natural product with potent herbicidal activity under field conditions makes these topics worthy

of further investigation

References

1 McLaren, J S., Biologically active natural substances from higher plants: status and future

potential, Pest Sci., 17, 559, 1986.

2 Putnam, A R., Allelochemicals from plants as herbicides, Weed Technol., 2, 510, 1988.