Allelochemicals: Role in Agriculture and Forestry - American Chemical

Chapter 36

Natural Plant Compounds Useful in Insect Control James A. Klocke

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NPI, University Research Park, Salt Lake City, UT 84108

Extracts of plants have been used as insecticides by humans since before the time of the Romans. Some of these extracts have yielded compounds useful as sources (e.g., pyrethrins, rotenoids, alkaloids), others as models (e.g., pyrethrins, physostigmine) of commercial insecticides. Recent technological advances which facilitate the isolation and identification of the bioactive constituents of plants should ensure the continued usefulness of plant compounds in commercial insect control, both as sources and models of new insect control agents and also as components in host plant resistance mechanisms. The focus in this paper will be on several classes of compounds, including limonoids, chromenes, ellagitannins, and methyl ketones, which were found to be components of the natural defenses of both wild and cultivated plants and which may be useful in commercial insect control. Insect resistance and environmental pollution due to the repeated application of persistent synthetic chemical insecticides have led to an increased interest in the discovery of new chemicals with which to control insect pests. Synthetic insecticides, including chlorinated hydrocarbons, organophosphorus esters, carbamates, and synthetic pyrethroids, will continue to contribute greatly to the increases in the world food production realized over the past few decades. The dollar benefit of these chemicals has been estimated at about $4 per $1 cost (I). Nevertheless, the repeated and continuous annual use in the United States of almost 400 million pounds of these chemicals, predominantly in the mass agricultural insecticide market (2), has become problematic. Many key species of insect pests have become resistant to these chemicals, while a number of secondary species now thrive due to the decimation of their natural enemies by these nonspecific neurotoxic insecticides. Additionally, these compounds sometimes persist in the environment as toxic residues, well beyond the time of their intended use. New chemicals are therefore needed which are not only effective pest 0097-6156/87/0330-0396$06.00/0 © 1987 American Chemical Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, G.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

36.

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Natural

Plant Compounds

Useful in Insect

Control

control agents, but which are safe, s e l e c t i v e , biodegradable, environmentally acceptable, economically v i a b l e , and suitable for use in programs of integrated pest management (3). One approach to the discovery of new i n s e c t i c i d e s which f u l f i l l the c r i t e r i a of e f f i c a c y , safety, s e l e c t i v i t y , etc., i s through the study of the natural chemical defenses of plants. Extracts of plants have been used as i n s e c t i c i d e s by humans since before the time of the ancient Romans, a practice that continues today with many of the 2000 species of plants known to have i n s e c t i c i d a l properties (4-5). The use of " i n s e c t i c i d a l plants i s e s p e c i a l l y prevalent among subsistence farmers since plants grown l o c a l l y are cheaper, and sometimes more accessible, than synthetic chemical p e s t i c i d e s . Commercially, however, only a few of these plants, including those containing pyrethrins, rotenoids, and a l k a l o i d s , have been used to any extent i n the United States as sources of i n s e c t i c i d e s (6-8). The most economically important group of natural plant i n s e c t i cides are the pyrethrins, a group of s i x c l o s e l y related esters extracted from pyrethrum (Chrysanthemum cinerariaefolium) flower heads (Figure 1). Pyrethrum has been used as an i n s e c t i c i d e since at least the early 1800*s i n Persia and Yugoslavia. By 1828 pyrethrum was being processed for commercial insect control, and by 1939 imports of pyrethrum into the United States reached a peak of 13.5 m i l l i o n pounds. Use of the natural product declined i n the early 1950's because of the advent of synthetic pyrethroid analogs (for example, a l l e t h r i n s ) , which were both more stable and more e f f e c t i v e i n the f i e l d . The present worldwide demand for pyrethrum flowers remains i n excess of 25,000 tons annually and i s s a t i s f i e d by the estimated 150 m i l l i o n flowers s t i l l hand-harvested d a i l y , predominantly i n natural stands and c u l t i v a t e d f i e l d s i n Kenya, Tanzania, and Ecuador (9). Rotenone and the rotenoids (Figure 2) have long been used as insecticides and p i s c i c i d e s ( f i s h poisons). By the early 1950's more than 7 m i l l i o n pounds of Leguminosae roots (Derris, Lonchocarpus, and Tephrosia spp.) containing these i n s e c t i c i d e s were imported annually into the United States. In 1972, about 1.5 m i l l i o n pounds of the roots were used i n the United States for pest control i n the home and garden markets and to control ectoparasites on animals (10). Among the most important of the natural alkaloids used i n insect control have been nicotine and the related compound nornicotine (Figure 3). The use of these i n s e c t i c i d a l alkaloids dates back to the 1600's and grew to 5 m i l l i o n pounds by the mid-1900's. Since then, the annual worldwide production of nicotine has dropped to about 1,250,000 pounds of nicotine sulfate and 150,000 pounds of nicotine a l k a l o i d because of the high cost of production, disagreeable odor, extreme t o x i c i t y to mammals, and l i m i t e d i n s e c t i c i d a l a c t i v i t y (10-11). The s t r u c t u r a l l y related compounds anabasine (neonicotine) (Figure 3) i s currently i n commercial use i n the Soviet Union (J5). Other less important i n s e c t i c i d a l alkaloids include veratrine [a mixture of alkaloids (cevadine, v e r a t r i d i n e , and s a b a c i l l i n e ) ] and ryanodine. Physostigmine (Figure 4), another alkal o i d that i s i s o l a t e d from the calabar bean (Physostigma venenosum), served as a model compound for the development of the carbamate insecticides (12-13)· 11


397


398

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

R=CH

R

3

CH =

CH

2

Pyrethrin 1 Pyrethrin II

R=CH OCO

R'=

C H — C H

R =

R'=

CH

3

Cinerin 1

R = CH OCO

R = CH

3

Cinerin II

R=

R

3


'=

CH

3

3

CH

3

R = CH OCO 3

2

'= C H

2

-CH

3

Jasmolin

1

R'= C H

2

-CH

3

Jasmolin

II

Figure 1. Structures of Six I n s e c t i c i d a l Esters Extracted from Pyrethrum (Chrysanthemum cinerariaefolium)

Figure 2 . Structure of Rotenone, an Insecticide and P i s c i c i d e Isolated from Leguminosae Roots

Ν R =CH R=H

3

NICOTINE NORNICOTINE

ANABASINE

Figure 3. Structures of Some Plant Alkaloids Used i n Insect Control



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Natural

Plant Compounds

Useful in Insect

Control

399

Insect growth regulators, including analogs and antagonists of endogenous hormones, have also been i d e n t i f i e d i n plants. Prominent among these are the analogs of two insect hormones (juvenile hormone and molting hormone) and the antagonist for juvenile hormone. Analogs of juvenile hormones found i n plants include the juvocimenes in Ocimum basilicum, juvabione i n Abies balsamea, and farnesol i n many plant o i l s (14). These natural plant products have never been used commercially as a source of i n s e c t i c i d e s , but they have served as model compounds for the development of synthetic juvenile hormone analogs such as kinoprene and methoprene (Figure 5). Chemicals s t r u c t u r a l l y similar or i d e n t i c a l to the insect molting hormone (ecdysterone) have been found i n many plants, especially in ferns and yews (15). One example i s ponasterone A, which d i f f e r s s t r u c t u r a l l y from the insect molting hormone only i n the absence of a hydroxyl group at C-25 (Figure 6). Ponasterone A caused severe disruption of the f i n a l stages of molting (termed ecdysis) when fed at 2 ppm i n a r t i f i c i a l diet to pink bollworm (Pectinophora gossypiella) larvae (16). Figure 7 i s an electron micrograph of _P. gossypiella fed 2 ppm ponasterone A i n a r t i f i c i a l d i e t . The insect possessed three head capsules because i t underwent two f a i l e d molting cycles before death. Even though feeding became impossible after the f i r s t inhibited ecdysis (because the adhering second head capsule covered the mouth parts) the larva produced a third head capsule before i t s death. While the above observation i s interesting and could possibly have some implications for the control of the pink bollworm, the complexity of the steroid nucleus of ponasterone A and other molting hormone analogs and their weak i n s e c t i c i d a l e f f e c t when applied t o p i c a l l y or administered o r a l l y to most species of economically important insects may preclude their commercialization. The only commercial use of the molting hormone analogs thus f a r has been i n the s e r i c u l t u r a l industry f o r the synchronization of cocoon spinning of silkworm colonies (17)· Juvenile hormone antagonists were f i r s t isolated from the bedding plant Ageratum houstonianum (Asteraceae) (18). The active constituents, termed precocenes I and I I , were shown to be chromenes (Figure 8)· Although these compounds are highly active against several species of insects (e.g., the large milkweed bug, Oncopeltus f a s c i a t u s ) , the precocenes have been found to be e f f e c t i v e on r e l a t i v e l y few economically important species of insects, and their potential for commercialization may therefore be l i m i t e d . Other chromenes, which d i f f e r s t r u c t u r a l l y only i n the moieties attached to C-6 and C-7 (Figure 8), have been isolated from other species i n the Asteraceae, including xerophytic Encelia species (19) and Hemizonia f i t c h i i (20). The H. f i t c h i i chromenes, including encecalin, eupatoriochromene, and 6-vinyl-7-methoxy-2,2dimethylchromene (Figure 8), were moderately toxic to house mosquito (Culex pipiens) larvae, with 6-vinyl-7-methoxy-2,2-dimethylchromene being the most active, and to large milkweed bug (0. fasciatus) nymphs, with encecalin being the most active (Tables I and I I , r e s p e c t i v e l y ) . Although these compounds are i n s e c t i c i d a l , they showed no antijuvenile hormone a c t i v i t y . Apparently, the presence of a v i n y l or a methyl ketone moiety, such as found i n the Hemizonia chromenes, rather than a methoxy substituent, such as found i n the


400


Ο


II

Figure 4 . Structure of Physostigmine, an I n s e c t i c i d a l A l k a l o i d Isolated from Physostigma venenosum as a Model Compound f o r the Synthetic Carbamate Insecticides

Juvenile Hormone 3 (JH 3)

Figure 5. Structures of the Insect Juvenile Hormone 3 and a Commercially Available Analog, Methoprene

OH

ECDYSTERONE PONASTERONE A

Figure 6. Stereostructures of the Insect Molting Hormone, Ecdysterone, and a Plant Analog, Ponasterone A



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Natural

Plant Compounds

Useful in Insect

Control

401

Figure 7. Electron Micrograph I l l u s t r a t i n g Ecdysis I n h i b i t i o n of a Larva of the Pink Bollworm, Pectinophora gossypiella after Ingestion of 2 ppm Ponasterone A i n an A r t i f i c i a l Diet (Magnification χ 100)

Jijl

Jl

2j>

C H

3

R_

2

Precocene 1 Precocene

II

Η OCH

3

Desmethoxyencecalin

COCH

Eupatoriochromene

COCH

Encecalin

COCH

6-Vinyl-7-methoxy2,2-dimethylchromene

CH = CH

OCH

3

OCH

3

Η

3

OH

3

OCH

3

2

3

OCH

3

Figure 8, Structures of some I n s e c t i c i d a l 2,2-Dimethylchromenes Isolated from Plant Species i n the Asteraceae



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FORESTRY

precocenes, r e s u l t s i n a loss of a n t i j u v e n i l e hormone a c t i v i t y . In f a c t , Bowers (21-22) found that alkoxy substitution of the chromene aromatic r i n g i n the C-6 and e s p e c i a l l y the C-7 positions was necessary for a n t i j u v e n i l e hormone a c t i v i t y . Although assays with other insects should be conducted, i t does not seem from an economic standpoint that the Encelia and Hemizonia chromenes themselves are of s u f f i c i e n t potency to warrant adaptation into pest management strategies. However, the r e l a t i v e ease of extraction of the H. f i t c h i i chromenes, as exemplified by their abundance (63%) i n the v o l a t i l e o i l (steam d i s t i l l a t e ) f r a c t i o n (Table I I I ) , coupled with the a v a i l a b i l i t y of the xerophytic Hemizonia plant material, make these compounds useful as models for new synthetic or semisynthetic i n s e c t i c i d e s . The a v a i l a b i l i t y and b i o l o g i c a l a c t i v i t y of xerophytic plants, such as H. f i t c h i i , make them a promising area of research for the discovery of new i n s e c t i c i d e s . Xerophytic plants are available on a large scale from marginal regions that generally cannot be used economically for food production (23-24). The f o l i a g e of many xerophytic plants has been found to be b i o l o g i c a l l y active, both as i n s e c t i c i d e s (19, 25) and as insect growth i n h i b i t o r s (26). E l l a g i c acid, a phenolic dilactone (Figure 9), was i s o l a t e d and spectrally i d e n t i f i e d as an insect growth i n h i b i t o r from the methanolic extracts of f i v e species of xerophytic plants, including Geranium viscosissimum var. viscosissimum, Erodium cicutarium, Tamarix chinensis, Quercus gambelii, and Cistus v i l l o s u s (26). Against the polyphagous herbivore, H e l i o t h i s virescens (tobacco budworm), the E C 5 0 (the e f f e c t i v e concentration for 50% growth i n h i b i t i o n ) was found to be 147 ppm or 0.49 mmol/kg diet wet weight (Table IV), well below the 0.77%-1.50% unbound e l l a g i c acid (dry weight basis) found i n the hot methanolic extracts of the a e r i a l parts of the f i v e plant species (26). Although the large amounts of unbound e l l a g i c acid could explain, for the most part, the a c t i v i t y of the methanol extracts against _H. virescens ( E C 5 0 = 0.0147%), i n the fresh plant very l i t t l e e l l a g i c acid i s found i n the unbound form, but i s instead bound into more complex molecules such as the ellagitannins (27-28). For instance, i n a number of species of Geranium, e l l a g i c acid i s predominantly bound i n an e l l a g i t a n n i n , geraniin (and a l l i e d compounds) (Figure 1 0 ) (29-30). A milder extraction methodology, that i s , one excluding heat, revealed this also to be the case for _G. viscosissimum var. viscosissimum. Geraniin was e a s i l y detected i n tissue which had been extracted at room temperature, while no geraniin was detected i n heated samples of s i m i l a r t i s s u e . Apparently, the large amounts of free (unbound) e l l a g i c acid detected, at least for £. viscosissimum var. viscosissimum, were a r t i f a c t s of the extraction methodology employed. Since e l l a g i c acid i s probably not encountered free i n large amounts i n fresh plant tissues, but rather as one or more bound forms, we i s o l a t e d and bioassayed geraniin. On a mmol/kg diet basis, geraniin was almost twice as active as a growth i n h i b i t o r of H. virescens larvae than was e l l a g i c acid ( E C 5 0 = 0.26 and 0.49, respectively) (Table IV). An increased a c t i v i t y of geraniin might be expected since complete hydrolysis of the compound would y i e l d , i n addition to e l l a g i c acid, equimolar amounts of b r e v i f o l i n (or other phenolic lactones), and g a l l i c acid (Figure 11) (31-32).


36.

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Natural

Plant Compounds

Useful in Insect

Control

403


Table I. Bioassay of Hemizonia f i t c h i i Chromenes with Larvae of Culex pipens Compound Tested

Instar Tested

LC5o(ppm)

6-Vinyl-7-methoxy2,2-dimethylchromene Encecalin

1st 3rd 1st 3rd 1st 3rd

1.8 3.8 3.0 6.6 6.4 13.0

Eupatoriochromene

Table I I .

Topical Bioassay of Hemizonia f i t c h i i Chromenes with Nymphs of Oncopeltus f a s c i a t u s ^

Compound Tested

Instar Tested

LD (yg)

2nd 3rd 2nd 3rd 2nd 3rd

10 11 23 35 b c

Encecalin 6-Vinyl-7-methoxy2,2-dimethylchromene Eupatoriochromene

50

Assay period was 8-10 days, s u f f i c i e n t time for control insects to ^undergo two molts. No e f f e c t was observed with 100 yg. No e f f e c t was observed with 200 yg.

Table I I I . Constituents of V o l a t i l e O i l of Hemizonia f i t c h i i Compound Class

Peak Area (% of total)

V o l a t i l e f a t t y acids Monoterpenes Sesquiterpenes Chromenes Miscellaneous constituents Alkanes

0.56 29.04 2.50 62.70 3.10 1.70

a

b

The monoterpene f r a c t i o n was predominantly made up of 1,8-cineole. The chromene f r a c t i o n was predominantly made up of encecalin and eup a t ο r io chr omene.


404


FORESTRY


Figure 9. Structure of E l l a g i c Acid, an Insect Growth Inhibitor Isolated from the Extracts of a Number of Xerophytic Plant Species

Figure 10. Structure of Geraniin, an Insect Growth Inhibitor Isolated from Geranium Species

Table IV.

Growth-Inhibitory A c t i v i t y of Some Bioactive Constituents Derived from Geranium viscosissimum var. viscosissimum Fed i n an A r t i f i c i a l Diet to F i r s t - I n s t a r Larvae of H e l i o t h i s virescens EC a (mmol/kg diet) 50

Test Compound E l l a g i c acid Geraniin G a l l i c acid

0.49 0.26 7.40

EC (ppm)

a

5 0

147 250 1262

Confidence limits

b

Slope

103- 140 195- 320 931-3843

b

1.78 1.68 2.03

EC50 i s the e f f e c t i v e concentration of additive necessary to reduce l a r v a l growth to 50% of the control values. Units are given i n both mmol/kg diet wet weight and ppm of diet wet weight. Confidence l i m i t s and slope were determined using the method of L i t c h f i e l d and Wilcoxon (66).



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Natural Plant Compounds

Useful in Insect

405

Control

A l t h o u g h t h e b i o l o g i c a l a c t i v i t y of b r e v i f o l i n as an i n s e c t growth i n h i b i t o r i s unknown, g a l l i c a c i d e x h i b i t e d some g r o w t h - i n h i b i t o r y a c t i v i t y a g a i n s t H . v i r e s c e n s l a r v a e (EC50 = 7.40 mmol/kg d i e t ) (Table IV). I n l i g h t o f the s u s c e p t i b i l i t y of a t l e a s t one s p e c i e s o f i n s e c t ( i . e . , Iî. v i r e s c e n s ) t o i n g e s t i o n of the f r e e and bound forms of e l l a g i c a c i d , c o u p l e d w i t h t h e l a r g e amounts o f such forms found i n a number of p l a n t s p e c i e s , e l l a g i c a c i d may be a c o n s t i t u e n t o f the c h e m i c a l d e f e n s e s of c e r t a i n p l a n t s a g a i n s t some i n s e c t s . As s u c h , i t c o u l d become a c a n d i d a t e model f o r compound d e s i g n , i . e . , f o r t h e s y n t h e s i s and s e m i s y n t h e s i s o f new i n s e c t i c i d e s , e s p e c i a l l y i f i t s water s o l u b i l i t y and p o t e n c y can be enhanced. Fortunately, ellagic a c i d i s n e i t h e r mutagenic (33) n o r a c u t e l y t o x i c i n t e s t s w i t h e x p e r i m e n t a l a n i m a l s (34-35) and humans ( 3 6 ) . Of c o u r s e , more a c t i v e compounds m o d e l l e d a f t e r e l l a g i c a c i d would a l s o have to be t e s t e d f o r m u t a g e n i c i t y , mammalian t o x i c i t y , e t c . Perhaps t h e most p r o m i s i n g o f the p l a n t s p e c i e s y i e l d i n g i n s e c t i c i d a l compounds a r e two s p e c i e s o f t r e e s i n the M e l i a c e a e , A z a d i r a c h t a i n d i c a (neem) and M e l i a a z e d a r a c h ( c h i n a b e r r y ) . Both s p e c i e s y i e l d the p o t e n t i n s e c t i c i d e a z a d i r a c h t i n , a h i g h l y d é r i v â t i z e d t e t r a n o r t r i t e r p e n o i d o f the l i m o n o i d t y p e . Although the s k e l e t a l s t r u c t u r e and s t e r e o c h e m i s t r y o f a z a d i r a c h t i n have not been t o t a l l y r e s o l v e d ( F i g u r e 12) ( 3 7 ) , the p o t e n t and s p e c i f i c e f f e c t s o f t h i s n a t u r a l p r o d u c t have w a r r a n t e d i t s e v a l u a t i o n as a s o u r c e o f (38) and a model f o r (39) new c o m m e r c i a l i n s e c t c o n t r o l a g e n t s . A z a d i r a c h t i n has a v e r y p r o m i s i n g p o t e n t i a l because of i t s p o t e n c y (comparable to t h e most p o t e n t c o n v e n t i o n a l s y n t h e t i c i n s e c t i c i d e s ) , s p e c i f i c i t y ( a f f e c t i n g b e h a v i o r a l and b i o c h e m i c a l and d e v e l o p m e n t a l p r o c e s s e s p e c u l i a r to i n s e c t s ) , n o n - m u t a g e n i c i t y ( i n the Ames t e s t ) ( 4 0 ) , and s y s t e m i c a c t i v i t y i n plants (being t r a n s l o c a t e d throughout t h e p l a n t f o l l o w i n g a b s o r p t i o n through the r o o t s y s t e m ) . However, the accumulated i n f o r m a t i o n on a z a d i r a c h t i n , w h i l e p r o m i s i n g , i s p r e s e n t l y much l e s s t h a n t h a t needed f o r i n s e c t i c i d a l p r o d u c t commercialization (41). The mode o f a c t i o n , s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s ( S A R ' s ) , f o r m u l a t i o n , and m e t a b o l i s m o f a z a d i r a c h t i n a r e not y e t w e l l u n d e r s t o o d . Furthermore, formulation studies are r e q u i r e d p r i o r to p r o d u c t development and c o m m e r c i a l i z a t i o n . C o n s e q u e n t l y , f u r t h e r i n v e s t i g a t i o n s a r e needed b e f o r e t h e f u l l p o t e n t i a l o f a z a d i r a c h t i n as an i n s e c t c o n t r o l agent o r i n s e c t i c i d e can be r e a l i z e d . I n o r d e r t o conduct s u c h i n v e s t i g a t i o n s , l a r g e amounts o f p u r i f i e d a z a d i r a c h t i n are needed. I s o l a t i o n schemes p r e v i o u s l y r e p o r t e d f o r a z a d i r a c h t i n have i n c l u d e d s o l v e n t e x t r a c t i o n and p a r t i t i o n f o l l o w e d by chromatography, e s p e c i a l l y open column and t h i n l a y e r chromatography (TLC) (42-44) a n d , more r e c e n t l y , h i g h performance l i q u i d chromatography (HPLC) ( 4 5 - 4 6 ) . While p r e p a r a t i v e HPLC i s an e x c e l l e n t t e c h n i q u e f o r g e n e r a t i n g a z a d i r a c h t i n of h i g h p u r i t y , a g e n e r a l method u t i l i z i n g the r a p i d and i n e x p e n s i v e t e c h n i q u e o f f l a s h chromatography (48) f o r the e f f i c i e n t p r e l i m i n a r y p u r i f i c a t i o n o f samples t h a t w i l l not r e a d i l y c o n t a m i n a t e e x p e n s i v e HPLC columns was d e v e l o p e d ( 4 7 ) . F u r t h e r p u r i f i c a t i o n ( g r e a t e r than 99%) was a c c o m p l i s h e d w i t h p r e p a r a t i v e HPLC. A z a d i r a c h t i n has s e v e r a l e f f e c t s on a number o f important species of i n s e c t p e s t s , i n c l u d i n g feeding

economically deterrency,



406


FORESTRY

growth i n h i b i t i o n , and ecdysis i n h i b i t i o n (49-50). Azadirachtin i s a p a r t i c u l a r l y potent feeding deterrent (or antifeedant) against the f a l l armyworm, Spodoptera frugiperda, having a PC95 (the concen t r a t i o n resulting i n 95% "protection" of treated disks) i n a cotton leaf disk assay of 0.1 yg/1.0 cm^ leaf disk (Table V). Against the corn earworm, H e l i o t h i s zea, the PC95 was 6.2 yg/1.0 cm^, which was s t i l l a potency greater than that found with other b i o l o g i c a l l y active natural plant compounds such as quassinoids (51) and other limonoids (52-53)· The growth-inhibitory a c t i v i t y of azadirachtin fed i n a r t i f i c i a l diet to three species of a g r i c u l t u r a l pests, _P. gossypiella, II. zea, and S_. frugiperda, was compared to the a c t i v i t y of a number of limonoids isolated from plants i n the Meliaceae and the Rutaceae (Table VI). After azadirachtin, the most active limonoid was cedrelone (Figure 13). Cedrelone was unique among the compounds tested i n Table VI since i t was the only limonoid, besides azadirachtin, to cause an i n h i b i t i o n i n ecdysis ( L C 5 0 = 150 ppm) when fed to pink bollworm larvae (54). Nakatani (55) observed that acetoxylation or ketonization of the 7-OH group rendered some Meliaceae limonoids ( i . e . , t r i c h i l i n s ) inactive as feeding deterrents. A similar phenomenon was observed when the growth-inhibitory a c t i v i t i e s of 7-deacetylgedunin, gedunin, and 7-ketogedunin, but not when the a c t i v i t i e s of azadiradione and deacetylazadiradione (Table VI and Figure 13), were compared. When comparing the a c t i v i t i e s of several Rutaceae limonoids, the most potent seem to have an a, ^-unsaturated 7-membered lactone (as i n obacunone) or at least have the p o t e n t i a l to form i t (as i n nomilin( (Figure 14). Apparently, at least i n v i t r o , nomilin eliminates i t s Α-ring a c e t y l group (forming obacunone) much more r e a d i l y than deacetylnomilin eliminates i t s Α-ring hydroxyl group (56). This fact might explain the much lowered growth i n h i b i t o r y a c t i v i t y of deacetylnomilin when compared to nomilin or obacunone (Table VI). L i t t l e i s yet known concerning the SAR's of b i o l o g i c a l l y active limonoids. However, many of the most potent of the growth-inhibitory limonoids from the Meliaceae and Rutaceae have one or more a l k y l a t i n g centers, including one almost invariably i n the Α-ring (e.g., a, 13-unsaturated ketone or lactone). Cedrelone has 2 a l k y l a t i n g centers, one i n the A-ring (α,β-unsaturated ketone) and one i n the B-ring (diosphenol) (Figure 13). Another example i s 6-O-acetylnimbandiol, which has an a,β-unsaturated ketone i n the Α-ring (Figure 13). 6-0-Acetylnimbandiol was i n s e c t i d i c a l ( E I 5 0 =21 ppm) when fed to the larvae of H. virescens, while the s t r u c t u r a l l y related salannin, which lacks the Α-ring ketone (Figure 13), was not (Table VII) (57). Nakanishi (58) has pointed out that natural products with e l e c t r o p h i l i c moieties tend to be cytotoxic and insect antifeedant. Possibly the growth-inhibitory a c t i v i t y of the limonoids may also be attributed to a nonspecific e l e c t r o p h i l i c e f f e c t . In addition to i t s a c t i v i t i e s of feeding deterrency and growth i n h i b i t i o n , azadirachtin disrupts the molting process of insects by i n h i b i t i n g ecdysis or the shedding of the " o l d " skin. The affected insects then die i n the pharate condition, unable to feed or locomote, The ecdysis-inhibitory a c t i v i t y of azadirachtin on three species of a g r i c u l t u r a l pests i s shown i n Table VIII. Although i t i s not known


36.

KLOCKE

Natural

Plant Compounds

Useful in Insect

Control

407

OH


Brevifolin

Gallic acid

Figure 11. Structures of Two Hydrolysis Products of Geraniin: B r e v i f o l i n and G a l l i c Acid

Figure 12. Upper: Azadirachtin (43). Azadirachtin (37)

Table V.

Previously Accepted Stereostructure of Lower: Recently Proposed Stereostructure of

Cotton Leaf Disk "Choice" Bioassay of Azadirachtin with Third-Instar Larvae of Two Species of A g r i c u l t u r a l Insect Pests Species

PC * (yg/leaf disk)

Spodoptera frugiperda H e l i o t h i s zea

0.1 6.2

95

*PC95 values are concentrationsjof azadirachtin r e s u l t i n g i n 95% "protection" of treated disks when compared to untreated disks.




408

Table VI.

Insect Growth-Inhibitory A c t i v i t y of Some Meliaceae and Rutaceae Limonoids. Values are the Dietary Concentrations for 50% Growth I n h i b i t i o n (EC50) Insect Species

Limonoid Azadirachtin Cedrelone Anthotheol Sendanin Methyl angolensate Nkolbisonin 7-Deacetylgedunin Gedunin Azadiradione 7-Ketogedunin Nomilin Obacunone Evodoulone 7-Deacetylproceranone Tecleanine Limonin 7-Deacetylazadiradione Deacetylnomilin

Pectinophora gossypiella 0.4 3 8 9 15 20 22 32 42 51

-96 175 210

290 950

Spodoptera frugiperda

Heliothis zea

0.4 2 3 11 40 65 60 47 130 800 72 70 120 350 320 756 5000

0.7 8 24 45 60 71 165 50 250 900 95 97 80 740

-

900 3500

*

*No a c t i v i t y was found with 2000 ppm.


*


KLOCKE

Natural Plant Compounds Useful in Insect Control

409

CEDRELONE

R=Ac AZADIRADIONE R=H 7-DEACETYLAZADIRADIONE

6-O-ACETYLNIMBANDIOL

SALANNIN

Figure 13 Structures of Some Insect Growth Inhibitory Limonoids Isolated from Plant Species i n the Meliaceae 0


410



Ο

R=Ac NOMILIN R=H

OBACUNONE

1-DEACETYLNOMILIN

Figure 14. Structures of Some Insect Growth Inhibitory Limonoids Isolated from Plant Species i n the Rutaceae

Table VII.

Ecdysis-and Growth-Inhibitory A c t i v i t i e s of Some Neem O i l Limonoids Fed i n an A r t i f i c i a l Diet to F i r s t - I n s t a r Larvae of H e l i o t h i s virescens b

a

Test Compound Deacetylazadirachtinol

El50 (ppm)

EC (ppm) 5 Q

0.80 0.17

Azadirachtin

0.80 0.07

6-0-Acetylnimbandiol

21.0 4.4

Salannin

95% Confidence Limits 0.660.120.460.0515.2 2.8 -

0.97 0.23 1.39 0.10 29.0 7.0

c 170

138

-210

EI50 values are the concentrations r e s u l t i n g i n 50% ecdysis inhibition. EC50 values are the e f f e c t i v e concentrations resulting i n 50% growth inhibition. No mortality was observed at 400 ppm.



36.

KLOCKE


Useful in Insect

Control

411

how a z a d i r a c h t i n i n h i b i t s e c d y s i s , i t a p p a r e n t l y does n o t do so by i n h i b i t i n g c h i t i n synthetase (49). P o s s i b l y the d i s r u p t i o n o f the m o l t i n g hormone t i t r e i n s e v e r a l s p e c i e s o f i n s e c t s t o which a z a d i r a c h t i n was a d m i n i s t e r e d (59-61) p r e v e n t e d e c d y s i s . Ultimately, the d i s r u p t e d hormone t i t r e and the i n h i b i t e d e c d y s i s may have been caused by an i n t e r f e r e n c e w i t h t h e n e u r o e n d o c r i n e s y s t e m , i n v o l v i n g the p r o t h o r a c i c o t r o p i c and a l l a t o t r o p i c hormones w h i c h c o n t r o l t h e b l o o d t i t r e s o f m o l t i n g hormone and j u v e n i l e hormone, r e s p e c t i v e l y (45). A n o t h e r l i m o n o i d i s o l a t e d from neem seeds and d e t e r m i n e d t o be as p o t e n t as a z a d i r a c h t i n as an e c d y s i s i n h i b i t o r has been i d e n t i f i e d as 3 - d e a c e t y l a z a d i r a c h t i n o l ( F i g u r e 15) ( 5 7 ) . B o t h compounds were l e t h a l t o 50% o f the t r e a t e d H . v i r e s c e n s l a r v a e (EI50) a t 0 . 8 ppm i n a r t i f i c i a l d i e t (Table V I I ) . S t r u c t u r a l l y , t h e r e a r e two d i f f e r e n c e s between the compounds. In 3 - d e a c e t y l a z a d i r a c h t i n o l , t h e C - l l - O - C - 1 3 e t h e r l i n k a g e o f a z a d i r a c h t i n i s r e d u c t i v e l y c l e a v e d a t the 11 p o s i t i o n and t h e a c e t o x y l group a t C - 3 i s h y d r o l y z e d t o a h y d r o x y l group. The i d e n t i f i c a t i o n of the n a t u r a l c h e m i c a l d e f e n s e mechanisms o f p l a n t s can be e x p l o i t e d by u t i l i z i n g t h e b i o l o g i c a l l y a c t i v e p l a n t c h e m i c a l c o n s t i t u e n t s as s o u r c e s and/or models o f new i n s e c t c o n t r o l agents. Examples h e r e i n c l u d e p y r e t h r i n s from Chry s an themum s p p . , chromenes from E n c e l i a and H . f i t c h i i , e l l a g i c a c i d and g e r a n i i n from CÎ. v i s c o s i s s i m u m v a r . v i s c o s i s s i m u m , and l i m o n o i d s from A . i n d i c a . A l t e r n a t i v e l y , a n a t u r a l c h e m i c a l d e f e n s e can be enhanced i n a p l a n t , such as i n an e c o n o m i c a l l y i m p o r t a n t c r o p p l a n t , i n o r d e r to a f f o r d endogenous p r o t e c t i o n from h e r b i v o r y . Such p r o t e c t i o n i s known as h o s t p l a n t r e s i s t a n c e . An example i s the w i l d tomato s p e c i e s L y c o p e r s i c o n h i r s u t u m f. g l a b r a t u m , w h i c h has been r e p o r t e d t o be r e s i s t a n t t o a wide range o f a r t h r o p o d p e s t s o f t h e c u l t i v a t e d tomato, L . e s c u l e n t u m . The t o x i c f a c t o r has been i d e n t i f i e d as 2 - t r i d e c a n o n e ( n - t r i d e c a n - 2 - o n e ) ( F i g u r e 16) (62) and r e l a t e d m e t h y l k e t o n e s (63) which accumulate i n g l a n d u l a r t r i c h o m e s on t h e l e a f s u r f a c e . In o r d e r t o enhance the low l e v e l s o f 2 t r i d e c a n o n e i n L^. e s c u l e n t u m , e x p i a n t t i s s u e d e r i v e d from s e g r e g a t i n g F - 2 p o p u l a t i o n s o f c r o s s e s between !L. e s c u l e n t u m and L^. h i r s u t u m f . g l a b r a t u m were c u l t u r e d and e v a l u a t e d f o r h i g h l e v e l s and accumul a t i o n of methyl ketones. C r o s s e s were e v a l u a t e d by a l e a f d i s k b i o a s s a y w i t h t h e t o b a c c o hornworm, Manduca s e x t a , and by gas c h r o m a t o g r a p h i c and s p e c t r o p h o t o m e t r y a n a l y s i s ( 6 4 ) . Several c r o s s e s have been i d e n t i f i e d thus f a r w h i c h accumulate h i g h l e v e l s of methyl ketones ( e s p e c i a l l y 2 - t r i d e c a n o n e ) . The c o r r e l a t i o n between the amount o f m e t h y l k e t o n e s and the r e s i s t a n c e to h e r b i v o r y by M. s e x t a i n t h e s e c r o s s e s was - 0 . 5 6 ( 6 5 ) . The g o a l o f t h i s r e s e a r c h i s t o g e n e r a t e a c o m m e r c i a l c u l t i v a r o f tomato which i s n a t u r a l l y r e s i s t a n t to i n s e c t a t t a c k . I n summary, n a t u r a l p l a n t compounds have been e x p l o i t e d c o m m e r c i a l l y as s o u r c e s ( e . g . , p y r e t h r i n s , r o t e n o i d s , a l k a l o i d s ) and models ( e . g . , p y r e t h r i n s , p h y s o s t i g m i n e ) o f i n s e c t i c i d e s . Other p l a n t compounds a r e c u r r e n t l y b e i n g e v a l u a t e d f o r s i m i l a r u s e s ( e . g . , chromenes, l i m o n o i d s ) . S t i l l o t h e r s a r e b e i n g e v a l u a t e d f o r use i n host p l a n t r e s i s t a n c e ( e . g . , l o n g - c h a i n methyl ketones). Such n o v e l c h e m i c a l s w i t h p o t e n t and o f t e n u n i q u e b i o l o g i c a l a c t i v i t i e s w i l l c o n t i n u e to be d i s c o v e r e d and e x p l o i t e d t h r o u g h b i o a s s a y and


412



Table VIII.

Ecdysis-Inhibitory A c t i v i t y of Azadirachtin Fed i n an A r t i f i c i a l Diet to F i r s t - I n s t a r Larvae of Three Species of A g r i c u l t u r a l Insect Pests Species

El95(ppm)

H e l i o t h i s zea Spodoptera frugiperda Pectinophora gossypiella

2 1 10

vb EC50(ppm)

a

0.7 0.4 0.4

EI95 values are the concentrations r e s u l t i n g i n 95% ecdysis inhibition. EC50 values are the e f f e c t i v e concentrations r e s u l t i n g i n 50% growth i n h i b i t i o n .

u

V

O C H l

OH

H

Figure 15. 3-Deacetylazadirachtinol, a Potent Insect Ecdysis Inhibitor Isolated from the Seeds of Azadirachta indica (57)

(n-Tridecan-2-one)

Figure 16. Structure of the Methyl Ketone, 2-Tridecanone, an Insecticide Found i n the Wild Tomato Species Lycopersicon hirsutum f. glabratum


36.

KLOCKE


Useful in Insect

Control

413

through recent advances in isolation chemistry (chromatography) and structural analysis (spectroscopy). The benefits of isolating and identifying these plant chemicals have been proven in the past and plants will continue to be invaluable as renewable resources or chemical progenitors of new insecticides. Acknowledgments


The author thanks Dr. M.F. Balandrin for helpful discussions. This work was supported in part by a grant awarded by the U.S. National Science Foundation (PCM-8314500). Literature Cited 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Pimentel, D. In "CRC Handbook of Natural Pesticides: Methods, Vol. I, Theory, Practice, and Detection"; Mandava, N.B., Ed.; CRC Press, Inc.: Boca Raton, Florida, 1985; pp. 3-19. Storck, W.J. Chem. Eng. News 1984, 62, 35-59. Staal, G.B. Ann. Rev. Entomol. 1975, 20, 417-460. Crosby, D.G. In "Natural Pest Control Agents"; Crosby, D.G., Ed.; Marcel Dekker, Inc.: New York, 1966; pp. 177-242. Jacobson, M. "Insecticides from Plants, a Review of the Literature, 1954-1971"; Agricultural Handbook 461, U.S. Department of Agricultural-Agricultural Research Service: Washington, D.C., 1975. Jacobson, M. Econ. Bot. 1982, 36, 346-354. Matsumura, F. "Toxicology of Insecticides"; Plenum Press: New York, 1975. Secoy, D.E.; Smith, A.E. Econ. Bot. 1983, 37, 28-57. Levy, L.W. Environ. Exp. Bot. 1981, 21, 389-395. Tyler, V.E.; Brady, L.R.; Robbers, J.E. "Pharmacognosy (ed. 7)"; Lea and Febiger: Philadelphia, 1976. Schmeltz, I. In "Naturally Occurring Insecticides"; Jacobson, M.; Crosby, D.G., Eds.; Dekker: New York, 1971; pp. 99-136. Stedman, E. Biochem. J. 1926, 20, 719-734. Gysin, H. Chimia 1954, 8, 205-220. Menn, J.J.; Pallos, F.M. Environ. Lett. 1975, 8, 71-88. Jones, C.G.; Firn, R.D. J. Chem. Ecol. 1978, 4, 138-177. Kubo, I.; Klocke, J.A. In "Plant Resistance to Insects"; Hedin, P.Α., Ed.; ACS Symposium Series No. 208, American Chemical Society: Washington, D.C., 1983; pp. 329-346. Nakanishi, K. In "Natural Products and the Protection of Plants"; Marini-Bettolo, G.B., Ed.; Elsevier Scientific Publ. Co.: New York, 1977; pp. 185-210. Bowers, W.S.; Ohta, T.; Cleere, J.S.; Marsella, P.A. Science 1976, 193, 542-547. Rodriguez, E. In "Plant Resistance to Insects"; Hedin, P.A., Ed.; ACS Symposium Series No. 208, American Chemical Society: Washington, D. C., 1983; pp. 291-302. Klocke, J.A.; Balandrin, M.F.; Adams, R.P.; Kingsford, E. J. Chem. Ecol. 1985, 11, 701-712. Bowers, W.S. Ent. Exp. Appl. 1982, 31,3-14. Bowers, W.S. In "Insecticide Mode of Action"; Coates, J.R., Ed.; Academic Press: New York, 1982; pp. 403-427.


414

23. 24. 25. 26.


27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

41.

42. 43. 44. 45. 46.


Campos-Lopez, E.; Roman-Alemany, A. J. Agric. Food Chem. 1980, 28, 171-182. Myers, N. Impact Sci. Soc., 1984, 34, 327-333. Jermy, T.; Butt, B.A.; McDonough, L.; Dreyer, D.; Rose, A.F. Insect Sci. Application 1981, 1, 237-242. Klocke, J.A.; VanWagenen, B.; Balandrin, M.F. Phytochemistry, 1985, in press. Bate-Smith, E.C. In "Chemistry in Botanical Classification"; Bendz, G.; Santesson, J., Eds.; Academic Press: New York, 1973; pp. 93-102. Okuda, T.; Mori, K.; Hatano, T. Phytochemistry 1980, 19, 547-551. Haddock, E.A.; Gupta, R.K.; Al-Shafi, S.M.K.; Layden, K.; Haslam, E.; Magnolato, D. Phytochemistry 1982, 21, 1049-1062. Okuda, T.; Yoshida, T.; Nayeshiro, H. Tetrahedron Letters 1976, 3721-3722. Okuda, T.; Yoshida, T.; Kazuko, M. Phytochemistry 1975, 14, 1877-1878. Okuda, T.; Nayeshiro, H.; Seno, K. Tetrahedron Letters 1977, 4421-4424. Wood, A.W.; Huang, Μ.Τ.; Chang, R.L.; Newmark, H.L.; Lehr, R.E.; Yagi, H.; Sayer, J.M.; Jerina, D.M.; Conney, A.H. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 5513-5517. Blumenberg, F.W.; Claus, C.; Ennecker, C.; Kessler, F.J. Arzneim.-Forsch 1960,10, 223-226 (Chem. Abstr. 54, 16653). Cliffton, E.E. Am. J. Med. Sci. 1967, 254, 117-123. Girolami, Α.; Cliffton, E.E. Throm. Diath. Haemorrh. 1967, 17, 165-175. Dilton, J.N.; Broughton, H.B.; Ley, S.V.; Lidert, Z.; Morgan, E.D.; Rzepa, H.S.; Sheppard, R.N. J. Chem. Soc. Chem. Commun. 1985, 968-971. Larson, R.O.; personal communication. Lidert, Z.; personal communication. Jacobson, M. In "Natural Pesticides from the Neem Tree (Azadirachta indica A. Juss.)"; Schmutterer, H.; Ascher, K.R.S.; Rembold, H., Eds.; German Agency for Technical Cooperation: Eschborn, Germany, 1980; pp. 33-42. Morgan, E.D. In "Natural Pesticides from the Neem Tree (Azadirachta indica A. Juss)"; Schmutterer, H.; Ascher, K.R.S.; Rembold, Η., Eds.; German Agency for Technical Cooperation: Eschborn, Germany, 1980; pp. 43-52. Butterworth, J.H.; Morgan, E.D. J. Insect Physiol. 1971, 17, 969-977. Zanno, P.R.; Miura, I.; Nakanishi, K.; Elder, D.L. J. Am. Chem. Soc. 1975, 97, 1975-1977. Uebel, E.C.; Warthen, J.D. Jr.; Jacobson, M. J. Liq. Chromatog. 1979, 2, 875-882. Rembold, H. In "Advances in Invertebrate Reproduction 3"; Engels, W.; Clark, W.H. Jr.; Fischer, Α.; Olive, P.J.W.; Went, D.F.; Elsevier Science Publishers: New York, 1984; pp. 481-491. Rembold, H.; Forster, H.; Czoppelt, Ch.; Rao, P.J.; Sieber, K.-P. In "Natural Pesticides from the Neem Tree (Azadirachta indica A. Juss) and Other Tropical Plants"; Schmutterer, H.; Ascher, K.R.S., Eds.; German Agency for Technical Cooperation: Eschborn, Germany, 1984; pp. 153-161.


36.

47. 48. 49. 50.


51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66.

KLOCKE

Natural

Plant Compounds

Useful in Insect

Control

415

Yamasaki, R.B.; Klocke, J.A.; Lee, S.M.; Stone, G.A.; Darlington, M.V., in preparation. S t i l l , W.C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 29232925. Kubo, I.; Klocke, J.A. Agric. Biol. Chem. 1982, 46, 1951-1953. Warthen, J.D. Jr. "Azadirachta indica: A Source of Insect Feeding Inhibitors and Growth Regulators"; Agricultural Reviews and Manuals ARM-NE-4, U.S. Department of Agriculture-Science and Education Administration: Beltsville, MD, 1979. Klocke, J.A.; Arisawa, M.; Handa, S.S.; Kinghorn, A.D.; Cordell, G.A.; Farnsworth, N.R. Experientia 1985, 41, 379-382. Klocke, J.A.; Kubo, I. Ent. Exp. Appl. 1982, 32, 299-301. Kubo, I.; Klocke, J.A. Les Colloques de L'I.N.R.A. 1982, 7, 117129. Klocke, J.A. Ph.D. Thesis, University of California, Berkeley, 1982. Nakatani, M.; James, J.C.; Nakanishi, K. J. Am. Chem. Soc. 1981, 103, 1228-1230. Dreyer, D.; personal communication. Kubo, I.; Matsumoto, Α.; Matsumoto, T.; Klocke, J.A. Tetrahedron 1985, in press. Nakanishi, K. Abstr. No. 2, Proc. Joint Meeting of the American Society of Pharmacognosy and the Society for Economic Botany: Boston, Mass., 1981. Rembold, H.; Sieber, K.-P. Z. Naturforsch. Sect. C. Biosci. 1981, 36, 466-469. Sieber, K.-P.; Rembold, H. J. Insect Physiol. 1983, 29, 523-527. Barnby, M.A.; Darlington, M.V.; Klocke, J.A.; unpublished results. Williams, W.G.; Kennedy, G.G.; Yamamoto, R.T.; Thakcer, J.D.; Bordner, J. Science 1980, 207, 888-889. Dimock, M.B., Kennedy, G.G., Williams, W.G. J. Chem. Ecol. 1982, 8, 837-842. Nienhuis, J.; Klocke, J.A.; Locy, R.; Butz, Α.; Balandrin, M.F. Hortscience 1985, 20, 112. Nienhuis, J.; Klocke, J.A.; Balandrin, M.F.; unpublished results. Litchfield, J.T. Jr.; Wilcoxon, F. J. Pharmacol. Exp. Ther. 1949, 96, 99-113.

RECEIVED December 17,1985


Allelochemicals: Role in Agriculture and Forestry - American Chemical

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