Plant Growth Regulators and Insect Control Agents from Marine

Chapter 50

Plant Growth Regulators and Insect Control Agents from Marine Organisms John H. Cardellina II, Michael F. Raub, and Bradford C. VanWagenen

Downloaded by COLUMBIA UNIV on October 31, 2017 | http://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch050

Department of Chemistry, Montana State University, Bozeman, M T 59717

In contrast to work in the burgeoning f i e l d of plant allelochemicals, very l i t t l e effort has been directed toward the study of plant growth regulators from the marine biosphere. We have initiated a program to determine whether there are plant growth regulators in marine organisms and, having detected such activity, to isolate and identify the active compounds. A number of extracts have exhibited plant growth promotion or inhibition. Three active compounds have been identified; one is the known auxin indole-3-acetamide. The other two are novel indoles. A number of marine organisms have been shown to avoid predation by the action of toxins or feeding deterrents upon potential predators. Testing of the activity of such compounds against insects has revealed some i n s e c t i c i d a l a c t i v i t y in several sesquiterpenes and diterpenes. Sessile marine organisms, much like their terrestrial counterparts, exist in an environment characterized by intense competition for living space. There is considerable evidence, amid some controversy (J_, 2), that many species have developed chemical expedients to insure their survival; survival may take the form of cohabitation with, domination over or eradication of competitors for space, as well as the use of toxins or feeding deterrents to repel predators. Plant growth regulators seem to play a major role in dominance/eradication situations in the terrestrial environment and might well play an important role in the marine biosphere as well. Periodic reports of the discovery of growth regulators in marine algae have appeared (3.-5.)» but these studies have a l l been comprised of qualitative analyses for known plant growth regulatory substances. More recently, a few articles have appeared that indicate the presence of growth regulatory substances of unknown structure in some algae (9-11). There are no previous reports of growth promoters or inhibitors in the extracts of sessile marine invertebrates. 0097-6156/87/0330-0562$06.00/0 © 1987 American Chemical Society

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


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N e a r l y s i x y e a r s o f c o l l e c t i n g work i n t h e B e r m u d i a n a r c h i p e l a g o has a f f o r d e d us an o p p o r t u n i t y f o r c o n t i n u i n g observation of the competition f o r space on the rocky substrates of the s h a l l o w water r e e f ecosystem. Algae, sponges, t u n i c a t e s , coelenterates and c o r a l s can be seen to p a r t i c i p a t e i n an intense c o m p e t i t i o n f o r the l i m i t e d space a v a i l a b l e . To s u r v i v e , an organism must e i t h e r a v o i d prédation or grow so r a p i d l y as t o o f f s e t l o s s e s t o p r e d a t o r s ; i n a d d i t i o n , the organism must have s u f f i c i e n t space f o r growth and r e p r o d u c t i o n . T h i s space can be a c q u i r e d by r a p i d growth, overgrowth of other organisms, or t o x i c i t y t o neighboring organisms; i n turn, space can be protected through prevention of overgrowth by other species. Organisms which t o l e r a t e overgrowth without apparent deleterious e f f e c t can also be observed; such overgrowth might, i n fact, be welcomed or encouraged as a means of camouflage protection. Such o b s e r v a t i o n s l e d us t o the h y p o t h e s i s t h a t t h e r e i s l i k e l y a d i v e r s i t y of growth promoters and i n h i b i t o r s to be found i n marine algae and invertebrates. We thus began an examination of selected extracts for plant growth regulatory behavior. Our view of the marine biosphere as a source of leads to novel i n s e c t c o n t r o l agents e v o l v e d from the d r a m a t i c developments i n marine c h e m i c a l e c o l o g y over the l a s t dozen y e a r s . A number of a n t i f e e d a n t s and t o x i n s have been i d e n t i f i e d d u r i n g s t u d i e s o f s p o n g e - n u d i b r a n c h (12) and a l g a - m o l l u s c and a l g a - f i s h (12) relationships. Reef communities are comprised of e x c e e d i n g l y complex i n t e r - s p e c i e s r e l a t i o n s h i p s and the s t r u g g l e f o r space, food and s u r v i v a l i s i n t e n s e ; i t thus stands t o reason t h a t chemical, as w e l l as physical, defenses are e s s e n t i a l for survival. At i s s u e i s whether or not the d e t e r r e n c e of or t o x i c i t y t o invertebrates observed i n the marine environment could be extended t o the t e r r e s t r i a l b i o s p h e r e . I f so, new i n s e c t c o n t r o l agents might be a v a i l a b l e from t h i s u n l i k e l y source and marine n a t u r a l p r o d u c t s would then r e p r e s e n t a l a r g e , a l m o s t w h o l l y untapped reservoir of p o t e n t i a l i n s e c t i c i d e s and feeding deterrents. Apart from the development of Padan, 1, from a l e a d p r o v i d e d by n e r e i s t o x i n , 2 , from the marine worm L u m b r i c o n e r e i s heteropoda (14) r l i t t l e attention has been rendered to secondary metabolites from marine organisms as insect control agents.

We i n i t i a t e d t h i s l i n e of r e s e a r c h by t e s t i n g i s o l a t e d compounds f o r t h e i r e f f e c t s upon two i n s e c t s , the grasshopper ( M e l a n o p l U S b i v i t a t t u s or XL sanguinipes) and the tobacco hornworm, Manduca sfixJtâ. We c o u l d t h e r e b y t e s t f o r a c t i v i t y a g a i n s t a d u l t


564

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY


and l a r v a l stage crop pests; the tobacco hornworm assay allows us t o t e s t f o r t o x i c i t y , f e e d i n g d e t e r r e n c e and j u v e n i l e hormone e f f e c t s i n a s i n g l e assay (15L). We have r e c e n t l y begun t e s t i n g crude extracts i n order t o focus our i s o l a t i o n e f f o r t s on compounds with p o t e n t i a l insect control a c t i v i t y ; the tobacco hornworm assay i s w e l l suited to bioassay-guided fractionation. The s e l e c t i o n p r o c e s s f o r c h o o s i n g organisms t o be screened for plant growth regulation i s based p r i m a r i l y on observations made i n the n a t u r a l marine h a b i t a t . Organisms which p r o l i f e r a t e i n competitive environments or which t o l e r a t e epiphytic organisms are l i k e l y sources of growth promoters, while organisms which survive by a v o i d i n g overgrowth or encroachment by other organisms might y i e l d growth i n h i b i t o r s . S i m i l a r l y , organisms which e x h i b i t no s i g n s of prédation are c o n s i d e r e d l i k e l y c a n d i d a t e s f o r i n s e c t i c i d a l screening.

M a t e r i a l s and Methods P l a n t Growth R e g u l a t i o n ftssayst i n i t i a l screening f o r a c t i v i t y involves the t e s t i n g of crude extracts i n a simple, quick bioassay u t i l i z i n g lettuce seeds. Concentrations of 0.5 to 5 mg/mL of the crude e x t r a c t s are t e s t e d ; 5 mL of s o l u t i o n a t each concentration are pipetted i n t o a small P e t r i dish. A Tfeflon ring i s p l a c e d i n the d i s h f o r support of a s m a l l s c r e e n , which i s p l a c e d atop the r i n g . Twenty l e t t u c e seeds a r e p l a c e d on the screen and allowed to germinate and grow under white l i g h t , usually f o r 72 hours. The g e r m i n a t i o n r a t e a t each c o n c e n t r a t i o n i s r e c o r d e d as a p e r c e n t a g e o f c o n t r o l s ; i n d i v i d u a l r o o t and c o l e o p t i l e lengths are determined, averaged f o r each concentration and r e c o r d e d as a r a t i o of average t e s t sample l e n g t h d i v i d e d by average control length. P u r i f i e d compounds are tested i n 3 s i m i l a r fashion, although the concentration range examined i s 10~ t o 10 M. I n s e c t i c i d a l Assays. The grasshopper assay has been described (16). Eggs of the tobacco hornworm, Manduca sejLfca, are o b t a i n e d from C a r o l i n a B i o l o g i c a l Supply and a r e hatched and r e a r e d on an a r t i f i c i a l agar-based d i e t (Black Cutworm) obtained from Bio-Serv Inc. Five to seven days a f t e r hatching, the larvae are weighed and transferred to s t e r i l e cups containing agar d i e t impregnated with t e s t compounds or e x t r a c t s . Dichloromethane, acetone or water s o l u t i o n s of t e s t m a t e r i a l s are added t o the warm d i e t and mixed thoroughly. A f t e r e v a p o r a t i o n of the s o l v e n t and s e t t i n g o f the agar, the d i e t i s c u t i n t o 25r-g wedges and f i v e l a r v a e are p l a c e d i n a cup w i t h the 25-g of impregnated d i e t m a t e r i a l . Controls consist of d i e t treated only with the solvents used to dissolve the t e s t materials. The tobacco hornworms are observed over a seven-day period f o r signs of t o x i c i t y , reduced feeding, r e l a t i v e weight gain (compared to controls) and a b i l i t y to shed exuvia (ecdysis). b

P l a n t Growth R e g u l a t i o n . Three plant growth promoters have been i d e n t i f i e d thus far. Caulerpin, 3, a unique bisindole pigment found i n some, but not a l l species of the green alga Caulerpa, was


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Organisms

565


the f i r s t compound to demonstrate a c t i v i t y i n our s t u d i e s (JLI). Both c a u l e r p i n and i t s h y d r o l y s i s product 4 e x h i b i t a c t i v i t y s i m i l a r to that of indole-3-acrylic acid, increasing root growth of lettuce seedlings to 115-118$ the length of controls. Equally i n t r i g u i n g i s the case of the sponge Dysidea etheria. D. etheria i s a successful competitor for space on the reef; i t i s also known to harbor a l g a l communities on i t s surface and within the sponge mass. Crude e x t r a c t s of the sponge e x h i b i t e d p l a n t growth r e g u l a t o r y a c t i v i t y . Our i n v e s t i g a t i o n s of the p o l a r organic soluble extracts yielded two growth promotors, i n d o l e - 3 acetamide and the novel i n d o l e 5 (18). The p r e v i o u s l y unknown 5 induced root l e n g t h extensions of 15$ over c o n t r o l s ; t h i s unique indole was subsequently found i n another sponge, Ulosa r u e t z l e r i .

Ο

These i n i t i a l successes led us to undertake a more organized and thorough study of marine i n v e r t e b r a t e s and algae f o r p l a n t growth r e g u l a t o r y a c t i v i t y . The l e t t u c e seed assay p e r m i t s an e v a l u a t i o n of e f f e c t s upon g e r m i n a t i o n and subsequent s e e d l i n g growth. Table I provides d e t a i l s of those extracts exhibiting some i n h i b i t i o n of germination.


566

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

Table I.

1

Plant Growth Regulation Assays - termination*' Extract Concentration

Organism C

BA-18 BS-2 BS-15 BS-24 BS-43 BS-70 BT-l BT-ll BT-21 BT-9

9/9 19/20 9/9 9/9 17/20 20/20 20/20 9/9 20/20 9/9

d

d

d d

d

e

e e


0 (control)

(mg/mL) 3.0

0.5

0.1

2.0

9/9 18/20 4/9 9/9 10/10 20/20 18/20 9/9 20/20 9/9

9/9 18/20 7/9 9/9 10/10 20/20 18/20 9/9 20/20 9/9

6/9 15/20 6/9 9/9 10/10 20/20 19/20 9/9 19/20 9/9

5.0

4.0

7/9 7/9 8/20 14/20 5/9 7/9 4/9 8/9 2/10 10/10 -9 18/20 10/20 10/20 6/9 6/9 7/20 17/20 6/9 6/9

3/9 8/20 -9 -9 2/10 0/20 5/20 -9 4/20 -9

NOTE: Some of these data appear i n ref. 26 reported as # seeds germinated/* seeds tested ^ o t active: BA-3 , BA-19 , BS-9 BS-32 , BS-65 ^alga "sponge ^tunicate coelenterate 9no measurement taken a

C

C

d

a

(

f

f

Table II. Plant Growth Regulation Assays - Root Length

a

Extract Concentrations (mg/mL) Organism b

BT-l , BT-ll BT-21 BS-2 BS-9 BS-15 BS-24 BS-32 BS-43 BS-65 BS-7Q BT-9 BT-15 b

b

C

C

C

C C C

C C

d

0 (control) 18.5 17.4 18.5 16.8 7.7 21.1 26.8 25 27.8 13.7 13.7 20.4 20.4

0.5

1.0

2.0

3.0

4.0

5.0

21.2 4.5 16.9 _e 6.7 16.5 18.4 17 10 12 9 15.5 16.3

18.1 2.7 13.6 5 3.9 7.9 12.2 15.6 8 8 7 11.1 17.3

13.6 2.3 7.8 3.4 3.2 7.1 14.5 9.3 3 7 4 11.9 18.7

4 1.6 9.8 3.4 2.8 6.2 14.4 8.8 3 6 _e 10.3 17

8 1.4 6.5 3.1 1.8 2.5 _e 7 1 3 0.5 9.5 16.8

4 _e 3.5 _e _e _e _e _e 1 3 0 10.4 11.1

NOTE: Some of these data appear in ref. 26 ^reported i n mm; measurements made 72 h after germination ^tunicate ^sponge coelenterate no measurement made: fungal contamination or concentration not run a

e


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Agents from

Manne

567

Organisms

More dramatic data were provided from measurements of root and coleoptile lengths in seedlings treated with test extracts. As i s evident from the data presented in Tables II and III inhibition or growth was observed i n many of the extracts tested. These preliminary results suggest that marine organisms, especially sponges, must be considered very l i k e l y sources of new growth regulatory substances. Efforts to identify the active constituents in a number of these organisms are now underway. Insect Control. Grasshoppers were chosen as an assay organism because they are a major crop pest in the grain producing plains and plateau states, they are readily available year round from the Agricultural Experiment Station at Montana State, and they are r e l a t i v e l y easy to maintain in the laboratory. Two species, Melanoplus b i v i t a t t u s and Melanoplus sanguinipes, have been used in these assays. Downloaded by COLUMBIA UNIV on October 31, 2017 | http://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch050

f

Table I I I . Plant Growth Regulatory Assays - C o l e o p t i l e Length

3

Extract Concentrations (mg/mL) Organism D

BT-1 BT-21 BS-2 BS-15 BS-43 BS-65 BS-7Q BT-9 BT-15

b

C

C C C C

d

0 (control) 3.75 4 2.5 9.25 3 3 3.2 3.2 3

0.5

1.0

2.0

3.0

4.0

5.0

3.7 3

3.1 2.4 1 16.2

Plant Growth Regulators and Insect Control Agents from Marine

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