Biologically Active Natural Products - American Chemical Society

Chapter 15

Allelopathy in the Florida Scrub Community as a Model for Natural Herbicide Actions 1

1

2

Nikolaus H. Fischer , Nesrin Tanrisever , and G. Bruce Williamson 1

Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803-1804 Department of Botany, Louisiana State University, Baton Rouge, LA 70803-1804

Downloaded by MONASH UNIV on May 2, 2017 | http://pubs.acs.org Publication Date: November 28, 1988 | doi: 10.1021/bk-1988-0380.ch015

2

We are investigating the hypothesis that allelochemicals released from plants of the Florida scrub community deter the invasion of fire-prone sandhill grasses. Bioassays guided our chemical isolations of phytotoxins from three scrub species. Constituents of the endemic scrub members, Ceratiola ericoides, Conradina canescens and Calamintha ashei, were tested for effects on the germination and radicle growth of lettuce (Lactuca sativa) and little bluestem (Schizachyrium scoparium), a native grass of the Florida sandhill community. From Ceratiola ericoides we isolated two triter penes, ursolic acid and erythrodiol, as well as flava nones, catechins, proanthocyanidins, a chalcone, and dihydrochalcones. Water leaf washes provided ursolic acid and a novel dihydrochalcone, ceratiolin, which exhibited no significant phytotoxic activity. However, ceratiolin spontaneously decomposed in water to hydro cinnamic acid, which showed considerable phytotoxic activity. From Conradina canescens we isolated copious amounts of ursolic acid and a series of monoterpenes. Major monoterpenes constituents were 1,8-cineole, camphor, borneol, myrtenal, myrtenol, α-terpineol, and carvone. We tested camphor, myrtenal, borneol, and carvone for their phytotoxic activity on lettuce and bluestem and found strong effects on lettuce. Calamintha ashei provided, besides ursolic acid and caryophyllene oxide, the monoterpenes menthofuran, epievodone, and calaminthone, all of which are phyto toxic. A saturated aqueous solution of epievodone had stimulatory effects on the germination of bluestem. Epievodone in a saturated aqueous solution of ursolic acid, a natural mild detergent, strongly inhibited the germination and growth of bluestem. Also, a mixture of epievodone, calaminthone, and caryophyllene oxide totally inhibited germination of bluestem but had only minor effects on lettuce. β

0097-6156/88/0380-0233$06.00/0 1988 American Chemical Society

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

BIOLOGICALLY ACTIVE NATURAL PRODUCTS

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Data obtained support our proposal that n a t u r a l s u r f a c t a n t s such as u r s o l i c a c i d enhance t h e s o l u b i l i z a t i o n o f a l l e l o p a t h i c l i p i d s v i a m i c e l l i z a t i o n and play a s i g n i f i c a n t role i n their transport to target seeds o r s e e d l i n g s . Two m e c h a n i s m s o f p r o d u c t i o n a n d d e l i v e r y o f n a t u r a l h e r b i c i d e s a r e emerging from these studies: (a) the formation of a highly phytotoxic d e r i v a t i v e from a r e l a t i v e l y n o n - t o x i c p r e c u r s o r , and (b) t h e production o f n a t u r a l s u r f a c t a n t s that a s s i s t i n transporting r e l a t i v e l y insoluble phytotoxic lipids into the s o i l .

I n F l o r i d a and t h e S o u t h e a s t e r n C o a s t a l P l a i n , t h e w e l l d r a i n e d s a n d y r i d g e s o f r e l i c t s h o r e l i n e s s u p p o r t two d i f f e r e n t p l a n t communities. T h e more p r e v a l e n t v e g e t a t i o n t y p e i s t h e s a n d h i l l , d o m i n a t e d by l o n g l e a f p i n e a n d o a k s w i t h a d e n s e h e r b a c e o u s c o v e r o f g r a s s e s and f o r b s . Throughout t h e s a n d h i l l v e g e t a t i o n a r e i s l a n d s and s t r a n d s o f t h e s c r u b v e g e t a t i o n , w h i c h a r e d o m i n a t e d b y a c l o s e d canopy o f sand p i n e w i t h a dense u n d e r s t o r y o f e v e r g r e e n o a k s , and n e a r l y devoid of herbaceous cover. The d i s t i n c t s p e c i e s s e t s a r e s e g r e g a t e d a c r o s s a b r u p t e c o t o n e s w i t h t h e l e s s common s c r u b s p e c i e s regarded as endemics. C h a r a c t e r i s t i c differences that d i s t i n g u i s h t h e two v e g e t a t i o n t y p e s a r e s u m m a r i z e d i n T a b l e I (_1) · Table

I.

Contrast

Shrub l a y e r : Herbaceous l a y e r : F o l i a g e phenology: Surface litter: Fire frequency: Plant relative growth rates: Age o f p l a n t s a t f i r s t reproduction:

i n scrub

and s a n d h i l l

vegetation

types

Scrub

Sandhill

Very dense N e a r l y none Evergreen Light 20-50 y e a r s

Open Complete cover Deciduous Heavy 3-8 y e a r s

Slow

Fast

Old

Young

I n 1 8 9 5 , Nash ( 2 ) s t a t e d , "The s c r u b f l o r a i s e n t i r e l y d i f f e r e n t from that o f t h e h i g h pine land ( s a n d h i l l ) , h a r d l y a s i n g l e p l a n t b e i n g common t o b o t h ; i n f a c t t h e s e two f l o r a s a r e n a t u r a l e n e m i e s a n d a p p e a r t o be c o n s t a n t l y f i g h t i n g e a c h o t h e r . " The r o l e o f f i r e i n m a i n t a i n i n g t h e two c o m m u n i t i e s h a s b e e n proposed as an a l t e r n a t i v e t o e d a p h i c f a c t o r s . I n t h e absence o f f i r e , s c r u b s p e c i e s w i l l invade s a n d h i l l communities and p r o l i f e r a t e (_3_,_4). O b s e r v a t i o n s a f t e r w i l d f i r e s i n d i c a t e t h a t i n v a d i n g s c r u b s p e c i e s a r e k i l l e d by t h e r e p e a t e d b u r n i n g n e c e s s a r y t o m a i n t a i n h e a l t h y s a n d h i l l c o m m u n i t i e s (_4-6_) · Scrub regenerates s u c c e s s f u l l y o n l y w i t h l o n g - i n t e r v a l , c a t a s t r o p h i c f i r e s (7_,_8), b u t t h i s f i r e c y c l e does n o t e x p l a i n t h e l a c k o f s a n d h i l l s p e c i e s c o l o n i z i n g t h e scrub communities.



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The two p l a n t c o m m u n i t i e s c o u l d be m a i n t a i n e d a s s e p a r a t e e n t i t i e s where f r e q u e n t s u r f a c e f i r e s i n the s a n d h i l l w o u l d preempt the success o f s c r u b i n v a d e r s , and d u r i n g the l o n g f i r e - f r e e i n t e r v a l s i n the s c r u b , a l l e l o p a t h y would p r e c l u d e s u c c e s s f u l sandhill colonizers. Once e s t a b l i s h e d , t h e p a t t e r n o f t h e c o m m u n i t i e s w o u l d be m a i n t a i n e d , t h o u g h e c o t o n e s m i g h t e x p a n d a n d c o n t r a c t e p i s o d i c a l l y w i t h n a t u r a l changes i n f i r e f r e q u e n c y o r habitat disruption. I n order to test t h i s a l l e l o p a t h y / f i r e h y p o t h e s i s (1_), o u r e c o l o g y a n d n a t u r a l p r o d u c t s c h e m i s t r y g r o u p s i n i t i a t e d a cooperative investigation. T h i s paper r e v i e w s o u r f i n d i n g s o f t h r e e e a r l y c o l o n i z e r s o f the F l o r i d a s c r u b , C e r a t i o l a e r i c o i d e s (A. G r a y ) H e l l e r (Empetraceae), Calamintha a s h e i (Weatherby) Shinner (Lamiaceae), and Conradina canescens T o r r . & Gray ( L a m i a c e a e ) . Preliminary results i n d i c a t e d that w a t e r - s o l u b l e a l l e l o t o x i n s r e l e a s e d from the l e a v e s of t h e s e s c r u b p l a n t s i n h i b i t g e r m i n a t i o n a n d g r o w t h o f s a n d h i l l s p e c i e s (9)· We p r e s e n t h e r e a summary o f o u r c h e m i c a l a n d b i o a c t i v i t y d a t a i n v o l v i n g a l l e l o t o x i n s r e l e a s e d from the above shrubs and t h e i r e f f e c t s on the g e r m i n a t i o n and growth o f l i t t l e b l u e s t e m ( S c h i z a c h y r i u m scoparium) (Michx.) Nash ( P o a c e a e ) . This n a t i v e s a n d h i l l g r a s s was u s e d t o t e s t f o r p o t e n t i a l b e n e f i t o f a l l e l o p a t h y by s c r u b p l a n t s t o d e t e r the i n v a s i o n o f f i r e - p r o n e s a n d h i l l grasses into the scrub. We a l s o i n c l u d e e v i d e n c e f o r t h e r o l e o f n a t u r a l detergents such as u r s o l i c a c i d as s o l u b i l i z a t i o n enhancers of b i o a c t i v e l i p i d s and d i s c u s s t h e i r f u n c t i o n i n t h e r e l e a s e and t r a n s p o r t o f a l l e l o p a t h i c l i p i d s i n r a i n washes from a n a l l e l o p a t h i c source p l a n t t o a t a r g e t s p e c i e s . Materials

and Methods

Bioassays. T h e p r e l i m i n a r y b i o a s s a y s w e r e p e r f o r m e d b y D.R. R i c h a r d s o n (9) a n d i n v o l v e d a p p l i c a t i o n o f t e s t w a s h e s d e r i v e d f r o m leaves of C e r a t i o l a e r i c o i d e s , Conradina canescens, and Calamintha a s h e i t o t a r g e t seeds ( S c h i z a c h y r i u m scoparium and L a c t u c a s a t i v a ) and subsequent m o n i t o r i n g o f g e r m i n a t i o n and growth ( T a b l e I I ) . Once e a c h m o n t h f r e s h l e a v e s w e r e c o l l e c t e d f r o m t a g g e d t e s t p l a n t s i n the s c r u b community, soaked i n d i s t i l l e d water a t a r a t i o o f 1 g o f f r e s h l e a f w e i g h t p e r 10 m l o f w a t e r f o r 24 h u n d e r r e f r i g e r a t i o n a t 8°C. S o a k i n g p e r m i t t e d r e l e a s e o f w a t e r s o l u b l e compounds f r o m the l e a f s u r f a c e o r through n a t u r a l l e a c h i n g as might occur d u r i n g r a i n f a l l , b u t the r e f r i g e r a t i o n prevented decay o f o r g a n i c m a t e r i a l s and r e l e a s e o f i n t e r n a l p r o d u c t s i n w h o l e l e a v e s . T h e w a t e r s o l u t i o n s w e r e d e c a n t e d , a n d 5 m l o f s o l u t i o n was a d d e d t o s t e r i l e p e t r i d i s h e s ( 1 0 cm d i a m e t e r ) l i n e d w i t h o n e s h e e t o f f i l t e r paper. Each d i s h c o n t a i n e d t h i r t y seeds o f t a r g e t p l a n t s w i t h t h r e e or f o u r r e p l i c a t e d i s h e s p e r t e s t wash. The t e s t s o l u t i o n s were m e a s u r e d f o r pH a n d o s m o l a r i t y a n d d i f f e r e d o n l y s l i g h t l y f r o m w a t e r c o n t r o l s , s o no a d j u s t m e n t s w e r e r e q u i r e d . The p e t r i d i s h e s w e r e k e p t i n the dark u n t i l c o n t r o l s e x h i b i t e d r a d i c l e growth o f s e v e r a l cm, 4 d a y s f o r l e t t u c e a n d 15 d a y s f o r l i t t l e b l u e s t e m . After the d i s h e s had been f r o z e n t o t e r m i n a t e g r o w t h , p e r c e n t g e r m i n a t i o n and r a d i c l e l e n g t h o f g e r m i n a t e d s e e d s w e r e r e c o r d e d (9_)·



236


Chemical

Analyses

C e r a t i o l a e r i c o i d e s . V o l a t i l e s f r o m t h e h e x a n e e x t r a c t o f _C. e r i c o i d e s were o b t a i n e d by p a s s i n g steam over t h e c o n c e n t r a t e o f t h e hexane e x t r a c t (3 g) c o a t e d on t h e i n s i d e w a l l s o f a d i s t i l l a t i o n column. The s t e a m d i s t i l l a t e ( 3 0 m l ) was e x t r a c t e d w i t h 10 m l o f n a n o g r a d e C H 2 C I 2 , a n d t h e e x t r a c t s o l u t i o n was s u b j e c t e d t o GC-MS analysis. The f l a v o n o i d s 1-3, 5, 6 a n d a m i x t u r e o f u r s o l i c a c i d (UA, 2 5 ) , a n d e r y t h r o d i o l ( 2 6 ) u s e d f o r t h e g e r m i n a t i o n a n d r a d i c l e g r o w t h s t u d i e s w e r e i s o l a t e d by p r e v i o u s l y d e s c r i b e d m e t h o d s ( 1 0 ) . C o m m e r c i a l l y a v a i l a b l e u r s o l i c a c i d and h y d r o c i n n a m i c a c i d (HCA) were a l s o used i n t h e b i o a s s a y s . The b i o a s s a y s o f t h e f l a v a n o i d s ( 1 - 3 , 5, 6 ) i n v o l v e d p r e p a r a t i o n o f a s a t u r a t e d a q u e o u s s o l u t i o n o f t h e n a t u r a l t r i t e r p e n e m i x t u r e by s o n i c a t i o n f o r 1 h o f a n e x c e s s o f t r i t e r p e n e m i x t u r e i n water and f i l t r a t i o n . Saturated solutions of t h e f l a v o n o i d s w e r e p r e p a r e d by a d d i n g 5 mg o f e a c h f l a v o n o i d t o 22 ml o f a q u e o u s s a t u r a t e d t r i t e r p e n e s o l u t i o n a n d s o n i c a t i o n o f t h i s m i x t u r e f o r 1 h. A f t e r f i l t r a t i o n , a m a x i m a l c o n c e n t r a t i o n o f 227 ppm o f e a c h compound was o b t a i n e d . The r e s u l t s o f t h e b i o a s s a y s p e r f o r m e d on l e t t u c e and b l u e s t e m a r e summarized i n T a b l e I I I . F l o r i d a s c r u b s o i l i s a c i d i c w i t h pH l e v e l s a s l o w a s 4 i n some areas (9). To t e s t f o r p o s s i b l e e f f e c t s o f a c i d o n c e r a t i o l i n , we c a r r i e d o u t a n a c i d t r e a t m e n t u n d e r r e f l u x f o r 2 h w i t h 4M H C 1 . H NMR s p e c t r a l a n a l y s i s o f t h e p r o d u c t m i x t u r e a s w e l l a s GC-MS c o m p a r i s o n s w i t h s t a n d a r d s s h o w e d t h a t HCA ( 1 1 ) a n d i t s m e t h y l e s t e r were formed under these c o n d i t i o n s . X

C a l a m i n t h a a s h e i and C o n r a d i n a canescens. The i s o l a t i o n s o f t h e mono- a n d s e s q u i t e r p e n e s a n d u r s o l i c a c i d f r o m _C. a s h e i u s e d i n t h e b i o a s s a y s t u d i e s (11,12) as w e l l as t h e methods f o r i s o l a t i o n and s t r u c t u r e d e t e r m i n a t i o n o f t h e _C. c a n e s c e n s t e r p e n o i d s h a v e b e e n p r e v i o u s l y d e s c r i b e d (13,14)· M i c e l l i z a t i o n s t u d i e s o f s c r u b p l a n t l e a c h a t e s and t h e i r pure c o n s t i t u e n t s by t h e W o l f f m e t h o d ( 1 5 ) . Measurements of r e l a t i v e a c r i d i n e f l u o r e s c e n c e i n t e n s i t y w e r e p e r f o r m e d o n a SLM 4 8 0 0 s p e c t r o f l u o r i m e t e r w i t h a n e x c i t a t i o n s l i t w i d t h o f 8 nm a n d a n e m i s s i o n s l i t w i d t h o f 2 nm. E m i s s i o n w a v e l e n g t h was 425 nm a n d e x c i t a t i o n w a v e l e n g t h was 3 6 0 nm o r 395 nm, d e p e n d i n g o n t h e absorbance (11,13,14). Results

and D i s c u s s i o n

B i o l o g i c a l a c t i v i t i e s of C e r a t i o l a e r i c o i d e s constituents. Certiola e r i c o i d e s i s a m o n o t y p i c g e n u s , one o f o n l y t h r e e g e n e r a o f t h e f a m i l y Empetraceae. An endemic o f t h e F l o r i d a s c r u b community, i t i s l o c a l l y dominant on d i s t u r b e d s i t e s and t h e r e i s an a b s e n c e o f herbaceous growth around C e r a t i o l a p l a n t s ( 9 ) . Preliminary results f r o m e x t e n s i v e m o n t h l y b i o a s s a y s ( T a b l e I I ) showed t h a t w a t e r s o l u b l e l i t t e r e x t r a c t s s t r o n g l y i n h i b i t e d g e r m i n a t i o n and r a d i c l e growth o f n a t i v e g r a s s e s o f t h e a d j a c e n t s a n d h i l l community; however, w a t e r s o l u b l e e x t r a c t s o f f r e s h l e a v e s were o n l y m i l d l y allelopathic (9). Although chromatographic patterns of the


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237


f l a v o n o i d s o f C e r a t i o l a h a d been p r e v i o u s l y c a r r i e d out i n c o n n e c t i o n w i t h a b i o c h e m i c a l s y s t e m a t i c study o f the Empetraceae ( 1 6 ) , we p e r f o r m e d a n e x t e n s i v e r e - i n v e s t i g a t i o n o f t h i s s p e c i e s i n search for possible a l l e l o p a t h i c constituents (10). Chemical a n a l y s i s o f t h e d i c h l o r o m e t h a n e e x t r a c t o f g r o u n d a e r i a l p a r t s o f _C. e r i c o i d e s y i e l d e d t w o known d i h y d r o c h a l c o n e s , a n g o l e t i n ( 1 ) a n d Table

II.

E f f e c t o f water washes o f s c r u b s p e c i e s o n S c h i z a c h y r i u m s c o p a r i u m g e r m i n a t i o n (G) a n d r a d i c l e l e n g t h ( R L ) 1


Conradina canescens G

March April May June July August September October November

74* 112 83 19* 52* 104 111 56* 161*

RL

126* 82 79 50* 84* 91 108 117 81*

Calamintha ashei G

66* 93 85 25* 20* 84 103* 63* 123

RL

Ceratiola ericoides fresh plant G RL

133 76 72* 44* 76* 72* 104* 83 83

80 103* 83 85 55* 84 93 74 147

Ceratiola ericoides litter G RL

114 91 99 83 80 102 129* 108 84

51* 133 81 9* 48* 100 85* 70 95

28 84* 81 79 93 108 101* 95 91

Numbers a r e g e r m i n a t i o n a s a p e r c e n t o f t h e c o n t r o l a n d r a d i c l e l e n g t h s as percent o f c o n t r o l . An a s t e r i x i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e ( p < 0 . 0 5 ) . ( D a t a f r o m r e f . 9.) 1

1

1

f

2 ,6 -dihydroxy-4-methoxy-3 ,5 -dimethyldihydrochalcone ( 2 ) , as w e l l as 2 , 4 - d i h y d r o x y c h a l c o n e ( 6 ) a n d t h r e e known f l a v a n o n e s , 7hydroxyflavanone, 8-methylpinocembrin ( 4 ) , and 6,8-dimethylpinocembrin ( 3 ) . Methanol e x t r a c t s o f ground leaves provided c a t e c h i n ( 8 ) , " e p i c a t e c h i n ( 9 ) a n d epicatechin-(43*8;2β-Κ)+7)e p i c a t e c h i n (A-2 d i m e r ) ( 1 0 ) . From w a t e r washes o f f r e s h l y h a r v e s t e d l e a v e s a n o v e l d i h y d r o c h a l c o n e , c e r a t i o l i n ( 7 ) , was i s o l a t e d ( 1 0 ) . (See F i g u r e I . ) F r e s h _C. e r i c o i d e s l e a v e s w e r e o d o r l e s s , b u t d r i e d p l a n t m a t e r i a l e m i t t e d a n o d o r much l i k e t h a t d e t e c t e d i n t h e s u r r o u n d i n g s of l i v i n g p l a n t s i n the n a t u r a l environment. Therefore, the a t m o s p h e r e a r o u n d d r i e d l e a v e s was f l u s h e d w i t h n i t r o g e n f o r e i g h t h o u r s , a n d t h e v o l a t i l e s w e r e t r a p p e d i n CH2CI2 a t -70°C. T h e s e a i r samples c o n t a i n e d a n g o l e t i n ( 1 ) , 2 , 6 ' - d i h y d r o x y -4 -methoxy - 3 * , 5 dimethyldihydrochalcone ( 2 ) , 2 ,4 -dihydroxychalcone ( 6 ) , a n d 6,8d i m e t h y l p i n o c e m b r i n ( 3 ) a s m a j o r c o n s t i t u e n t s . A l l f o u r compounds w e r e a l s o f o u n d i n t h e C H 2 C I 2 e x t r a c t o f _C. e r i c o i d e s ( 1 0 ) . B i o l o g i c a l a c t i v i t i e s o f the above C e r a t i o l a f l a v o n o i d s had n o t been p r e v i o u s l y r e p o r t e d except f o r a s s e s s m e n t s o f the c y t o t o x i c a n d a n t i m i c r o b i a l p o t e n t i a l o f a n g o l e t i n (1) and i t s b e n z y l d e r i v a t i v e s , w h i c h e x h i b i t e d no a c t i v i t y ( 1 7 ) . F o r r e a s o n s t h a t w i l l be d i s c u s s e d l a t e r , f i v e f l a v o n o i d s (1-4,6) were t e s t e d i n a s a t u r a t e d ?

T

1

1

T

!


?

238


1 , R = H , R- Me

31

OH

4 , OH , H

2 , R= Me,R-H

Me

H H

Me H


5

Me

Ο

11, R = O H 12, R = C H 13, R - C H - ( C O ) - C H 3

s

2

Figure

I.

2

3

C o n s t i t u e n t s of C e r a t i o l a e r i c o i d e s : angoletin ( 1 ) , 2 jo'-dihydroxy-A-methoxy-S ,5 -dimethyldihydrochalcone ( 2 ) , 6,8-dimethylpinocembrin ( 3 ) , 8-methylpinocembrin ( 4 ) , 5-desoxypinocembrin ( 5 ) , 2 ^'-dihydroxychalcone (6), ceratiolin ( 7 ) , catechin (8), epicatechin (9), epicatechin( 4 3 + 8 ; 2 3 - K ) + 7 ) - e p i c a t e c h i n (Â-2 d i m e r ) ( 1 0 ) hydrocinnamic a c i d ( 1 1 ) , 4-phenyl-2-butanone ( 1 2 ) , and t r i k e t o n e ( 1 3 ) . T

1

1

T


15. FISCHER ET AL.

239


aqueous s o l u t i o n s o f an u r s o l i c a c i d ( U A ) - e r y t h r o d i o l m i x t u r e (ED) (Table I I I ) . The l a t t e r a r e two t r i t e r p e n e s ( 2 5 a n d 2 6 ) i s o l a t e d f r o m t h e t h e c u t i c a l wax o f _C. e r i c o i d e s l e a v e s . We o b s e r v e d o n l y m i n o r i n h i b i t i o n o f g e r m i n a t i o n w i t h l e t t u c e a n d no s i g n i f i c a n t e f f e c t s w i t h b l u e s t e m . Comparison o f t h e a c t i v i t y o n r a d i c l e g r o w t h o f e a c h compound i n a n a q u e o u s s o l u t i o n of the t r i t e r p e n e m i x t u r e w i t h the t r i t e r p e n e m i x t u r e c o n t r o l y i e l d e d o p p o s i t e e f f e c t s o n _L. s a t i v a a n d S. s c o p a r i u m . T h e c o m p o u n d s m i l d l y s t i m u l a t e d g r o w t h o f _L. s a t i v a w h i l e i n h i b i t i o n s a s s t r o n g a s 52% f o r 6 , 8 - d i m e t h y l p i n o c e m b r i n ( 3 ) were o b s e r v e d f o r j ^ .


Table

I I I . E f f e c t s o f _C. e r i c o i d e s f l a v o n o i d s o n t h e g e r m i n a t i o n a n d r a d i c l e l e n g t h o f _L. s a t i v a a n d _S. s c o p a r i u m d i s s o l v e d i n a s a t u r a t e d aqueous s o l u t i o n o f an u r s o l i c acid-erythrodiol mixture 1

L. Compound

2

sativa

G

97 100 88* 86* 90*

RL

(97) (100) (88*) (86*) (90*) (100)

UA-ED(25-26)

101 120* 136* 115 132*

(90*) (107) (121) (103) (117) (89*)

G

86 94 106 97 100

_S. s c o p a r i u m RL

(81) (89) (100) (92) (95) (95)

66* 70 71 52* 57*

(85) (90) (91) (66*) (73) (128)

t e r m i n a t i o n (G) a n d r a d i c l e l e n g t h s ( R L ) a r e e x p r e s s e d a s percentages of t h e i r controls. E a c h s a m p l e was c o m p a r e d t o a n u r s o l i c a c i d - e r y t h r o d i o l m i x t u r e (UA-ED) c o n t r o l . Data i n p a r e n t h e s e s i n d i c a t e c o m p a r i s o n t o H2O c o n t r o l s . An a s t e r i x i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from the c o n t r o l a t ρ < 0.05. Angoletin (1); 2 ,6Wihydroxy^'-methoxy-S',5 -dimethyldihydrochalcone ( 2 ) ; 2 ,4'-dihydroxychalcone ( 6 ) ; 6,8-dimethylpinocembrin ( 3 ) ; 8-methylpinocembrin ( 4 ) .

2

!

T

1

scoparium r a d i c l e growth. When c o m p a r e d w i t h w a t e r c o n t r o l s , w h i c h m i g h t be a b e t t e r c o m p a r i s o n f o r a s s e s s i n g t h e b e h a v i o r o f t h e n a t u r a l system, o n l y 6 , 8 - d i m e t h y l p i n o c e m b r i n shoved a s t a t i s t i c a l l y s i g n i f i c a n t i n h i b i t i o n (66%). A l t h o u g h the o t h e r f l a v o n o i d s caused some decrease i n t h e r a d i c l e l e n g t h s o f S. scopariuL' t e s t s e e d l i n g s , n o n e o f t h e s e a c t i v i t i e s was s t a t i s t i c a l l y s i g n i f i c a n t . T a n n i n s i n h i b i t b a c t e r i a ( 1 7 ) a n d t h e i r e f f e c t s c a n be s i g n i f i c a n t i n a l l e l o p a t h i c i n t e r a c t i o n s when n i t r i f y i n g b a c t e r i a are involved (18,19). R i c e a n d P a n c h o l y d e m o n s t r a t e d t h a t a 2 ppm c o n c e n t r a t i o n o f condensed t a n n i n s added t o s o i l t o t a l l y i n h i b i t s o x i d a t i o n o f NH^ w i t h i n t h r e e weeks ( 2 0 ) . T h i s i n h i b i t i o n c o u l d i n d i r e c t l y i n f l u e n c e the growth o f p l a n t s w i t h r o o t s near the s o i l surface. S y n e r g i s t i c i n h i b i t o r y e f f e c t s o f t a n n i n s were o b s e r v e d o n Avena s a t i v a c o l e o p t i l e segments. Condensed t a n n i n a l o n e caused minor i n h i b i t i o n s , whereas i n the presence o f l o wc o n c e n t r a t i o n s o f i n d o l e a c e t i c a c i d r o o t s e g m e n t e l o n g a t i o n was c o m p l e t e l y b l o c k e d (21). +


240



C a t e c h i n ( 8 ) , e p i c a t e c h i n ( 9 ) , a n d t h e A-2 d i m e r ( 1 0 ) a t 62 a n d 620 ppm h a d m i n o r t o m o d e r a t e e f f e c t s on t h e g e r m i n a t i o n o f _L. s a t i v a , a n d o n l y c a t e c h i n a t 62 ppm e x h i b i t e d a s i g n i f i c a n t g e r m i n a t i o n i n h i b i t i o n ( 6 6 % o f c o n t r o l ) o n _S. s c o p a r i u m ( T a b l e IV). The g r o w t h i n h i b i t i o n s o f _L. s a t i v a b y t h e two c a t e c h i n s w e r e low to moderate. The s t r o n g e s t i n h i b i t o r y e f f e c t s ( 6 4 % ) w e r e e x h i b i t e d by e p i c a t e c h i n a t 620 ppm u p o n _L. s a t i v a , a n d s i g n i f i c a n t g r o w t h p r o m o t i o n ( 1 3 6 % ) was f o u n d o n _S. s c o p a r i u m a t t h e 62 ppm l e v e l of e p i c a t e c h i n . I n summary, t h e g e r m i n a t i o n a n d g r o w t h i n h i b i t i o n s c a u s e d by t h e f l a v o n o i d s , c a t e c h i n s , and t h e A-2 d i m e r ( 1 0 ) do n o t r e a c h l e v e l s t h a t w e r e o b s e r v e d w i t h p l a n t l e a c h a t e s a n d c h r o m a t o g r a p h i c f r a c t i o n s o f _C. e r i c o i d e s t h a t c o n t a i n e d m i x t u r e s o f t h e above compounds. Table

IV.

E f f e c t s o f C e r a t i o l a e r i c o i d e s c a t e c h i n s on t h e g e r m i n a t i o n ( G ) a n d r a d i c l e l e n g t h ( R L ) o f _L. s a t i v a a n d scoparium 1

Compound

c a t e c h i n (8) c a t e c h i n (8) e p i c a t e c h i n (9) epicatechin (9) A-2 d i m e r ( 1 0 ) A-2 d i m e r ( 1 0 )

Cone. (ppm)

62 620 62 620 62 620

_L. s a t i v a G

RL

95 97 94 97 85 92

87 92* 75* 64* 96 70*

j>_. s c o p a r i u m G

66* 84 77 73 114 114

RL

122 107 136* 113 102 76

t e r m i n a t i o n (G) a n d r a d i c l e l e n g t h s ( R L ) a r e e x p r e s s e d i n p e r c e n t o f c o n t r o l s . An a s t e r i x i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e f r o m c o n t r o l (p < 0.05). W h o l e l e a f w a t e r w a s h e s o f _C. e r i c o i d e s o b t a i n e d a t a m b i e n t temperature were e x t r a c t e d w i t h e t h y l a c e t a t e - c h l o r o f o r m ; the e x t r a c t s p r o v i d e d , b e s i d e s u r s o l i c a c i d , one m a j o r c o m p o u n d , c e r a t i o l i n ( 7 ) , w h i c h i s a new a n d n o v e l d i h y d r o c h a l c o n e ( 1 0 ) . C e r a t i o l i n was s u s p e c t e d t o p l a y a s i g n i f i c a n t r o l e i n t h e a l l e l o p a t h i c a c t i v i t y o f C e r a t i o l a , s i n c e p l a n t p r o d u c t s on t h e l e a f s u r f a c e a r e more l i k e l y t h a n a r e i n t e r n a l l e a f c o n s t i t u e n t s t o be washed i n t o t h e s o i l d u r i n g r a i n . F o r t h i s r e a s o n , c e r a t i o l i n was i n v e s t i g a t e d f u r t h e r as a p o t e n t i a l s o u r c e f o r a l l e l o p a t h i c a c t i v i t y against l i t t l e bluestem. H o w e v e r , c e r a t i o l i n h a d no e f f e c t o n t h e g e r m i n a t i o n o f _S. s c o p a r i u m a t t h e 125 ppm l e v e l . In fact, i t promoted the r a d i c l e growth of t h i s t a r g e t s p e c i e s (145%). These d a t a were c o n t r a r y t o t h e r e s u l t s i n t h e s e a s o n a l s t u d i e s o f w a t e r washes o f C e r a t i o l a l e a v e s and, i n p a r t i c u l a r , i t s l i t t e r (Table II). D u r i n g t h e i s o l a t i o n a n d s t r u c t u r a l s t u d i e s , we c o u l d n o t d e t e c t c e r a t i o l i n i n t h e c r u d e e x t r a c t s o f g r o u n d d r i e d l e a v e s o f C_. e r i c o i d e s , b u t we d i d f i n d i t i n t h e l e a f w a t e r w a s h e s o f f r e s h a e r i a l p a r t s and i n t h e s o l u t i o n s f r o m s h o r t - t e r m whole p l a n t



15. FISCHER ET AL.

241


d i p p i n g s i n m e t h a n o l , i n w h i c h i t r e p r e s e n t e d the major component (10). A l s o , t h e amount o f c e r a t i o l i n e x t r a c t e d f r o m d r y p l a n t m a t e r i a l , w h i c h h a d b e e n s t o r e d a t room t e m p e r a t u r e f o r s i x m o n t h s , was c o n s i d e r a b l y l o w e r t h a n t h e y i e l d f r o m f r e s h p l a n t m a t e r i a l . T h i s i n d i c a t e d t h a t c e r a t i o l i n may be t h e r m a l l y u n s t a b l e a n d underwent d e c o m p o s i t i o n d u r i n g s t o r a g e o f the p l a n t m a t e r i a l . T h e r e f o r e , d e c o m p o s i t i o n e x p e r i m e n t s were c a r r i e d out w i t h w a t e r s o l u t i o n s o f pure c e r a t i o l i n . One s o l u t i o n was e x p o s e d t o s u n l i g h t , a n d t h e o t h e r was k e p t i n t h e d a r k . A f t e r 3 days h y d r o c i n n a m i c a c i d (HCA) ( 1 1 ) was d e t e c t e d a s a p r o d u c t i n b o t h e x p e r i m e n t s . T h e sample w h i c h h a d been k e p t i n the d a r k a l s o c o n t a i n e d t r a c e amounts o f t h e t r i k e t o n e ( 1 3 ) . S i n c e t h e f o r m a t i o n o f t h e s e c o m p o u n d s was f a c i l e i n w a t e r , t h e s e d e c o m p o s i t i o n p r o d u c t s must a l s o f o r m u n d e r n a t u r a l c o n d i t i o n s , e s p e c i a l l y i n d e c a y i n g C. e r i c o i d e s l i t t e r . A s a t u r a t e d a q u e o u s s o l u t i o n o f c e r a t i o l i n showed no 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 t h e two t e s t s e e d s ( T a b l e V ) . On t h e o t h e r h a n d , HCA e x h i b i t e d h i g h g e r m i n a t i o n a n d g r o w t h i n h i b i t i o n s o n b o t h t a r g e t seeds. A t 63 ppm, HCA showed s i g n i f i c a n t i n h i b i t o r y a c t i v i t y o n S* s c o p a r i u m g e r m i n a t i o n and g r o w t h , w h i l e e q u i m o l a r amounts o f c e r a t i o l i n ( 1 2 5 ppm) e x h i b i t e d no a c t i v i t y o n g e r m i n a t i o n a n d s t i m u l a t e d growth. T h i s d i f f e r e n c e may e x p l a i n e a r l i e r f i n d i n g s t h a t l i t t e r w a s h e s o f C. e r i c o i d e s s h o w e d c o n s i d e r a b l e a c t i v i t y w h i l e f r e s h l e a f washes were s i g n i f i c a n t l y l e s s e f f e c t i v e ( T a b l e II). S i n c e s o i l u n d e r C. e r i c o i d e s i s a c i d i c a n d t h e d e g r a d a t i o n o f c e r a t i o l i n i n t o HCA i s p r o m o t e d u n d e r a c i d i c c o n d i t i o n s , a c t i v i t y i s l i k e l y t o be g r e a t e r i n t h e s a n d y s o i l a n d d e c o m p o s i n g l i t t e r t h a n i n f r e s h l e a f r a i n washes. Low c o n c e n t r a t i o n s o f HCA ( 2 - 1 0 ppm) have been d e t e c t e d i n the s o i l under C e r a t i o l a . Presently, q u a n t i t a t i v e a n a l y s e s are i n progress t o determine s e a s o n a l T a b l e V.

E f f e c t s o f h y d r o c i n n a m i c a c i d (HCA) a n d c e r a t i o l i n o n g e r m i n a t i o n and r a d i c l e growth o f L a c t u c a s a t i v a and Schizachyrium scoparium *

Compound

Cone. (ppm)

_L. s a t i v a G

HCA

(11)

Ceratiolin(7)

1000 500 250 125 63 31 16 125

Schizachyrium

RL

G

0* 0* 4* 95 109 100 104

40* 56* 72* 76*

0* 2* 50* 68* 74* 113 109

102

83

102

9*

scoparium RL

17* 32* 46* 65 88 85 145

G e r m i n a t i o n s (G) and r a d i c l e l e n g t h s (RL) are i n p e r c e n t o f t h e controls. An a s t e r i x i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from the c o n t r o l (p < 0.05).



242


c o n c e n t r a t i o n c h a n g e s o f HCA a n d o t h e r d e c o m p o s i t i o n p r o d u c t s o f ceratiolin. The t r i k e t o n e ( 1 3 ) has n o t y e t b e e n t e s t e d f o r i t s a l l e l o p a t h i c p o t e n t i a l due t o i n s u f f i c i e n t a m o u n t s o f m a t e r i a l . F u t u r e b i o a s s a y s w i l l i n c l u d e compound 13 as w e l l as t h e 4 - p h e n y l - 2 b u t a n o n e ( 1 2 ) , w h i c h was f o u n d i n t h e v o l a t i l e s o f _C. e r i c o i d e s . The l a t t e r compound a l s o a p p e a r s t o be a d e g r a d a t i o n p r o d u c t o f c e r a t i o l i n , p o s s i b l y b e i n g formed v i a a c i d c l e a v a g e of the triketone (13). B i o l o g i c a l a c t i v i t i e s o f C a l a m i n t h a a s h e i and C o n r a d i n a c a n e s c e n s constituents. The m i n t s C a l a m i n t h a a s h e i a n d C o n r a d i n a c a n e s c e n s are endemic to the F l o r i d a s c r u b community. Previously neither s p e c i e s h a d b e e n i n v e s t i g a t e d c h e m i c a l l y . W a t e r w a s h e s o f f r e s h _C. a s h e i and _C. c a n e s c e n s l e a v e s had s i g n i f i c a n t i n h i b i t o r y e f f e c t s o n S_. s c o p a r i u m g e r m i n a t i o n and g r o w t h ( T a b l e I I ) ( 9 ) . The c u t i c a l waxes c o n t a i n e d c o p i o u s amounts of u r s o l i c a c i d ( U A , 2 5 ) (11). F u r t h e r c h e m i c a l a n a l y s i s o f t h e a e r i a l p a r t s o f _C. a s h e i p r o v i d e d t h e known m o n o t e r p e n e m e n t h o f u r a n ( 1 7 ) and t h e s e s q u i t e r p e n e caryophyllene epoxide. I n a d d i t i o n , t h e new menthofurans, c a l a m i n t h o n e ( 1 5 ) i t s d e s a c e t y l d e r i v a t i v e ( 1 6 ) , and epievodone (14) were i s o l a t e d ( 1 1 , 1 2 ) . The e f f e c t s o f t h e _C. a s h e i c o n s t i t u e n t s on t h e g e r m i n a t i o n and g r o w t h o f _L. s a t i v a a n d _S. s c o p a r i u m a r e summarized i n T a b l e V I . (See F i g u r e I I . ) Table VI.

E f f e c t s of s a t u r a t e d aqueous s o l u t i o n s of C a l a m i n t h a a s h e i c o n s t i t u e n t s o n t h e g e r m i n a t i o n and r a d i c l e g r o w t h o f _S. s c o p a r i u m a n d _L. s a t i v a 1

S.

Compound(s)

m i x t u r e o f 14, 1 5 , caryophyllene oxide calaminthone (15) epievodone(14) caryophyllene oxide ursolic acid (UA) e p i e v o d o n e + s a t d . aq. s o i n o f UA calaminthone ( v o l a t i l e s ) epievodone (volatiles)

cone,

RL

G

(ppm)

0*

285

L.

scoparium

sativa

G

RL

97

61*

*

+

25θ! 250j 625 T

+

250

1

129^ 185 128 86

*

57*

88 93 91 1 3 6

*

33

113 96 104 104 97

138

A

66 81

+

11

91 77

Numbers a r e g e r m i n a t i o n (G) and r a d i c l e l e n g t h s ( R L ) as p e r c e n t o f water c o n t r o l s o l u t i o n s . An a s t e r i x i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e f r o m t h e w a t e r c o n t r o l (p < 0.05). T h i s c o n c e n t r a t i o n r e p r e s e n t s t h e m a x i m a l c o n c e n t r a t i o n b a s e d on the t o t a l amounts weighed. In a l l cases o n l y p a r t of each sample dissolved.


FISCHER ET AL.

Allelopathy as a Modelfor Herbicide Actions

15, R = 0 A c

20,R = H,R'=0H


16, R = 0 H

17, R = H 4 - d e s o x y ;

25,R=R = M e , R = H; R = - C O O H 2

,

3

2 6 ^ = Η · , ^ =R = Me;R =- C H O H 2

3

f

Figure

II.

C o n s t i t u e n t s of Conradina canescens and Calamintha ashei: epievodone ( 1 4 ) , calaminthone ( 1 5 ) , desacetylcalaminthone ( 1 6 ) , menthofuran ( 1 7 ) , 1 , 8 - c i n e o l e ( 1 8 ) , camphor ( 1 9 ) , b o r n e o l ( 2 0 ) , myrtenal (21), myrtenol ( 2 2 ) y ot-terpineol (23), carvone ( 2 4 ) , u r s o l i c a c i d ( 2 5 ) , e r y t h r o d i o l ( 2 6 , i s o l a t e d from_C. e r i c o i d e s ) .


243


244


A s a t u r a t e d aqueous s o l u t i o n of a c h r o m a t o g r a p h i c fraction c o n t a i n i n g c a l a m i n t h o n e , e p i e v o d o n e , and c a r y o p h y l l e n e o x i d e c o m p l e t e l y s u p p r e s s e d g e r m i n a t i o n o f _S. s c o p a r i u m b u t h a d no s i g n i f i c a n t e f f e c t s on L. s a t i v a g e r m i n a t i o n . In contrast, s a t u r a t e d a q u e o u s s o l u t i o n s o f t h e p u r e compounds w e r e s t i m u l a t o r y t o S* s c o p a r i u m g e r m i n a t i o n ( 1 8 5 % o f c o n t r o l f o r e p i e v o d o n e ) . S i g n i f i c a n t i n h i b i t o r y e f f e c t s w e r e o b s e r v e d when e p i e v o d o n e was a d m i n i s t e r e d as a v o l a t i l e o r i n a s a t u r a t e d aqueous s o l u t i o n o f UA ( 2 5 ) , w h i c h e x h i b i t e d no s i g n i f i c a n t e f f e c t s when t e s t e d a l o n e . Two f a c t s a r e o f i n t e r e s t i n t h e a b o v e b i o a s s a y r e s u l t s : ( a ) The h i g h l y s p e c i f i c a c t i v i t y of a mixture of calaminthone, epievodone, and c a r y o p h y l l e n e o x i d e toward l i t t l e b l u e s t e m c o n s t r a s t s w i t h i n s i g n i f i c a n t g e r m i n a t i o n i n h i b i t i o n on l e t t u c e ; ( b ) t h e s t r o n g i n h i b i t o r y e f f e c t s e x h i b i t e d by t h e t e r p e n e m i x t u r e t o w a r d bluestem c o n t r a s t w i t h t h e g e r m i n a t i o n p r o m o t i o n s of b l u e s t e m caused by i n d i v i d u a l compounds. A s i m i l a r dramatic s y n e r g i s t i c phytotoxic e f f e c t by a m i x t u r e o f c a m p h o r , p u l e g o n e , a n d b o r n e o l was p r e v i o u s l y o b s e r v e d b y A s p l u n d ( 2 2 ) . Our i n t e r p r e t a t i o n s o f t h e s y n e r g i s t i c p r o p e r t y o f UA w i l l be d i s c u s s e d i n t h e f o l l o w i n g s e c t i o n .

Water w a s h e s o f f r e s h C o n r a d i n a c a n e s c e n s leaves w e r e strongly i n h i b i t o r y due t o t h e p r e s e n c e o f a s e r i e s o f m o n o t e r p e n e s t o g e t h e r w i t h c o p i o u s a m o u n t s o f UA ( 2 5 ) ( 1 3 ) . I n a d i r e c t e d , b i o a s s a y m o n i t o r e d s e a r c h f o r t h e a c t i v e f r a c t i o n s and t h e i r c o n s t i t u e n t s o f £.* c a n e s c e n s , we a n a l y z e d a h i g h l y a c t i v e f r a c t i o n o f t h e d i e t h y l e t h e r e x t r a c t by GC-MS a n a l y s i s ( 1 4 ) . S e v e r a l m i n o r m o n o t e r p e n e s were p r e s e n t , b u t 1 , 8 - c i n e o l e ( 1 8 ) , camphor (1,9), borneol ( 2 0 ) , myrtenal ( 2 1 ) , myrtenol ( 2 2 ) , cPterpineol

( 2 3 ) , and

c a r v o n e ( 2 4 ) were the major c o n s t i t u e n t s . I n t h e w a t e r l e a f soak o f f r e s h _C. c a n e s c e n s l e a v e s m o n o t e r p e n e s 1 8 , 1 9 , 2 0 , a n d 23 w e r e present. A l l o f t h e s e m o n o t e r p e n e s a r e known c o m p o u n d s , a n d s e v e r a l > 1 , 8 - c i n e o l e , camphor, and b o r n e o l ) a r e p o t e n t p l a n t g e r m i n a t i o n and g r o w t h i n h i b i t o r s ( 2 4 ) . B i o l o g i c a l a c t i v i t i e s o f f o u r C. c a n e s c e n s m o n o t e r p e n e s o n l e t t u c e and o f t h r e e on _S. s c o p a r i u m w e r e d e t e r m i n e d ( T a b l e V I I ) . I n g e n e r a l , l e t t u c e was c o n s i d e r a b l y more s e n s i t i v e t o s a t u r a t e d a q u e o u s s o l u t i o n s o f t h e m o n o t e r p e n e s t h a n was S c h i z a c h y r i u m , d e s p i t e the f a c t that both species s u f f e r e d complete germination i n h i b i t i o n when t r e a t e d w i t h a s a t u r a t e d a q u e o u s s o l u t i o n o f t h e h i g h l y a c t i v e f r a c t i o n of the d i e t h y l ether e x t r a c t . R e l e a s e mechanisms o f a l l e l o p a t h i c p l a n t l i p i d s . The F l o r i d a s c r u b c o m m u n i t y s h a r e s many c h a r a c t e r i s t i c s ( T a b l e I ) o f t h e c h a p a r r a l v e g e t a t i o n of Southern C a l i f o r n i a (25-28), except the scrub c o n s i s t s of c o m p l e t e l y d i f f e r e n t s p e c i e s t h a t grow i n a sandy s o i l u n d e r d i f f e r e n t c l i m a t i c c o n d i t i o n s . I n t h e C a l i f o r n i a c h a p a r r a l and o t h e r e c o s y s t e m s two m a j o r r e l e a s e a n d t r a n s p o r t m e c h a n i s m s o f a l l e l o p a t h i c agents have been proposed ( 1 9 ) . W a t e r - s o l u b l e a l l e l o t o x i n s s u c h as low m o l e c u l a r w e i g h t o r g a n i c a c i d s , p h e n o l i c a c i d s , a n d p l a n t p h e n o l i c s c a n be w a s h e d o f t h e l e a f s u r f a c e i n t o t h e s o i l by f o g d r i p a n d r a i n . A l t e r n a t i v e l y , a l l e l o t o x i n s c a n be v o l a t i l i z e d f r o m t h e p l a n t l e a f s u r f a c e and c a r r i e d t o t h e g r o u n d t o be a b s o r b e d by o r g a n i c s o i l p a r t i c l e s , s e e d s , o r r o o t s . A l l e l o p a t h i c p l a n t p r o d u c t s s u c h as l o w m o l e c u l a r w e i g h t a l c o h o l s , a l d e h y d e s , c a r b o x y l i c a c i d s , k e t o n e s , and c e r t a i n p h e n o l i c s , as w e l l a s mono- a n d s e s q u i t e r p e n e s , c o u l d be r e l e a s e d by t h i s "volatility


15. FISCHER ET AL. Table V I I .

245

Allelopathy as a Modelfor Herbicide Actions

Major monoterpenes i n the a c t i v e f r a c t i o n o f a d i e t h y l e t h e r e x t r a c t o f C o n r a d i n a c a n e s c e n s and t h e i r e f f e c t s on t h e g e r m i n a t i o n a n d r a d i c l e g r o w t h o f L a c t u c a s a t i v a and S c h i z a c h y r i u m s c o p ii r i u m 1

2

Compounds

L.

sativa


G

c a m p h o r (19) borneol (20) m y r t e n a l (21) c a r v o n e (24)

37** 7**

S.

RL

G

7**

95

8** 8**

77

59*

scoparium RL

100 54* 112

o**

t c c o n d i t i o n s i n t h e GC-MS a n a l y s i s w e r e : 30m b o n d e d s i l i c a c a p i l l a r y c o l u m n ; i n j e c t i o n temp. 250°C; 60° f o r 1 m i n . ; then 5°/min. t o 210°. R e t e n t i o n t i m e s a n d mass s p e c t r a o f a l l c o m p o u n d s g i v e n i n the t a b l e were c o n f i r m e d by c o r r e l a t i o n w i t h standards. t e r m i n a t i o n (G) a n d r a d i c l e l e n g t h s ( R L ) a r e i n p e r c e n t o f t h e d i s t i l l e d water c o n t r o l s : * t r e a t m e n t was s i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l a t p O . 0 5 ; * * t r e a t m e n t was s i g n i f i c a n t l y d i f f e r e n t f r o m t h e c o n t r o l a t p O . 0 1 .

mechanism". The a b o v e m e c h a n i s m s e x c l u d e t h e w i d e h o s t o f n o n v o l a t i l e , w a t e r - i n s o l u b l e plant l i p i d s from p a r t i c i p a t i o n i n allelopathic actions. F u r t h e r m o r e , o p e r a t i o n o f the " v o l a t i l i t y m e c h a n i s m " i n t h e F l o r i d a s c r u b may be d o u b t e d f o r a number o f reasons: F i r s t l y , i n F l o r i d a r e g u l a r winds would c a r r y a l l e l o p a t h i c v o l a t i l e s away f r o m t h e s o u r c e p l a n t s , n o t d e p o s i t i n g t h e m i n t h e immediate v i c i n i t y o f the p l a n t f o r a l l e l o p a t h i c a c t i o n . Secondly, the sandy s o i l i n the s c r u b l a c k s the l i p o p h i l i c o r g a n i c matter n e c e s s a r y f o r a b s o r p t i o n o f the v o l a t i l e s from the a i r . T h i r d l y , the r e l a t i v e l y h i g h temperature o f the s u r f a c e sands would prevent absorption of v o l a t i l e l i p i d s . W a t e r - s o l u b i l i z a t i o n o f l i p i d s c a n be f a c i l i t a t e d b y t h e i n v o l v e m e n t o f n a t u r a l d e t e r g e n t s s u c h a s UA ( 2 5 ) t h r o u g h m i c e l l i z a t i o n (23). This detergent e f f e c t allows release and t r a n s p o r t i n r a i n washes o f v o l a t i l e and n o n - v o l a t i l e h y d r o p h o b i c plant products. T h e r e f o r e , one a s p e c t o f o u r r e s e a r c h o n t h e F l o r i d a s c r u b community i n v o l v e d the a n a l y s e s o f l e a f washes f o r t h e d e t e c t i o n of m i c e l l e s (1,11,13,14). We u s e d t h e m e t h o d by W o l f f f o r d e t e c t i o n o f m i c e l l i z a t i o n and d e t e r m i n a t i o n o f c r i t i c a l m i c e l l e c o n c e n t r a t i o n s , because i t has a wide a p p l i c a b i l i t y i n m i c e l l e d e t e c t i o n o f a n i o n i c , c a t i o n i c , and n o n - i o n i c t e n s i d e s ( 1 5 ) . This method i s based on the f a c t t h a t below the c r i t i c a l m i c e l l e c o n c e n t r a t i o n (CMC) the f l u o r e s c e n c e o f a c r i d i n e i s independent o f s u r f a c t a n t c o n c e n t r a t i o n , w h i l e a b o v e t h e CMC, q u a n t u m y i e l d s decrease d r a s t i c a l l y with i n c r e a s i n g surfactant concentration. This change i s due t o the b e h a v i o r o f a c r i d i n e , w h i c h o n l y f l u o r e s c e s i n p r o t i c s o l v e n t s a n d i s s u f f i c i e n t l y h y d r o p h o b i c t o be l o c a t e d m a i n l y


246


w i t h i n the hydrocarbon-like i n t e r i o r of the m i c e l l e . Preliminary t e s t s w i t h t h e s y n t h e t i c t e n s i d e sodium d o d e c y l s u l f a t e (SDS) and n a t u r a l s u r f a c t a n t s such as u r s o l i c a c i d ( 2 5 ) and h y d r o c i n n a m i c a c i d ( 1 1 ) d e m o n s t r a t e d t h e u s e f u l n e s s o f t h e W o l f f method f o r o u r s t u d i e s ( F i g u r e I I I ) . We u n a m b i g u o u s l y e s t a b l i s h e d t h a t t h e h i g h l y i n h i b i t o r y water leachates of C e r a t i o l a e r i c o i d e s , Calamintha a s h e i , and C o n r a d i n a c a n e s c e n s ( n o t shown) f o r m m i c e l l e s . A l l three s p e c i e s c o n t a i n UA i n t h e l e a f w a x e s , t h u s p r o v i d i n g a p o s s i b l e matrix f o r micellization. I n each case t h e t y p i c a l a b r u p t change i n fluorescence y i e l d of a c r i d i n e a t the c r i t i c a l m i c e l l e concentration was o b s e r v e d ( F i g u r e I I I ) . We e s t i m a t e d t h e c o n c e n t r a t i o n o f t h e l e a c h a t e s o l u t i o n b y a s s u m i n g a r e a s o n a b l e a v e r a g e m o l e c u l a r w e i g h t o f 3 0 0 . The d r a m a t i c d e c r e a s e i n f l u o r e s c e n c e i n t e n s i t y n e a r 10~"^ a n d 1 0 ~ m o l a r c o n c e n t r a t i o n s c l e a r l y i n d i c a t e d m i c e l l i z a t i o n of the leachate s o l u t i o n of a l l three scrub species. This r e s u l t strongly suggested t h a t UA a c t s a s a m i c e l l a r h o s t f o r t h e b i o a c t i v e t e r p e n o i d s i n C a l a m i n t h a and C o n r a d i n a w a t e r w a s h e s , most l i k e l y i n c r e a s i n g t h e i r r a t e o f s o l u b i l i z a t i o n i n aqueous f o l i a r l e a c h a t e s and/or a i d i n g t h e i r t r a n s p o r t t o t a r g e t seeds or p l a n t s . This e f f e c t i s complementary t o the " v o l a t i l i t y " mechanism of a l l e l o p a t h i c t e r p e n e s , w h i c h h a d been p r o p o s e d by M u l l e r ( 2 8 ) . The " s y n e r g i s t i c " e f f e c t s o f u r s o l i c a c i d a s e x e m p l i f i e d by t h e b i o a s s a y s d e s c r i b e d i n Table VI are presently not understood. Experiments are i n progress t h a t w i l l a l l o w s e p a r a t i o n o f t h e c o n t r i b u t i o n s due t o p o s s i b l e micelle-mediated increases i n concentration of a l l e l o p a t h i c agents a n d / o r t o i m p r o v e d t r a n s p o r t m e c h a n i s m s t h a t f a c i l i t a t e movement o f a l l e l o p a t h i c a g e n t s t o a n d t h r o u g h membranes o f t a r g e t s e e d s .


3

I n summary, u r s o l i c a c i d a p p e a r s t o s e r v e s e v e r a l i m p o r t a n t e c o l o g i c a l functions i n the three scrub species studied: (a) I t s f i x a t i v e a c t i o n f o r C a l a m i n t h a and C o n r a d i n a c o n s t i t u e n t s reduces the v o l a t i l i z a t i o n l o s s of the a l l e l o p a t h i c t e r p e n e s , w h i c h w o u l d o t h e r w i s e be r a p i d . (b) I t s d e t e r g e n t e f f e c t enhances w a t e r - s o l u b i l i z a t i o n o f nonp o l a r t e r p e n e s and o t h e r v o l a t i l e and n o n v o l a t i l e l i p i d s i n r a i n washes. (c) I t f a c i l i t a t e s t r a n s p o r t of p h y t o t o x i n s t o and e n t r y i n t o t h e membranes o f t a r g e t s e e d s o r s e e d l i n g s . Conclusions

and O u t l o o k

D e t a i l e d knowledge of a l l e l o p a t h i c a c t i o n s i n n a t u r a l p l a n t c o m m u n i t i e s c a n p r o v i d e e x c e l l e n t m o d e l s f o r new s t r a t e g i e s i n d e v e l o p i n g h i g h l y s e l e c t i v e h e r b i c i d e s . The c o l l a b o r a t i v e e f f o r t o f écologiste a n d c h e m i s t s i n t h e p r e s e n t s t u d y h a s p r o d u c e d c o n s i d e r a b l e i n f o r m a t i o n r e l a t e d to a l l e l o p a t h i c i n t e r f e r e n c e i n a n a t u r a l p l a n t community. The F l o r i d a s c r u b r e p r e s e n t s a n i d e a l n a t u r a l ecosystem f o r s t u d i e s o f t h i s t y p e , s i n c e t h e sandy s o i l o f the s c r u b c o n t a i n s l i t t l e o r g a n i c m a t t e r . This absence s i m p l i f i e s considerably s o i l analyses f o r a l l e l o t o x i n s , which are frequently o b s c u r e d by c h e m i c a l s r e l e a s e d f r o m t h e o r g a n i c m a t t e r i n t h e soil. F u r t h e r m o r e , u n c o n t r o l l e d r e v e r s i b l e and i r r e v e r s i b l e a b s o r p t i o n o f s o u r c e p l a n t a l l e l o c h e m i c a l s by o r g a n i c s o i l p a r t i c l e s are a l s o reduced i n the sandy scrub s o i l .



15. FISCHER ET AL.

Figure

III.


Dependence o f r e l a t i v e f l u o r e s c e n c e i n t e n s i t y o f 1.2 χ 10 M a c r i d i n e s o l u t i o n vs. c o n c e n t r a t i o n o f l e a c h a t e s o f C a l a m i n t h a a s h e i (CA) a n d C e r a t i o l a e r i c o i d e s (CE) a s w e l l a s aqueous s o l u t i o n s o f hydrocinnamic a c i d (HCA), u r s o l i c a c i d (UA) a n d s o d i u m d o d e c y l s u l f a t e ( S D S ) .

American Chemical Society. Library 1155

15th St., N.W.

Cutler; Biologically Active Natural Products Washington, D.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1988.

247



248

Our studies in the Florida scrub have provided insight into new mechanisms of action that might also occur in other natural ecosystems. Chemical, microbial, or spontaneous decomposition of a nontoxic plant product into highly phytotoxic derivatives provides a possible mechanism for specific directed toxicity toward a target species. In Ceratiola ericoides the nontoxic ceratiolin represents an allelotoxin precursor, and its decomposition product HCA, which is mainly formed in the litter, acts as the specific allelotoxin for the sandhill target species. A promising practical aspect of this finding includes the synergistic allelopathic effects, exemplified by the dramatic increase in inhibition of bluestem germination caused by a mixture of volatile terpenes from Calamintha ashei. Further advances in the knowledge of bioactivity enhancements due to synergistic component mixtures could be of immense value in the development of natural biodegradable herbicides. The observation of highly selective phytotoxicity of Calamintha terpenes 9 distinctly higher germination inhibition of bluestem than of lettuce - represents another useful guide in the development of selective herbicides (30). Nonpolar cutical waxes and resins can possibly function as fixatives to prevent the loss of allelopathic volatiles by rapid evaporation. This property as a slow-release solvent for volatile lipids could be facilitated by a wide host of constituents in plant leaf waxes: triterpenes, long-chain fatty acids, hydrocarbons, alcohols, and esters, including fats. This fixative property, which is commonly exploited in the perfume industry to enhance long-term retention of active components, undoubtedly plays a significant role in the fixation of allelopathic plant volatiles. Finally, the function of natural detergents such as triterpene acids and long-chain fatty acids as micelle-forming matrixes for volatile as well as nonvolatile bioactive lipids has distinct practical implications. Natural or synthetic surfactants enhance the solubilization of lipids in rain washes and thus facilitate the transport of allelopathic lipids into the soil to reach target seeds or seedlings. Acknowledgments This material is based upon work supported by the U.S. Department of Agriculture, Competitive Research Grants Program for Forest and Rangeland Renewable Resources under Agreement No. 85-FSTY-9-0139. Literature Cited 1. Williamson, G. B.; Richardson, D. R.; Fischer, Ν. H. In Frontiers of Allelochemical Research; Rizvi, S.J.N., Ed.; Elsevier: Amsterdam, 1988 (in press). 2. Nash, G. V. Bull. Torrey Bot. Club 189 5 , 22,. 141-161. 3. Laessle, A. M. Ecol. Monogr. 1958, 28, 361-387. 4. Veno, P. A. Ecology 1976, 57, 498-508. 5. Williamson, G. B.; Black, Ε. M. Nature 1981, 293, 643-644. 6. Hebb, E. A. Southern J. Appl. Forestry 1982, 6, 144-147. 7. Harper, R. M. In Florida Geological Survey; Sixth Annual Report, 1914, pp.163-391.



15. FISCHER ET AL.

Allelopathy as a Model for Herbicide Actions 249

8. Webber, H. J. Amer. J. Bot. 1935, 22, 344-361. 9. Richardson, D. R. Ph.D. Dissertation, University of South Florida, Tampa, 1985. 10. Tanrisever, Ν.; Fronczek, F. R.; Fischer, N. H.; Williamson, G. B. Phytochemistry 1987, 26, 175-179. 11. Tanrisever, N.; Fischer, N. H.; Williamson, G. B. Phytochemistry 1988, 27, 000-000. (in press). 12. Macias, F. Α.; Fronczek, F. R.; Fischer, N. H. Phytochemistry, submitted. 13. Williamson, G. B.; Fischer, N. H.; Richardson, D. R.; De la Peña, A. J. Chem. Ecol., submitted. 14. De la Pena, A. Master's Thesis, Louisiana State University, Baton Rouge, 1985. 15. Wolff, T. J. Colloid and Interface Science 1981, 83, 658-660. 16. Moore, D. M.; Harborne, J. B. and Williams, C. A. Bot. J. Linn. Soc. 1970, 63, 277-283. 17. Hufford, C. D.; Oguntimein, B. O. Phytochemistry 1980, 19, 20362038. 18. Waage, S. K.; Hedin, P. Α.; Grimley, E. Phytochemistry 1984, 23, 2795-2787. 19. Rice, E. L. Allelopathy; Second edition; Academic Press: New York, 1984. 20. Rice, E. L.; Pancholy, S. K. Amer. J. Bot. 1973, 60, 691-702. 21. Zinsmeister, H. D. Planta 1964, 61, 130-141. 22. Asplund, R. O. Weed Science 1969, 17, 454-455. 23. Turro, N. J.; Graetzel, M.; Braun, A. M. Angew. Chem., Int. Ed. Engl. 1980, 19, 675-696. 24. Fischer, Ν. H. In The Science of Allelopathy; Putnam, A. R. and Tang, C., Eds.; Wiley: New York, 1986, pp.203-218. 25. Muller, C. H.; Muller, W. H.; Haines, B. L. Science 1964, 143, 471-473. 26. Muller, C. H. Bull. Torrey Bot. Club 1966, 93, 332-351. 27. Muller, C. H.; Hanawalt, R. B.; McPherson, J. K. Bull. Torrey Bot. Club 1968, 95, 225-231. 28. Muller, C. H. Vegetacio 1969, 18, 348-357. 29. Muller, W. H.; Muller, C. H. Bull. Torrey Bot. Club 1964, 91, 327-330. 30. Duke, S. O. Rev. Weed Science 1986, 2, 15-44. RECEIVED April

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