Biologically Active Natural Products - ACS Publications - American

Chapter 23

Allelochemical Properties of Nicotiana tabacum Leaf Surface Compounds 1

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D. R. Lawson , D. A. Danehower , D. G. Shilling , M. L. Menetrez , and H. W. Spurr, Jr. 3

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North Carolina State University, Raleigh, NC 27695-7620 University of Florida, Gainesville, F L 32611 Crops Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Oxford, NC 27565 2

Downloaded by UNIV OF PITTSBURGH on May 4, 2015 | http://pubs.acs.org Publication Date: November 28, 1988 | doi: 10.1021/bk-1988-0380.ch023

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The chemical constituents on the leaf surface of green tobacco (Nicotiana tabacum) are diverse in their com position. They include, among other compounds, alkanes, fatty alcohols, wax esters, diterpenes, and sucrose esters. In addition to contributing to the flavor quality of the cured leaf, evidence has been reported that some of these compounds, primarily the diterpenes and the sucrose esters, have biological ac tivity in tobacco-insect and tobacco-microbe interac tions. Evidence is less definitive for such activity in tobacco-plant relationships, in that allelopathic potential for these compounds is strictly inferred from in vitro bioassays. In order to further investigate the allelochemi cal potential of these compounds, preparative chromato graphic procedures for isolating the major leaf surface components of Nicotiana tabacum, were developed. Com ponents were then assayed for phytotoxic and microbial activity. The results of this work support previously reported results and suggest additional biological ac tivity which has not been reported to date. Research over the past 25 years on the allelochemical potential of Nicotiana tabacum, L. leaf surface compounds has identified many of these compounds to be active as insect deterrents and attractants (1-5), antimicrobial agents (4,6-9), and potential al lelopathic agents (4,10). The principal leaf surface constituents responsible for most of the reported biological activities are the following secondary metabolites: a- and β-4,8,13-duvatriene-l,3diols (ADVT and BDVT); a- and p-4,8,13-duvatriene-l-ols (DVTmonols), (12Z)-labda-12,14-diene-8a-ol (Z-AB, Z-abienol) and 6-0acetyl-2,3,4-tri-0-acyl-a-D-glucopyranosyl-3"D-fructofuranoside (sucrose esters) (Figure 1). The sucrose esters consist of six groups, each group consisting of a mixture of structural isomers 0097-6156/88/0380-0363$06.00/0 © 1988 American Chemical Society

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


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BIOLOGICALLY ACTIVE NATURAL PRODUCTS

F i g u r e 1. Major J^. tabacum l e a f s u r f a c e secondary m e t a b o l i t e s : (a) ADVT, BDVT-4 (b) DVT-monols; (c) Z-AB; (d) Sucrose E s t e r s (R = C~-C~ normal and methyl branched f a t t y a c i d s ) .



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of equal m o l e c u l a r weight. These isomers d i f f e r i n the chain l e n g t h and/or p o s i t i o n of the short chain f a t t y a c i d r e s i d u e s at t h e 2, 3 , o r 4 p o s i t i o n o f t h e g l u c o s e m o i e t y . Each of the s i x g r o u p s o f s u c r o s e e s t e r s d i f f e r s f r o m e a c h o t h e r by one m e t h y l e n e u n i t ( 1 4 AMU) or m u l t i p l e s thereof. Several of these metabolites a r e f o r m e d by t h e t r i c h o m e h e a d s o f t h e g l a n d u l a r h a i r s f o u n d o n the l e a f surface (14). The e f f e c t s of N i c o t i a n a leaf surface constituents on m i c r o o r g a n i s m s and o t h e r p l a n t s has been i n v e s t i g a t e d less thoroughly t h a n t h e i r e f f e c t s on i n s e c t s . I n 1963, S h e p h e r d and Mandryk (6) a t t r i b u t e d the l a c k of spore ( c o n i d i a ) g e r m i n a t i o n of P e r o n o s p o r a t a b a c i n a Adam, t h e b l u e m o l d p a t h o g e n , on t h e l e a f s u r f a c e o f N_. t a b a c u m L . c v " V i r g i n i a G o l d " i n p a r t t o the p r e s e n c e o f a w a t e r s o l u b l e t o x i n . Much l a t e r , C r u i c k s h a n k e t a l . ( 1 1 ) i s o l a t e d two f u n g i t o x i c c o m p o u n d s , ADVT a n d BDVT, f r o m t h e l e a f s u r f a c e o f " V i r g i n i a G o l d " t o b a c c o , w h i c h a c c o u n t e d f o r most of the i n h i b i t o r y a c t i v i t y . P o s s i b l e i n h i b i t o r y e f f e c t s o f N i c o t i a n a l e a f s u r f a c e com p o u n d s on p l a n t g r o w t h h a v e a l s o b e e n i d e n t i f i e d . U s i n g a wheat ( T r i t i c u m a e s t i v u m L., cv Wakeland) c o l e o p t i l e segment bioassay, ADVT, BDVT, a n d t h e s u c r o s e e s t e r s ( 4 , 1 2 ) h a v e d e m o n s t r a t e d p o t e n t i a l plant growth i n h i b i t o r y a c t i v i t y . W h i l e t h i s work has p i q u e d interest i n the a l l e l o p a t h i c p o t e n t i a l of t h e s e compounds, b i o l o g i c a l e f f e c t s on p l a n t s h a v e b e e n i n f e r r e d s t r i c t l y f r o m i n v i t r o bioassays. No i n v i v o e f f e c t s h a v e y e t b e e n r e p o r t e d . The p u r p o s e o f t h i s s t u d y was to further characterize the a l l e l o c h e m i c a l p o t e n t i a l o f N. t a b a c u m l e a f s u r f a c e c o m p o u n d s o n t h e g r o w t h o f f u n g i and p l a n t s . S p e c i f i c a l l y , the e f f e c t s of t h i s s u c h c o m p o u n d s o n P_. t a b a c i n a s p o r e g e r m i n a t i o n and in vivo b i o l o g i c a l e f f e c t s o n two common w e e d s p e c i e s , E c h i n o c h l o a crusg a l l i L. B e u v o i s ( b a r n y a r d g r a s s ) a n d S e s b a n i a e x a l t a t a ( R a f ) C o r y (hemp s e s b a n i a ) w e r e i n v e s t i g a t e d . M A T E R I A L S AND

METHODS

I s o l a t i o n o f L e a f S u r f a c e Components. W h o l e , g r e e n , bud l e a v e s o f N i c o t i a n a tabacum ( B r a z i l i a n d o m e s t i c v a r i e t y , G a l p a o ) were washed with gentle a g i t a t i o n 4 5 - 6 0 s e c o n d s w i t h HPLC g r a d e m e t h y l e n e c h l o r i d e i n a 25 cm B u c h n e r f u n n e l f i t t e d w i t h Whatman No. 1 f i l t e r paper. G a l p a o was c h o s e n f o r t h i s w o r k a s i t c o n t a i n s a l l o f t h e m a j o r c u t i c u l a r c h e m i c a l s o f i n t e r e s t . The r e s u l t i n g e x t r a c t was f i l t e r e d u n d e r a s p i r a t o r vacuum u n t i l t h e p l a n t m a t e r i a l was f r e e o f e x c e s s s o l v e n t . The f i n a l f i l t r a t e f r o m s e v e r a l e x t r a c t i o n s was concentrated i n v a c u o a t 35°C, and t h e n d r i e d o v e r sodium s u l f a t e . The c r u d e e x t r a c t was s t o r e d i n a n a m b e r b o t t l e u n d e r n i t r o g e n a t m o s p h e r e a t -80°C u n t i l n e e d e d . F r a c t i o n a t i o n by C o m p o n e n t C l a s s . P r e p a r a t i v e chromatography of t h e c r u d e e x t r a c t was c a r r i e d o u t o n a 2.5 χ 68.5 cm b e d of S e p h a d e x LH-20 g e l ( 2 ) . Ten m l o f a 300 mg/ml s o l u t i o n o f the c r u d e e x t r a c t w e r e l o a d e d o n t o t h e c o l u m n and e l u t e d a t 5 m l / m i n w i t h a 99:1 m e t h y l e n e c h l o r i d e - m e t h a n o l mobile phase. One h u n d r e d f i f t y 5 ml f r a c t i o n s w e r e c o l l e c t e d and w e r e f o l l o w e d by a s i n g l e , 500 m l c o l l e c t i o n . A f t e r e a c h r u n , t h e c o l u m n was w a s h e d w i t h 1



366


l i t e r o f 97:3 m e t h y l e n e c h l o r i d e - m e t h a n o l a t 5 m l / m i n a n d t h e n r e e q u i l i b r a t e d w i t h 99:1 m e t h y l e n e c h l o r i d e - m e t h a n o l . The c o m p o s i t i o n o f i n d i v i d u a l t e s t t u b e f r a c t i o n s o r g r o u p e d f r a c t i o n s was d e t e r m i n e d u s i n g t h i n - l a y e r c h r o m a t o g r a p h y ( T L C ) o n s i l i c a g e l l a y e r s and gas c h r o m a t o g r a p h y (GC) o f s i l y l a t e d d e r i v a t i v e s o n a DB-5 c o l u m n (JL3). T h i n l a y e r p l a t e s were d e v e l o p e d i n 9:1 c h l o r o f o r m - m e t h a n o l . F o r v i s u a l i z a t i o n , TLC p l a t e s w e r e s p r a y e d w i t h 10% e t h a n o l i c phosphomolybdic a c i d and t h e n h e a t e d a t 100°C f o r 1-3 m i n u t e s . Gas c h r o m a t o g r a p h i c p e a k i d e n t i f i c a t i o n was b a s e d o n r e t e n t i o n t i m e s o f a u t h e n t i c s t a n d a r d s a n d GC/MS d a t a obtained from p r e v i o u s experiments. The S e p h a d e x L H - 2 0 p r o c e d u r e p r o v i d e d a n e x c e l l e n t means o f f r a c t i o n a t i n g t h e l e a f s u r f a c e com p o n e n t s by c h e m i c a l c l a s s . E l u t i o n o f t h e column p r o v i d e d the following fractions ( i n order of t h e i r e l u t i o n ) : hydrocarbons, wax e s t e r s , Z - A B / D V T - m o n o l s , D V T - d i o l s , m i s c e l l a n e o u s o x y g e n a t e d duvanes, and s u c r o s e e s t e r s . The s e c o n d a r y m e t a b o l i t e f r a c t i o n s were then s u b j e c t e d t o a d d i t i o n a l p r e p a r a t i v e chromatographic p r o cedures i n order to o b t a i n pure m a t e r i a l s . I s o l a t i o n o f Z-AB/DVT-monols. Preparative separation of the S e p h a d e x LH-20 g e l f r a c t i o n c o n t a i n i n g t h e s e c o m p o u n d s was c a r r i e d o u t o n a 21.5 χ 2.5 cm b e d o f M e r c k S i l i c a G e l 6 0 . Two h u n d r e d mg of sample were l o a d e d i n a 1 ml i n j e c t i o n volume onto t h e column a n d e l u t e d a t 4 m l / m i n w i t h a 7:3 m e t h y l e n e c h l o r i d e - h e x a n e m o b i l e phase. E i g h t y 5 ml t e s t tube f r a c t i o n s were c o l l e c t e d . TLC a n d GC e v a l u a t i o n s o f i n d i v i d u a l t e s t t u b e f r a c t i o n s o r c o m b i n e d f r a c t i o n w e r e c a r r i e d o u t u s i n g t h e TLC a n d GC p r o c e d u r e s d e s c r i b e d above. Thin layer p l a t e s w e r e d e v e l o p e d w i t h 7:3 methylene chloride-hexane. I s o l a t i o n of DVT-diols. P r e p a r a t i v e chromatography of the DVTd i o l m i x t u r e w a s c a r r i e d o u t o n a 2 1 . 5 χ 2.5 cm b e d o f M e r c k S i l i c a G e l 60. Two h u n d r e d mg o f c o m b i n e d S e p h a d e x L H - 2 0 f r a c t i o n s c o n t a i n i n g t h e d i o l s were l o a d e d onto the column i n a 1 ml i n j e c t i o n v o l u m e , a n d t h e c o l u m n was e l u t e d a t 5 m l / m i n w i t h 1:1:8 isopropanol-chloroform-hexane. E i g h t y 5 ml f r a c t i o n s were c o l lected. Chemical composition of i n d i v i d u a l t e s t tube f r a c t i o n s or g r o u p e d f r a c t i o n c o l l e c t i o n s was d e t e r m i n e d a s d e s c r i b e d above. Thin layer plates were developed with 1:1:8 isopropanolc h l o r o f orm-hexane . I s o l a t i o n of Sucrose Esters. Preparative i s o l a t i o n or enrichment of i n d i v i d u a l s u c r o s e e s t e r g r o u p s was c a r r i e d o u t o n a Whatman Magnum 20 P a r t i s i l 10 ODS-3 h i g h - p e r f o r m a n c e p r e p a r a t i v e c o l u m n . One h u n d r e d t w e n t y - f i v e mg o f s a m p l e w e r e l o a d e d o n t o t h e c o l u m n in a 1 ml injection volume. The m o b i l e phase was 6:4 a c e t o n i t r i l e - w a t e r e l u t e d a t 5 ml/min. E i g h t y 5 ml f r a c t i o n s were collected. An IBM Instruments 9430 ultra-violet/visible spectro p h o t o m e t e r , s e t a t a w a v e l e n g t h o f 225 nm, was u s e d f o r d e t e c t i o n . A b s o r b a n c e d a t a o f t h e e l u a t e o f e a c h t e s t t u b e was p l o t t e d as a h i s t o g r a m . C o m b i n a t i o n s o f f r a c t i o n s w e r e b a s e d on h i s t o g r a m profiles.



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Peronospora tabacina C o n i d i a l Germination I n h i b i t i o n Assays. M i c r o b i a l bioassays f o r the determination of spore germination i n h i b i t o r y a c t i v i t y o f t h e l e a f s u r f a c e compounds w e r e c a r r i e d o u t b y t h e m e t h o d o f M e n e t r e z e t a l . ( 2 3 ) . S t o c k s o l u t i o n s ( 2 . 0 mg/ml) o f t h e v a r i o u s l e a f s u r f a c e c o m p o n e n t s w e r e p r e p a r e d a n d 10 f o l d s e r i a l d i l u t i o n s performed. F i f t y m i c r o l i t e r s o f each s o l u t i o n , including methylene c h l o r i d e and water c o n t r o l s , were then p i p e t t e d o n t o a 1.5% w a t e r - a g a r m e d i u m ( t o t a l v o l u m e o f a g a r = 2 m l ) c o n t a i n e d i n a 10 χ 3 5 mm p e t r i d i s h , a n d t h e m e t h y l e n e c h l o r i d e was a l l o w e d t o e v a p o r a t e . T h r e e h u n d r e d m i c r o l i t e r s o f a f r e s h l y h a r v e s t e d s p o r e s u s p e n s i o n c o n t a i n i n g 1 χ 10^ c o n i d i a / m l of water were t h e n p i p e t t e d onto t h e w a t e r - a g a r s u r f a c e . The p e t r i d i s h e s were c o v e r e d and p l a c e d i n t o a c o v e r e d t r a y c o n t a i n ing moistened f i l t e r paper. The s a m p l e s were i n c u b a t e d f o u r h o u r s at 18°C a n d d e t e r m i n a t i o n s o f p e r c e n t g e r m i n a t i o n w e r e made b y c o u n t i n g a m i n i m u m o f 100 s p o r e s a n d c o m p a r i n g w i t h b o t h m e t h y l e n e c h l o r i d e and water c o n t r o l s . A l l experiments were performed i n d u p l i c a t e and were r e p l i c a t e d t h r e e t i m e s . P l a n t Growth I n h i b i t i o n Assays. The p h y t o t o x i c a c t i v i t y o f t h e v a r i o u s i s o l a t e d f r a c t i o n s o r p u r i f i e d compounds i s o l a t e d from N i c o t i a n a t a b a c u m was d e t e r m i n e d b y a p r e v i o u s l y d e s c r i b e d b i o a s s a y m e t h o d ( 1 5 ) . Some m o d i f i c a t i o n s w e r e made f o r t h i s r e s e a r c h as d e s c r i b e d below. The p u r e compounds o r m i x t u r e s w e r e d i s s o l v e d in e i t h e r methylene c h l o r i d e or acetone t o e s t a b l i s h the d e s i r e d concentrations. U n d e r a l a m i n a r - f l o w h o o d , 3.5 m l o f e a c h s o l u t i o n w e r e p l a c e d i n t o 60 m l g l a s s j a r s . A f t e r e v a p o r a t i o n , 0.22 g (60 s e e d s ) o f E c h i n o c h l o a c r u s g a l l i L. B e a u v o i s ( b a r n y a r d g r a s s ) or 0.40 g ( 3 0 s e e d s ) o f S e s b a n i a e x a l t a t a ( R a f ) C o r y (hemp s e s b a n i a ) seed were p l a c e d i n t o t h e j a r s . Under s t e r i l e c o n d i t i o n s , 3.5 m l o f 15 mM MES [ 2 - m o r p h o l i n o e t h a n e s u l f o n i c a c i d ] b u f f e r ( 0 . 1 % v / v e t h a n o l ) , a d j u s t e d t o pH 6.0, w e r e a d d e d t o e a c h jar. The j a r s were t h e n c o v e r e d , a n d t h e s e e d s w e r e i n c u b a t e d i n g r o w t h c h a m b e r s w i t h a 1 2 - h p h o t o p e r i o d a n d a 28/23°C d a y / n i g h t temperature regime, r e s p e c t i v e l y . T h e e f f e c t s o f t h e v a r i o u s com p o u n d s w e r e e v a l u a t e d a f t e r 84 h . A t t h i s t i m e any q u a l i t a t i v e s y m p t o m s w e r e n o t e d , a n d t h e t o t a l p l a n t f r e s h w e i g h t (TFW) was determined. T h i s w e i g h t was t h e n u s e d t o d e t e r m i n e t h e p r e d i c t e d s h o o t - p l u s - r o o t f r e s h w e i g h t (PSRFW) a s d e t e r m i n e d f r o m t h e f o l lowing models: b a r n y a r d g r a s s PSRFW = -0.24 + 0.741 (TFW) hemp s e s b a n i a PSRFW = -0.48 + 0.727 (TFW) T h e p r e d i c t e d s h o o t - p l u s - r o o t f r e s h w e i g h t was u s e d t o i n c r e a s e the s e n s i t i v i t y of the bioassay. A l l e x p e r i m e n t s were conducted two o r t h r e e t i m e s w i t h t h r e e r e p l i c a t i o n s . Data were i n i t i a l l y s u b j e c t e d t o a n a l y s i s o f v a r i a n c e ( 1 6 ) . S i n g l e degree o f freedom comparisons between t h e c o n t r o l and f r a c t i o n s e l u t e d f r o m t h e S e p h a d e x LH-20 c o l u m n w e r e u s e d t o d e t e r m i n e significant effects f o r the i n i t i a l p u r i f i c a t i o n (Table I ) . S t a n d a r d d e v i a t i o n s were used t o s t a t i s t i c a l l y e v a l u a t e t h e e f f e c t s o f c o n c e n t r a t i o n f o r e a c h o f t h e p u r i f i e d compounds on t h e g r o w t h o f t h e two b i o a s s a y s p e c i e s .


367

368


T a b l e I. P h y t o x i c i t y o f the t o t a l l e a f - s u r f a c e e x t r a c t and p a r t i a l l y separated components ( f r a c t i o n s ) i s o l a t e d from N i c o t i a n a tabacum F r a c t ion number

Hemp s e s b a n i a Barnyard g r a s s C o n c e n t r â t ion TFW PSRFW TFW PSRFW (PPm) -% i n h i b i t ! o n 3 2

2k 2k

100 10 1

3k 3k

+9.0 9.6

+18.7* 13.2

3.4 2.2 4.9

4.7 3.0 7.6

100 10

+8.9 6.7

+18.5* 9.4

7.4 6.7

3.2 9.9

33 33

100 10

23.1* 12.8*

34.0* 18.2*

8.4 1.1

13. 1.

kk kk

100 10

2.8 0

6.8 +5.1

10.6 +8.6

49 49 49

100 10 1

21.6* 30.8*

35.0* 45.3*

11..7 +5..5 8..0

64 64 64

100 10 1

38.1* 2.6 +28.9*

17.6 +9.6 12.3

61..2* 7-.8 +53.. 1*

11.2 . 11.2 .

17.2 17.3

69 69 69 69

100J 10* 1 0.1

43.6* 38.6* +15.7* +55.5*

69.0* 59.2* +29.1* +101 .4 .

23.,2* 13..5*

36.4* 21.0*

98 98

100 10

19.9 7-7

32.,8* 11.3 .

17..6* +9..0

27.0* +14.8

109 109

100 10

26.8* +9.8

113 113 113

100 10 1

+10.2 +14.4

100* 10 1 0.1

36.2* 15.9* +32.3* +56.7


24

column column column column

wash wash wash wash

-

-

43-.3* +17..4

18., 1* 8.,2

+18. 5 +24.,9*

18..2* +6..3 3. 5

57., 1* 19.,2* +58.• 9* +103.,8*

14. , 1* 8. 3

-

-

28.3* 9-3 27.6* +10.4 5.4 22.1* 13.1

-

10005 crude e x t r a c t 84. ,9* 43.2* 58.7* 27. 5* crude e x t r a c t 100 43.4* 68., 1* 30.2* 19. 5* crude e x t r a c t 10 0.4 0. 7 21.1* 13. 7 crude e x t r a c t 1 11.8 + 14.7 +27. 1* 7.,6 crude e x t r a c t 0.1 +34.6* 0.8 1.2 +63..it* crude e x t r a c t 0.01 +42.1 +77. 0* ' T o t a l f r e s h we î ght Predicted shoot-plus-root fresh weight. •^Values f o l l o w e d by an a s t e r i s k a r e s i g n i f i c a n t l y d i f f e r e n t from the ^ c o n t r o l at the P=0.05 l e v e l . Growth S t i m u l a t i o n i n d i c a t e d by +. Shoots o f barnyard g r a s s were bleached w h i t e and root growth of both s p e c i e s was e i t h e r v e r y i n h i b i t e d a n d / o r abnormal. -'No root growth and v e r y l i t t l e shoot g r o w t h .


23.

LAWSON ET AL.


R E S U L T S AND

N. tabacum Leaf Surface Compounds

369

DISCUSSION

Chromatographic I s o l a t i o n s . Preparative chromatography on Sephadex L H - 2 0 g e l o f G a l p a o l e a f s u r f a c e e x t r a c t s y i e l d e d en r i c h e d d i t e r p e n e and s u c r o s e e s t e r f r a c t i o n s as i n d i c a t e d i n Figure 2. The g e l f r a c t i o n s a n d t h e c r u d e e x t r a c t w e r e e v a l u a t e d for biological activity by determining t h e i r e f f e c t on fungal s p o r e g e r m i n a t i o n and p l a n t g r o w t h . Decisions to further frac t i o n a t e s e l e c t e d g e l f r a c t i o n s w e r e b a s e d on t h e s e i n i t i a l b i o a s say r e s u l t s . S u b s e q u e n t a d s o r p t i o n and r e v e r s e p h a s e p r e p a r a t i v e c h r o m a t o g r a p h i c p r o c e d u r e s y i e l d e d a t l e a s t 9 8 % p u r i t y o f ADVT, BDVT, D V T - m o n o l s , and sucrose e s t e r s ( g r o u p V) (Figure 2). E i g h t y p e r c e n t p u r i t y o f Z-AB was obtained. Such p u r i t i e s were r o u t i n e l y obtained from the crude l e a f s u r f a c e e x t r a c t a f t e r j u s t two p r e p a r a t i v e c h r o m a t o g r a p h i c s t e p s . These pure i s o l a t e s were also evaluated for b i o l o g i c a l a c t i v i t y . Peronospora tabacina Spore Germination Inhibition. Spore ( c o n i d i a l ) g e r m i n a t i o n a s s a y s w e r e c o n d u c t e d on b o t h t h e component m i x t u r e s o b t a i n e d from Sephadex L H - 2 0 g e l chromatography as w e l l as on t h e p u r e compounds i s o l a t e d f r o m t h e s e c o n d c h r o m a t o g r a p h i c steps. Although a l l of the major chemical classes represented w e r e t e s t e d , o u r d i s c u s s i o n w i l l be l i m i t e d t o t h o s e f r a c t i o n s o r components w h i c h showed 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 . Bioassays o f t h e D V T - m o n o l f r a c t i o n s s h o w e d no a c t i v i t y e v e n a t t h e h i g h e s t c o n c e n t r a t i o n t e s t e d ( 5 0 ppm-data not shown). Z-AB, however, i n h i b i t e d c o n i d i a l germination b y 3 7 % a t 5 0 ppm. This activity d e c r e a s e d r a p i d l y a t l o w e r c o n c e n t r a t i o n s ( 5 ppm), and e v e n t u a l l y showed m i l d stimulatory activity at the lowest concentrations (Figure 3c). The phenomenon o f g e r m i n a t i o n i n h i b i t i o n at high concentrations and s t i m u l a t i o n at lower concentrations was r e p r o d u c i b l e a n d was o b s e r v e d f o r v i r t u a l l y a l l t h e c o m p o n e n t s a s sayed. A s s a y s o f ADVT a n d BDVT ( F i g u r e 3 a a n d b ) c o n f i r m e d the previous observations of Cruickshank e t a l . (_Π). Activity was h i g h e s t a t 5 0 ppm a n d , o n c e a g a i n , d e c r e a s e d r a p i d l y a t l o w e r c o n c e n t r a t i o n s , e v e n t u a l l y l e a d i n g to s l i g h t s t i m u l a t i o n of germina tion. A p p a r e n t l y no s i g n i f i c a n t d i f f e r e n c e e x i s t s b e t w e e n t h e two isomers with regard to t h e i r b i o l o g i c a l a c t i v i t y versus concentra tion. This contrasts w i t h the report of Cruickshank e t al.» who observed s l i g h t l y higher a c t i v i t y f o r the β isomer. Bioassays of c o n c e n t r a t i o n s b e t w e e n 5 a n d 5 0 ppm a r e p l a n n e d i n o r d e r t o a s c e r t a i n the a c t u a l E D ^ Q ( e s t i m a t e d dose r e q u i r e d f o r 50% germination inhibition). The s u c r o s e e s t e r m i x t u r e ( F i g u r e 3 d ) s h o w e d v e r y s l i g h t i n h i b i t o r y a c t i v i t y a t 5 0 ppm. Further f r a c t i o n a t i o n of t h i s mix t u r e i n t o Groups I-VI has been accomplished and b i o a s s a y s are planned i n order to a s c e r t a i n the r e l a t i v e a c t i v i t y of the i n d i v i d u a l groups. P l a n t Growth I n h i b i t i o n . The p l a n t g r o w t h - r e g u l a t i n g activities o f t h e compounds i s o l a t e d f r o m t o b a c c o w e r e e v a l u a t e d p r i m a r i l y t o d e t e r m i n e any p o s s i b l e e c o l o g i c a l s i g n i f i c a n c e . The r o l e o f a l lelopathic compounds in both natural ecosystems and the


370



Green Bud Leaf Tissue

|cH2CI2 WASH Crude Leaf Surface Wash SEPHADEX LH-20

1

Fxn # 2 4 - 2 6

1 39-41 1

34-36

Hydrocarbons and waxes

44-46

1

64-67

1 Wash 1

69-76

SILICA GEL

SILICA e g

DVT-monols Z-AB

ADVT

BDVT

REVERSED PHASE

I

I

I

I

II III IV ν VI Sucrose

I

I

I

Esters

F i g u r e 2. E x t r a c t i o n and s e p a r a t i o n scheme f o r t h e i s o l a t i o n o f p r i n c i p l e N_. tabacum l e a f s u r f a c e components.


23.

LAWSONETAL.


100

Ν. tabacum Lea/Surface Compounds

371

a

η

50

τ

τ

(

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r

.

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.05

.5

5

50

100

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.005

.05

.5

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Rate (ppm)

F i g u r e 3 . E f f e c t s o f IN. tabacum l e a f s u r f a c e components on _P. tabacina germination: (a) ADVT; (b) BDVT; ( c ) Z-AB (d) Sucrose E s t e r s .



372


a g r o e c o s y s t e m h a s b e e n w e l l d o c u m e n t e d ( 1_7 ) · P r o v i n g a compound phytotoxic i s not only important f o r e c o l o g i c a l considerations but can a l s o have t e c h n o l o g i c a l i m p l i c a t i o n s i n terms o f t h e develop ment o f p e s t i c i d e s t h a t a r e b a s e d on n a t u r a l p r o d u c t chemistry (18). With these c o n s i d e r a t i o n s i n mind, a whole-plant bioassay was u s e d . T h i s b i o a s s a y a l l o w e d a compound t o be e v a l u a t e d f o r i t s a b i l i t y t o i n f l u e n c e t h e growth o f an i n t a c t p l a n t . Thus, any observable b i o l o g i c a l a c t i v i t y has g r e a t e r p o t e n t i a l i m p l i c a t i o n s than a c t i v i t y observed u s i n g an i n v i t r o bioassay. The b i o a s s a y used i n these s t u d i e s c o n t r a s t s w i t h t h e wheat c o l e o p t i l e b i o a s s a y u s e d by C u t l e r e t a l . ( 4 ) t o e v a l u a t e s i m i l a r compounds a l s o i s o l a t e d f r o m N. t a b a c u m . The i n i t i a l e v a l u a t i o n o f t h e c r u d e l e a f - s u r f a c e t o b a c c o e x t r a c t i n d i c a t e d a s i g n i f i c a n t amount o f i n h i b i t o r y a n d s t i m u l a t o r y a c t i v i t y on b o t h b i o a s s a y s p e c i e s ( T a b l e I ) . The q u a n t i t a t i v e a n d q u a l i t a t i v e a c t i v i t y o f t h e c r u d e e x t r a c t i n d i c a t e d t h a t compounds were present t h a t had p l a n t g r o w t h - r e g u l a t i n g a c t i v i t y and war ranted further i n v e s t i g a t i o n . Therefore, t h e c r u d e e x t r a c t was f r a c t i o n a t e d a s p r e v i o u s l y d e s c r i b e d i n o r d e r t o d e t e r m i n e what c o m p o u n d ( s ) was c a u s i n g t h e b i o l o g i c a l a c t i v i t y o f i n t e r e s t . T a b l e I shows t h e e f f e c t s o f t h e v a r i o u s f r a c t i o n s s e p a r a t e d by S e p h a d e x LH-20 g e l c h r o m a t o g r a p h y o n t h e g r o w t h o f b a r n y a r d g r a s s a n d hemp s e s b a n i a . F r a c t i o n s 64 a n d 69 a n d t h e c o l u m n w a s h (CW) c a u s e d t h e g r e a t e s t a m o u n t o f p l a n t g r o w t h i n h i b i t i o n . F r a c t i o n 6 9 c a u s e d 6 9 % a n d 3 6 % i n h i b i t i o n o f PSRFW a t 1 0 0 ppm i n b a r n y a r d g r a s s a n d hemp s e s b a n i a , r e s p e c t i v e l y . T h e CW n o t o n l y c a u s e d a s i m i l a r l e v e l o f i n h i b i t i o n a t 100 ppm, b u t a l s o c a u s e d 1 0 4 % s t i m u l a t i o n i n b a r n y a r d g r a s s PSRFW a t 0.1 ppm. Although t h e q u a n t i t a t i v e a c t i v i t y o f t h e s e f r a c t i o n s was s i g n i f i c a n t , o f m o r e i n t e r e s t was t h e q u a l i t a t i v e characteristic o f f r a c t i o n 69 a n d t h e CW t o c a u s e b l e a c h i n g ( i . e . , w h i t e s h o o t t i s s u e ) i n barnyard grass. This q u a l i t a t i v e c h a r a c t e r i s t i c not o n l y s t r e s s e d t h e importance o f u s i n g a whole p l a n t bioassay, but a l s o a c t e d a s a u n i q u e b i o l o g i c a l m a r k e r t h a t was u s e d t o f o l l o w b i o l o g i c a l a c t i v i t y of i n t e r e s t during the chemical purification process. By u s i n g a b i o a s s a y t h a t p r o v i d e d b o t h q u a l i t a t i v e and q u a n t i t a t i v e information during the chemical separation process, the time-consuming t a s k o f chemical p u r i f i c a t i o n and s t r u c t u r a l e l l u c i d a t i o n was d e v o t e d t o c o m p o u n d s t h a t c a u s e d t h e b i o l o g i c a l activity of interest. F i g u r e s 4 a n d 5 show t h e p l a n t g r o w t h - r e g u l a t i n g a c t i v i t y o f t h e p u r i f i e d compounds w h i c h were i d e n t i f i e d a s components o f t h e b i o l o g i c a l l y a c t i v e f r a c t i o n s p r e s e n t e d i n T a b l e I . Of t h e t e r p e n e s e v a l u a t e d , BDVT s h o w e d t h e g r e a t e s t l e v e l o f p l a n t g r o w t h i n h i b i t i o n ( F i g u r e s 4b a n d 5 b ) , f o l l o w e d by t h e α i s o m e r ( F i g u r e 4a and 5 a ) . The β i s o m e r c a u s e d 3 0 % i n h i b i t i o n a n d t h e α i s o m e r c a u s e d 5 % i n h i b i t i o n a t 1 ppm when a s s a y e d a g a i n s t b a r n y a r d grass. S p r i n g e r e t a l . (12) a l s o s h o w e d t h a t t h e β i s o m e r was m o r e a c t i v e t h a n t h e α isomer i n t h e wheat c o l e o p t i l e a s s a y system. These compounds a l s o c a u s e d b l e a c h i n g o f t h e s h o o t t i s s u e i n b a r n y a r d grass. The o t h e r t e r p e n e s t h a t were e v a l u a t e d , DVT-monols, ( d a t a n o t s h o w n ) a n d Z-AB ( F i g u r e 4 c a n d 5 c ) a l s o c a u s e d significant l e v e l s o f i n h i b i t i o n o f b a r n y a r d g r a s s PSRFW. I n a d d i t i o n , b o t h o f t h e s e compounds c a u s e d g r o w t h s t i m u l a t i o n a t l o w e r c o n c e n t r a t i o n s .


23.

LAWSONETAL.

373

Ν. tabacum Leaf Surface Compounds


100

1

1

10

Rate (ppm)

100

.1

1

Rate

10

100

(ppm)

F i g u r e 4. E f f e c t s o f N. tabacum l e a f s u r f a c e components E c h i n o c h l o a c r u s g a l l i Xbarnyard g r a s s ) growth: (a) ADVT; (b) BDVT; (c) Z-AB; (d) Sucrose E s t e r s .


on


374


.1

1

10

Rate (ppm)

100

.1

1

10

100

Rate (ppm)

F i g u r e 5. E f f e c t s o f N_. tabacum l e a f s u r f a c e components on Sesbania e x a l t a t a (hemp s e s b a n i a ) growth: (a) ADVT; (b) BDVT; (c) Z-AB; (d) Sucrose E s t e r s .



23. LAWSON ET AL.


375

The q u a n t i t a t i v e a n d q u a l i t a t i v e a c t i v i t y o f t h e p u r e compounds accounted f o r the b i o l o g i c a l a c t i v i t y associated with the i n i t i a l l y separated f r a c t i o n s presented i n Table I . C u t l e r and Cole (10) h a v e p r e v i o u s l y d e s c r i b e d t h e i n h i b i t o r y p r o p e r t i e s o f t h e s e compounds, b u t n o t t h e g r o w t h s t i m u l a t i o n o r t h e b l e a c h i n g a c tivity. The second most a c t i v e i s o l a t e s were t h e s u c r o s e esters ( F i g u r e A d a n d 5d). The sucrose esters not only i n h i b i t e d b a r n y a r d g r a s s PSRFW c o m p a r a b l e t o BDVT, b u t a l s o c a u s e d s l i g h t l y m o r e i n h i b i t i o n o f PSRFW ( a t 10 ppm) o f hemp s e s b a n i a a s c o m p a r e d t o BDVT. The i n h i b i t o r y e f f e c t s o f t h e s u c r o s e e s t e r s o f t o b a c c o h a v e b e e n p r e v i o u s l y d e m o n s t r a t e d (A) i n a w h e a t c o l e o p t i l e a s s a y . I n a d d i t i o n , t h e s u c r o s e e s t e r s c a u s e d 2A% a n d 32% s t i m u l a t i o n o f b a r n y a r d g r a s s PSRFW a t 1 a n d 0.1 ppm, r e s p e c t i v e l y . The s u c r o s e e s t e r s a l s o caused bleaching o f barnyard grass shoot t i s s u e . The q u a l i t a t i v e and q u a n t i t a t i v e e f f e c t s o f t h e p u r i f i e d sucrose e s t e r s a g r e e d c l o s e l y w i t h t h e a c t i v i t y a s s o c i a t e d w i t h t h e CW (Table I ) . CONCLUSIONS The i n v e s t i g a t i o n s d e s c r i b e d i n t h i s p a p e r c o n f i r m a n d e x p a n d u p o n p r e v i o u s work c o n d u c t e d on t h e b i o l o g i c a l a c t i v i t y o f N i c o t i a n a tabacum l e a f s u r f a c e exudates. The c h r o m a t o g r a p h i c p r o c e d u r e s a l low the isolation of relatively large quantities of these m a t e r i a l s , as w e l l as other surface c o n s t i t u e n t s (hydrocarbons, wax e s t e r s , e t c . ) , w i t h a m i n i m u m n u m b e r o f c h r o m a t o g r a p h i c s t e p s , which include simple low pressure chromatographic systems. The S e p h a d e x LH-20 s e p a r a t i o n i s a p a r t i c u l a r l y p o w e r f u l m e t h o d . I t has h i g h l o a d i n g c a p a c i t y , i s a v e r y m i l d s e p a r a t i o n method based on b o t h p a r t i t i o n i n g a n d g e l p e r m e a t i o n m o d e s , a n d i t r e s u l t s i n a c l e a r - c u t f r a c t i o n a t i o n i n t o component c l a s s e s . Bioassays f o rPeronospora tabacina c o n i d i a l germination in h i b i t i o n i n d i c a t e d t h a t , i n a d d i t i o n t o t h e known a c t i v i t y o f ADVT a n d B D V T , Z_-AB a l s o h a d s i g n i f i c a n t b i o l o g i c a l a c t i v i t y . T h e weaker a c t i v i t y d i s p l a y e d by t h e complex m i x t u r e o f s u c r o s e e s t e r s d e s e r v e s f u r t h e r work. A s s a y s o f i n d i v i d u a l s u c r o s e e s t e r groups o r i n d i v i d u a l i s o m e r s may d e m o n s t r a t e t h a t t h e b i o l o g i c a l a c t i v i t y i n d i c a t e d f o r t h e sucrose e s t e r s as a group i s concentrated in a l i m i t e d number o f i s o m e r s . F o r e x a m p l e , C u t l e r e t a l . (A) t e s t e d t h e s e compounds a g a i n s t b o t h gram p o s i t i v e a n d gram n e g a t i v e b a c teria. G r o w t h i n h i b i t o r y a c t i v i t y was i n d i c a t e d a g a i n s t gram p o s i t i v e o r g a n i s m s a n d a c t i v i t y w h i c h was c o n c e n t r a t e d i n the G r o u p V e s t e r s i n w h i c h t h e 2,3, a n d A h y d r o x y p o s i t i o n s o n t h e g l u c o s e m o i e t y a r e e s t e r i f i e d w i t h β-methylvaleric a c i d . Plant growth-regulating a c t i v i t y was a l s o d e m o n s t r a t e d i n the whole p l a n t growth bioassay. O n c e a g a i n , b o t h a n d BDVT 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 . Growth i n h i b i t i o n a t higher concentrations, and t h e o b s e r v a t i o n o f b l e a c h i n g a c t i v i t y w i t h t h e s e compounds a n d the sucrose e s t e r s i s s i g n i f i c a n t . Moreover, t h e s t i m u l a t i o n o f p l a n t g r o w t h a t l o w e r c o n c e n t r a t i o n s i s i n d i c a t i v e o f more complex biological properties. T h i s r e s e a r c h confirms t h a t N i c o t i a n a tabacum c o n t a i n s p l a n t growth-regulating compounds. The u s e o f t h e w h o l e p l a n t bioassay



376


a l l o w e d b o t h q u a n t i t a t i v e and q u a l i t a t i v e e v a l u a t i o n s of i s o l a t e s d u r i n g the p u r i f i c a t i o n p r o c e s s . Thus, not only were the app r o p r i a t e c o m p o u n d s i d e n t i f i e d b u t t i m e s p e n t s e p a r a t i n g and identifying chemicals of no b i o l o g i c a l s i g n i f i c a n c e was reduced. Questions s t i l l remain c o n c e r n i n g the r o l e of t h e s e compounds i n p o s s i b l e e c o l o g i c a l i n t e r a c t i o n s ( i . e . , a l l e l o p a t h y ) . I n a number o f e x a m p l e s , n a t u r a l p r o d u c t s h a v e b e e n shown t o l e a c h f r o m p l a n t s and e l i c i t an a l l e l o p a t h i c r e s p o n s e t o s u r r o u n d i n g v e g e t a t i o n (17, 18). However, the plant g r o w t h - r e g u l a t i n g activity o f t h e compounds evaluated i n t h i s s t u d y was d e m o n s t r a t e d i n glass jars. The e x t e n t o f t h e i r a c t i v i t y i n s o i l h a s n o t b e e n s t u d i e d . In add i t i o n , a l t h o u g h r a i n f a l l has b e e n shown to reduce the l e v e l s of t h e s e compounds on tobacco leaves, which would indicate that leaching i s p o s s i b l e , the concentration that u l t i m a t e l y reaches t h e s o i l has not been d e t e r m i n e d ( 2 1 ) . Therefore, the e c o l o g i c a l s i g n i f i c a n c e of t h e s e compounds relative to p l a n t s has not yet been a s c e r t a i n e d . Once b i o l o g i c a l a c t i v i t y h a s b e e n d e m o n s t r a t e d f o r a n a t u r a l product, s e v e r a l approaches to t h e i r use come to mind. First, plant b r e e d e r s and g e n e t i c i s t s a r e a l r e a d y actively involved in the i n t r o d u c t i o n of s e v e r a l of t h e s e c h e m i c a l t r a i t s i n t o t o b a c c o f o r the purpose of p r o t e c t i n g the p l a n t from i n s e c t prédation o r disease. I n t h e f u t u r e , b i o t e c h n o l o g y may s u p p l e m e n t t h i s c l a s s i c genetic approach. These approaches h o l d promise f o r the in vivo a p p l i c a t i o n o f N. t a b a c u m l e a f s u r f a c e c h e m i c a l s i n t h e c o n t r o l o f pests. In a d d i t i o n , the sucrose esters i n p a r t i c u l a r , represent an u n u s u a l c l a s s of n a t u r a l p r o d u c t s t h a t have not been e x t e n s i v e l y examined. A s i d e f r o m t h e work of S e v e r s o n e t a l . (13), there i s o n l y one a d d i t i o n a l r e p o r t o f t h e o c c u r r e n c e o f t h e s e c o m p o u n d s i n plants (22). The w i d e range of b i o l o g i c a l a c t i v i t y , (apparent) s t r u c t u r a l s i m p l i c i t y o f i n d i v i d u a l i s o m e r s , and p o t e n t i a l l y i n e x pensive nature of s y n t h e t i c s t a r t i n g m a t e r i a l s make t h e s e comp o u n d s an i n t r i g u i n g p o s s i b i l i t y for lead chemistry i n the synt h e s i s of n o v e l p e s t i c i d e s . Acknowledgment s P a p e r No. 11504 of the J o u r n a l S e r i e s of the N o r t h C a r o l i n a Agricultural Research S e r v i c e , R a l e i g h , NC 2 7 6 9 5 - 7 6 0 1 . Florida A g r i c u l t u r a l E x p e r i m e n t S t a t i o n J o u r n a l S e r i e s No. 8886 . The u s e o f t r a d e names i n t h i s p u b l i c a t i o n d o e s n o t i m p l y e n d o r s e ment by t h e N o r t h Carolina A g r i c u l t u r a l Research Service of the p r o d u c t s named, n o r c r i t i c i s m o f s i m i l a r ones n o t m e n t i o n e d . The a u t h o r s would l i k e t o t h a n k Mr. C l y d e W. A d c o c k , Mr. H e n r y ( B u d d y ) Q u i c k , Mr. M a r k M o s s i e r , M r . D a n a i Chow, M r . W e s l e y A d c o c k , M r . A l b e r t B u t l e r , M r . J o h n W e e k s , and Ms. Marie H a l l for their excellent technical assistance. A p p r e c i a t i o n i s extended to the North C a r o l i n a Tobacco F o u n d a t i o n and the R. J. Reynolds T o b a c c o Company f o r p a r t i a l f i n a n c i a l s u p p o r t o f t h i s w o r k . Literature 1.

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