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A a r d v a n d e s t o f f e n e n h u n w e r k i n g 1 A n a l y t i s c h - c h e m i s c h e b e p a l i n g 4 C o n c l u s i e 9 L i t e r a t u u r 1 0 B i j l a g e 1 ( n a g e k o m e n l i t e r a t u u r ) 1 1 B i j l a g e 2 ( t e s t m e t h o d e n ) 1 2
1 1 . INLEIDING I n d e u i t s c h e i d i n g s p r o d u k t e n v a n w o r t e l s v a n p l a n t e n komen s t o f f e n v o o r , d i e z i c h b i j l a n g d u r i g t e l e n o p d e z e l f d e o p l o s s i n g k u n n e n o p h o p e n . Een a a n t a l v a n d e z e s t o f f e n z i j n f e n o l i s c h e v e r b i n d i n g e n . Soms komen o o k a l d e h y d e - e n c a r b o x y l z u u r g r o e p e n i n d e v e r b i n d i n g e n v o o r . I n p r i n c i p e k u n n e n h e t e c h t e r z e e r v e r s c h i l l e n d e t y p e n o r g a n i s c h e v e r b i n d i n g e n z i j n . T e n o p z i c h t e v a n d e g e w a s g r o e i k u n n e n d e z e s t o f f e n z o w e l remmend a l s s t i m u l e r e n d z i j n . Ook k a n e r s p r a k e z i j n v a n s t o f f e n d i e d e opname v a n s p o o r e l e m e n t e n b e ï n v l o e d e n . Deze i n v l o e d k a n v o o r sommi g e p o s i t i e f e n v o o r a n d e r e n e g a t i e f z i j n . H i e r w o r d e n d e r e s u l t a t e n v a n e e n a a n t a l p u b l i k a t i e s o p d i t g e b i e d v e r m e l d . D e z e l f d e s o o r t s t o f f e n z i j n o o k g e v o n d e n i n w a t e r i g e e x t r a c t e n v a n v e e n - , m o e r a s - o f b o s g r o n d e n . V i a s p e c t r o f o t m e t r i s c h e r e a c t i e s o f g a s c h r o m a t o g r a f i e i s h e t m o g e l i j k d e f e n o l i s c h e e n a n d e r e v e r b i n d i n g e n t e b e p a l e n . V i a g e w a s e x p e r i m e n t e n z a l h e t m o g e l i j k z i j n d e i n v l o e d v a n o p h o p i n g b i j g e s l o t e n t e e l t e n b e t e r i n t e s c h a t t e n . A l s s n e l l e r e t e s t m e t h o d e z i j n e c h t e r e e n a a n t a l b i o t e s t m e t h o d e n o n t w i k k e l d . I n e e n e e r d e r v e r s l a g (PTG ' 9 0 - 6 2 ) w e r d o o k a a n d a c h t b e s t e e d a a n u i t s c h e i d i n g s p r o d u k t e n v a n p ^ a n t e w o r t e l s . D a a r g i n g h e t m e e r o v e r s t o f f e n d i e s p o o r e l e m e n t e n a l s Fe c o m p l e x b i n d e n e n d e i n v l o e d op d e opname. I n d a t v e r s l a g w e r d o o k a a n d a c h t g e s c h o n k e n a a n e n k e l e g e g e v e n s o v e r o n d e r l i n g remmende i n v l o e d v a n p l a n t e n v a n d e z e l f d e s o o r t e n v a n v e r s c h i l l e n d e s o o r t e n o n d e r l i n g ( a l l e l o p a t h i e ) . Zo w o r d t d a a r remming v a n a s p e r g e d o o r d e z e l f d e s o o r t v e r m e l d . H i e r g a a t h e t m e e r s p e c i a a l o m f e n o l i s c h e v e r b i n d i n g e n e n o r g a n i s c h e z u r e n .
2 . AARD VAN DE STOFFEN EN HUN WERKING
Zo o n d e r z o c h t e n J a l a l e n Read ( 1 9 8 8 ) e e n a a n t a l p h y t o t o x i s c h e s t o f f e n u i t h e i d e g r o n d , d i e d o o r C a l l u n a u i t g e s c h e i d e n w o r d e n . I n t a b e l 1 w o r d t e e n a a n t a l d o o r h e n v e r m e l d e s t o f f e n genoemd. T a b e l 1 . V e r b i n d i n g e n a a n g e t o o n d i n d e C a l l u n a g r o n d . I n ^ i g / 1 0 0 g d r o g e g r o n d . A r o m a t i s c h e z u r e n A l i f a t i s c h e z u r e n b e n z o ë z u u r 30 - J 50 3 - h y d r o x y o c t a a n z u u r 50 - 2 0 0 0 p - m e t h o x y b e n z o ë z u u r 9 0 • 540 8 - h y d r o x y o c t a a n z u u r 0 - 1 2 5 0 o - h y d r o x y b e n z o ë z u u r 50 • 1200 h y d r o x y d e c a a n z u u r 1 5 - 740 v a n i l l i n e z u u r 1C0 • 550 8 - h y d r o x y n o n a a n z u u r 30 - 770 s y r i n g i n e z u u r 35 • D20 8 - e t h o x y d e c a a n z u u r 0 - 2 5 0 0 p - c u m a r i n e z u u r 0 • 280 8 - h y d r o x y d e c a a n z u u r 1 2 5 - 3 0 0 0 f e r u l i n e z u u r 0 - 800 U i t J a l a l a n d R e a d , 1 9 8 3 . G r o e i r e d u c t i e s m e t d i t s o o r t s t o f f e n i s g e c o n s t a t e e r d . Zo i s d a t h e t g e v a l v o o r s a l i c y l z u u r d a t b i j e e n c o n c e n t r a t i e v a n 0 , 0 1 mM b i j t a r w e
remming v e r o o r z a a k t . O n d e r v a r e n s kwamen g e h a l t e n v o o r v a n 0 , 0 4 ; 0 , 0 5 ; 0 , 0 4 e n 0 , 0 0 4 mM p -h y d r o x y b e n z o ë z u u r , v a n i l l i n e z u u r , c u m a r i n e z u u r e n f e r u l i n e z u u r v o o r . G r i m v a l l e t a l . ( 1 9 9 0 ) c i t e e r t o n d e r z o e k , w a a r b i j f e n o l i s c h e a l d e h y d e n e r g f y t o t o x i s c h genoemd w o r d e n ( e x t r a c t i e s i n w a t e r v o o r a l v a n m o e r a s g r o n d e n c o n i f e r e n b o s b l e e k s t e r k g r o e i r e m m e n d i n e e n w o r t e l t e s t ) . P a u l & Mc L a r e n ( 1 9 8 1 ? ) g e v e n d e s t r u c t u u r v a n e e n g r o o t a a n t a l f e n o l e n u i t v e e n e x t r a c t e n , w a a r v a n sommige g r o e i r e m m e n d z i j n . E n k e l e s t r u c t u r e n w o r d e n i n f i g u u r 1 w e e r g e g e v e n . D i t s o o r t s t o f f e n k a n i n p o t g r o n d e n e e n i n v l o e d o p p l a n t e n u i t o e f e n e n . Wang, e t a l . ( 1 9 6 7 ) g e v e n o o k e e n opsomming v a n f e n o l i s c h e v e r b i n d i n g e n , d i e d e p l a n t e g r o e i remmen. O f o s u - B u d u ( 1 9 9 0 ) noemt ammonia a l s u i t s c h e i d i n g s p r o d u k t b i j s o j a b o o n . W o j c i k - W o j t k o w i a k e t a l ( 1 9 9 0 ) noemen o o k e e n a a n t a l f e n o l e n a l s t o x i s c h e u i t s c h e i d i n g s p r o d u k t e n b i j v e r t e r i n g v a n w e e f s e l s v a n r o g g e . Van d e z e s t o f f e n w o r d t e e n s t e r k remmende i n v l o e d b e s c h r e v e n . S c e h o v i c ( 1 9 9 0 ) b e s c h r i j f t f e n o l e n i n w e i d e p l a n t e n . I n sommige p l a n t e n z i j n d e g e h a l t e n a a n o p l o s b a r e f e n o l e n e n p o l y m e r e f e n o l e n e r g h o o g ( m e e r d a n 1 0 g / k g ) . A l s d e z e p l a n t e n v e r g a a n komen d e z e f e n o l e n i n h e t o r g a n i s c h m a t e r i a a l . De f e n o l i s c h e v e r b i n d i n g e n d i e d o o r d e z e a u t e u r v e r m e l d w o r d t z i j n l i g n i n e n , t a n n i n e e n e e n v o u d i g e f e n o l e n . G ö h l e r b e s c h r e e f r e e d s i n 1 9 6 7 remming b i j l a n g d u r i g e t e e l t v a n t o m a a t e n komkommer. I n d e g e b r u i k t e g r i n d c u l t u u r t r a d b i j t o m a a t w o r t e l s c h a -d e o p . De remming w e r -d g e v o n -d e n b i j t o e v o e g i n g v a n v e r s e w o r t e l s e n v a n e x t r a c t e n . T o e v o e g i n g v a n v e r d u n d w a t e r s t o f p e r o x y d e k o n d e remming g r o t e n d e e l s t e n i e t d o e n . Ook c h l o o r k a l k ( C a O C l - ) h a d e e n d e r g e l i j k e f f e c t . W o r t e l e x u d a t e n v a n p l a n t e n b e v a t t e n e e n g r o o t a a n t a l s t o f f e n , w a a r o n d e r remmende k u n n e n z i j n . Zo b e s c h r i j f t V a n c u r a e t a l ( 1 9 7 2 ) s t o f f e n i n w o r t e l e x u d a t e n . N a a s t e e n v r i j g r o o t a a n t a l p l a n t e v o e d e n d e s t o f f e n a l s a m i n o z u r e n e n s u i k e r s , z i j n e r o o k o r g a n i s c h e z u r e n . Sommige h i e r v a n z o a l s o x a a l z u u r d a t i n w o r t e l e x u d a a t v a n komkommer i n r e l a t i e f h o g e c o n c e n t r a t i e s v o o r k o m t , k u n n e n i n p r i n c i p e d e g e w a s g r o e i remmen. U r e n e n R e i s e n a u e r ( 1 9 8 8 ) b e s c h r i j v e n i n w o r t e l e x u d a t e n v a n p l a n t e n o r g a n i s c h e z u r e n , s i d e r o p h o r e n e n a l l e l o p a t h i s c h e v e r b i n d i n g e n . Young e t a l . ( 1 9 8 9 ) b e s c h r i j f t e e n a a n t a l f e n o l i s c h e v e r b i n d i n g e n -m e e s t r e e d s e e r d e r genoe-md - i n g r o n d e n w a a r o p t a r w e s t r o i s t o e g e p a s t .
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4 p^vnyipropan« sit«i«ton of i tgmn n 5 '«sorcir^l 6 phlorogluonol y '0 om»fK0wycortche( OCH, <9 orctnoi CHO 2 *ant||,o CHO OH . )J ^ 0 H « . C O ^ ^ O C H , OH I $yr»ngold«hyd« Ç". CO O u OH 20 ocviovanitloi 3 p hyd»o«y-benzo^vhyö« COOH OH OH 17 pro'oeof«cku»e ood COOH HO '0 H 16 a-r«*orcyl«e add OH 16 p hydroiy-b«ntoiC oeid HJCO^^^^OCH, OH 14 fynngic ocid OH '3 p.coumarte oe<d COOH CH 0 L , OH 12 faruhc oad COOH I CO O " i j r 9 r.6-<i»m»tho*y p^*nol CH. H diçuaiocylmfhone OH OCH OM OH 21 3.4-dihydfo*r ptoptoptonone CH OH ' 15 «anilltc oc'd-oc
0 scission probuct f'om B ' inq o' Hovonoid 22 P ^vdro*ypKeovi -pyruvic ocid CO ' T 0 r 2 3 2 , 4 , 6 I n h y d f O * ' oce'opHenoo« F i g u u r 1 . S t r u c t u u r v a n e n k e l e a r o m a t i s c h e v e r b i n d i n g e n u i t v e e n P a u l & Mc L a r e n , 1 9 8 1 .I n f i g u u r 2 e n 3 z i j n r e s u l t a t e n w e e r g e g e v e n v a n d e t o x i c i t e i t v a n e e n a a n t a l f e n o l i s c h e c a r b o n z u r e n e n d e remming i n d e g r o e i v a n e e n a a n t a l g e w a s s e n . C o n c e n t r a t i e s v a n 1 0 0 m g . k g i n w a t e r c u l t u u r g a v e n e e n a a n z i e n l i j k e remming t e z i e n . H e t o n d e r l i n g v e r s c h i l i n d e i n v l o e d v a n d e s t o f f e n i s n i e t g r o o t .
3 . ANALYTISCH-CHEMISCHE BEPALING VAN FENOLEN EN ORGANISCHE ZUREN
I n d i e n o r i ë n t e r e n d o n d e r z o e k a l s r e s u l t a a t z o u h e b b e n d a t i n g e s l o t e n t e e l t i n b e p a a l d e g e v a l l e n remming o p t r e e d t d a n h e e f t h e t z i n om a n a l y s e s t e d o e n i n d e g e c o n c e n t r e e r d e v o e d i n g s o p l o s s i n g e n . H e t z a l b e l a n g r i j k z i j n om m e t h o d e n t e r b e s c h i k k i n g t e h e b b e n d i e m e t e e n v o u d i g e m i d d e l e n e e n b e e l d g e v e n v a n d e f e n o l e n e n o r g a n i s c h e z u r e n . A a n g e z i e n h e t z u r e s t o f f e n b e t r e f t k a n e e n e e r s t e s t a p z i j n a a n z u r e n v a n d e v o e d i n g s o p l o s s i n g e n e x t r a c t i e m e t e e n o r g a n i s c h o p l o s m i d d e l . S u b s t r a t e n a l s v e e n k u n n e n h e t b e s t e m e t N A O H - o p l o s s i n g e n g e ë x t r a h e e r d w o r d e n . S c h e i d i n g k a n p l a a t s v i n d e n v i a g a s c h r o m a t o g r a f i e , v l o e i s t o f c h r o m a t o g r a f i e , p a p i e r c h r o m a t o g r a f i e e n d u n n e l a a g c h r o m a t o -g r a f i e . Ook k a n e e n t o t a a l f r a c t i e ' f e n o l i s c h e v e r b i n d i n -g e n ' n u t t i -g z i j n om t e b e p a l e n . D e t e c t i e k a n b i j v o o r b e e l d g e b e u r e n v i a u v a b s o r p -t i e e n v i a k l e u r i n g m e -t e e n r e a g e n s e n m e -t i n g v a n d e l i c h -t a b s o r p -t i e . I n f r a r o o d a b s o r p t i e k a n i n f o r m a t i e g e v e n o v e r f e n o l g r o e p e n , c a r b o x y l -z u u r g r o e p e n , e n -z o v o o r t . Een v r i j u i t g e b r e i d s c h e m a v o o r e x t r a c t i e e n g l o b a l e s c h e i d i n g v a n f e n o l e n , z o a l s b e s c h r e v e n d o o r S c e h o v i c ( 1 9 9 0 ) , i s i n f i g u u r 4 w e e r g e g e v e n . Voor d e d e t e c t i e w o r d t h i e r g e b r u i k g e m a a k t v a n k l e u r r e a c t i e s . J a l a l e n R e a d ( 1 9 8 3 ) b e s c h r i j v e n e e n m e t h o d e d i e t o e g e p a s t w o r d t op h e i d e g r o n d . E x t r a c t i e g e b e u r t m e t a l c o h o l i s c h e l o o g . Z u i v e r i n g k a n p l a a t s h e b b e n d o o r o v e r b r e n g e n i n e e n o r g a n i s c h o p l o s m i d d e l , e x t r a c t i e m e t N a H C O ^ - o p l o s s i n g e n o p n i e u w e x t r a c t i e m e t e e n o r g a n i s c h o p l o s m i d d e l n a a a n z u r e n . D u n n e - l a a g c h r o m a t o g r a f i e w o r d t t o e g e p a s t om t e s c h e i d e n . De d e t e c t i e g e b e u r t h i e r m e t u v - l i c h t . B i j k w a n t i t a t i e v e s c h e i d i n g e n w o r d t g a s c h r o m a t o g r a f i e t o e g e p a s t . V a n c u r a e t a l . ( 1 9 7 2 ) b e p a l e n e e n r e e k s o r g a n i s c h e z u r e n , w a a r o n d e r o x a a l z u u r m e t p a p i e r c h r o m a t o g r a f i e .
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A » Solution d'albumine de sérum bovin (1.5 g/1 dan* tampon pH5 • Na('l)
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F i g u u r 4 . A n a l y s e s c h e m a v o o r d e b e p a l i n g v a n e e n a a n t a l f e n o l f r a c t i e s . S c e h o v i c ( 1 9 9 0 ) .
Young e t a l . ( 1 9 8 9 ) analvseren t a r w e e n r i j s t g r o n d e n i n v e r b a n d met f y t o t o x i c i t e i t . H e t door hen t o e g e p a s t e schema i s i n f i g u u r 5 weerge g e v e n . V o o r a l m e t d e extractie b i j pH 8 worden f e n o l e n m e e g e ë x t r a -h e e r d . De d e t e c t i e die in dat onderzoek w o r d t g e b r u i k t i s een b i o t e s t .
7 s o i l r e s i d u e 2 0 0 K t o i l 4 0 0 m i d i s t i l l e » ! w a t e t a d j u s t t o p H 5 . 5 • 0 . 2 w i i h I N , O. l N I K " ! o r p H 8 . 0 • 0 . 2 w i t h I N , 0 . 1 N \ a ( ; M s h a k e ( o f 4 8 h i n c o l d r o o m ( 4 ° f . ) c e n t r i f u g e a t 1 5 * 1 0 r p r n , ? 0 n u n s u p e i n a t a n t f i l t e r t h r o u g h M i l h p « . r e ( u . 4 5 ; n n ) c l e a r a q u e o u s e x t r a c t A d j u s t p H t o 7 2 0 0 m l f o r I v o p h i h ? i t t o n d r y c r u d e e x t r a c t s e e d h i o a s s a y Ft* Sotl function procedure for r»cr paddy ar>d wheat-rice rouuoo m»W
l y o p h i l i s a t i o n
F i g u u r 5 . E x t r a c t i e m e t h o d e v o o r d e b e p a l i n g v a n f y t o t o x i s c h e v e r b i n d i n g e n i n r i j s t e n t a r w e g r o n d e n .
Wang, Y a n g , Chuang ( 1 9 6 7 ) b e s c h r i j v e n e e n p a p i e r c h r o m a t o g r a f i s c h e me t h o d e . I d e n t i f i c a t i e h e e f t p l a a t s v i a b e s t r a l i n g m e t u l t r a v i o l e t l i c h t o f b e s p u i t i n g m e t g e d i a z o t e e r d p n i t r a l i n e o f g e d i a z o t e e r d s u l f a n y l -z u u r . Ook W o j c i k - W o j t k o w i a k ( 1 9 9 0 ) t o n e n f e n o l e n o p p a p i e r o n d e r u v - l i c h t a a n . B a u e r e n T r e u t t e r ( 1 9 9 0 ) g e b r u i k e n v l o e i s t o f c h r o m a t o g r a f i e v o o r s c h e i d i n g . D i t i s e e n v r i j b e w e r k e l i j k e m e t h o d e . B i j p a p i e r c h r o m a t o g r a f i e g e b r u i k e n z e o n d e r a n d e r e d i p h e n y l b o o r z u u r - 3 - a m i n o - e t h y l e s t e r 1% i n m e t h a n o l p l u s 5% p o l y e t h y l e e n g l y c o l 4 0 0 . H e t i s d u s m o g e l i j k d o o r e x t r a c t i e s d i e g e b r u i k maken v a n d e z u r e e i g e n s c h a p p e n v a n f e n o l e n e e n ruwe a f s c h e i d i n g t e k r i j g e n . D e t e c t i e k a n g e b e u r e n d o o r k l e u r r e a c t i e s a l s d e * ) F o l i n - C i o a l t e u - r e a c t i e . V e r d e r k a n g e b r u i k w o r d e n g e m a a k t v a n d e s p e c i f i e k e a d s o r p t i e i n h e t u v v a n d e f e n o l g r o e p . Door h e t g r o t e a a n t a l s t o f f e n i n d e r g e l i j k e e x t r a c t e n i s d e z e m e t h o d e e c h t e r n i e t e r g s e l e c t i e f . De e n i g e m e t h o d e i s d a n e e n v e r d e r e z u i v e r i n g . Met s e l e c t i e v e a b s o r p t i e k o l o m m e n k a n d i t n o g v r i j e e n v o u d i n g , v e r d e r g a a n d e s c h e i d i n g m e t g a s - o f v l o e i s t o f c h r o m a t o g r a f i e i s v r i j b e w e r k e l i j k . * ) H e t r e a g e n s v o l g e n s F o l i n - C i o c a l t e u i s e e n r e a g e n s o p f e n o l e n , d a t b e r u s t o p o x y d a t i e . De f e n o l e n w o r d e n d a a r b i j o m g e z e t v a n f e n o l i n c h i n o n . De k l e u r g a a t v a n g e e l n a a r b l a u w . H e t i s p h o s p h o w o l f r a a m m o l y b -d e e n r e a g e n s .
CONCLUSIE I n d i t v e r s l a g w o r d e n g e g e v e n s v e r m e l d o v e r f e n o l i s c h e v e r b i n d i n g e n . D i t z i j n a r o m a t i s c h e v e r b i n d i n g e n m e t e e n -OH g r o e p , v a a k i n c o m b i n a t i e m e t a l d e h y d e g r o e p e n ( - o f c a r b o x y l z u u r g r o e p e n (-COOH). Deze v e r b i n d i n g e n k u n n e n d e p l a n t e g r o e i remmen, v o o r b e e l d e n h i e r v a n z i j n v e r m e l d . De f e n o l i s c h e k u n n e n v r i j g e m a k k e l i j k g e ï s o l e e r d w o r d e n g e b r u i k m a k e n d v a n d e z u r e e i g e n s c h a p p e n v a n d e f e n o l g r o e p . E x t r a c t i e k a n g e b e u r e n m e t v e r d u n d e a l k a l i , b i j v o o r b e e l d v e r d u n d e N a O H - o p l o s s i n g . H i e r u i t k u n n e n d e f e n o l e n w e e r g e ï s o l e e r d w o r d e n n a a a n z u r e n d o o r e x t r a c t i e m e t e e n o r g a n i s c h o p l o s m i d d e l a l s e t h e r . De f e n o l e n k u n n e n m e t h i e r v e r m e l d e k l e u r r e a c t i e s w o r d e n b e p a a l d . De remming k a n w o r d e n g e t e s t m e t k i e m t e s t e n , g r o e i v a n z a a i l i n g e n o f w o r t e l t e s t m e t h o d e n . De p r a k t i s c h e b e t e k e n i s h i e r v a n b i j h e t l a n g d u r i g t e l e n o p d e z e l f d e o p l o s s i n g i s n o g n i e t d u i d e l i j k . Met e e n v o u d i g e b i o l o g i s c h e t e s t m e t h o d e n e n g l o b a l e i s o l a t i e z o u h i e r e c h t e r g e m a k k e l i j k e e n b e e l d v a n v e r k r e g e n w o r d e n . I n d i e n w e r k e l i j k e f f e c t e n z o u d e n w o r d e n g e v o n d e n , z o u u i t g e b r e i d e r o n d e r z o e k z i n v o l z i j n .
Literatuur B a u e r , H. e n D. T r e u t t e r ( 1 9 9 0 ) . I d e n t i f i c a t i o n o f P e l a r g o n i u m g e n o t y p e s b y p h e n o l i c " f i n g e r p r i n t s " . I . S e p r a t i o n a n d i d e n t i f i c a t i o n o f p h e n o l i c compounds i n l e a v e s . G a r t e n b a u w i s s e n s c h a f t 5 5 : 1 1 3 - 1 1 8 . G ö h l e r , F . ( 1 9 6 7 ) . U n t e r s u c h u n g e n ü b e r d i e U r s a c h e n v o n W a c h s t u r n s s c h a d e n b e i m e h r j ä h r i g e r D u r c h f ü h r u n g . A r c h i v f ü r G a r t e n b a u XV: 1 7 -3 0 . G o o r , B . J . v a n , ( 1 9 9 0 ) . E x u d a t e n v a n p l a n t e w o r t e l s e n m i c r o b e n i n d e r h i z o s f e e r . S a m e n s t e l l i n g e n w e r k i n g b i j d e o p n a m e . I n t e r n V e r s l a g PTG, N a a l d w i j k , n r . 6 2 . G l a s s , A.D.M. ( 1 9 7 6 ) . T h e a l l e l o p a t h i c p o t e n t i a l o f p h e n o l i c a c i d s a s s o c i a t e d w i t h t h e r h i z o s p h e r e o f P t e r i d i u m a q u i l i n u m . C a n . J . B o t , 5 4 : 2 4 4 0 - 2 4 4 4 . G r i m v a l l , A . , M.B. B e n g t s s o n , H. B o r é n e n D. W a h l s t r ö m , ( 1 9 9 0 ) . P h y t o -t o x i c s u b s -t a n c e s i n r u n o f f f r o m f o r e s -t e d c a -t c h m e n -t a r e a s . P r o c e e d i n g s o f " I n t e r n a t i o n a l symposium o n h u m i c s u b s t a n c e s i n t h e a q u a t i c a n d t e r r e s t r i a l e n v i r o n m e n t " . S p r i n g e r V e r s l a g , B e r l i j n , i n b e w e r k i n g . J a l a l , M . A . F . e n D . J . R e a d , ( 1 9 8 3 ) . T h e o r g a n i c a c i d c o m p o s i t i o n o f C a l l u n a h e a t h l a n d s o i l w i t h s p e c i a l r e f e r e n c e t o p h y t o a n d f u n g i -t o x i c i -t y . I . I s o l a -t i o n a n d i d e n -t i f i c a -t i o n o f o r g a n i c a c i d a n d I I . M o n t h l y q u a n t i t a t i v e d e t e r m i n a t i o n o f t h e o r g a n i c a c i d c o n t e n t o f C a l l u n a a n d s p r u c e d o m i n a t e d s o i l s . P l a n t a n d S o i l 7 0 : 257 - 272 a n d 2 7 3 - 2 8 6 . O f o s u - B u d u , K . G . , K. F u j i t a e n S . O g a t a , ( 1 9 9 0 ) . E x c r e t i o n o f ureide a n d o t h e r n i t r o g e n o u s compounds b y t h e r o o t s y s t e m o f s o y b e a n a t d i f f e r e n t g r o w t h s t a g e s . P l a n t a n d S o i l 1 2 8 : 1 3 5 - 1 4 2 . P a u l . E . A . e n A . D . Mc L a r e n , ( ? ) S o i l B i o c h e m i s t r y , M a r c e l D e k k e r I n c . , N . Y . : 1 6 7 - 1 8 3 . S c e h o v i c , J . , 1 9 9 0 . T a n i n e s e t a u t r e s p o l y m è r e s p h é n o l i q u e s d a n s l e s p l a n t e s d e p r a i r i e s : d é t e r m i n a t i o n d e l e u r t e n e u r e t d e l e u r a c t i v i t é b i o l o g i q u e . R e v u e S u i s s e A g r i c . 2 2 : 1 7 9 - 1 8 4 . U r e n , N . C . e n H.M. R e i s e n a u e r , ( 1 9 8 8 ) . T h e r o l e o f r o o t exudates in nutrient aquisition. In: B. Tinker and A. Lauchli, Advances in p l a n t n u t r i t i o n 3 , P r a e g e r N . Y . , W e s t p o r t , p . 79 - 1 1 4 .
Vancura, V. and A. H a n z l i k o v a , ( 1 9 7 2 ) . R o o t e x u d a t e s o f plants. IV. Differences in chemical composition of seed and seedlings exuda tes. Plant and S o i l 3 6 : 2 7 1 - 2 8 2 .
Wang, T.S., T.K. Yang a n d T . T . Chuang, ( 1 9 6 6 ) . S o i l p h e n o l i c a c i d s a s plant growth i n h i b i t o r s . S o i l S c i e n c e 1 0 3 : 239 - 2 4 6 .
Wojcik-Wojtkowiak, D . , B. P o l i t y c k a , M. S c h n e i d e r a n d J . P e r k o w s k i ,
( 1 9 9 0 ) . P h e n o l i c s u b s t a n c e s a s a l l e l o p a t h i c a g e n t s a r i s i n g d u r i n g t h e d e g r a d a t i o n o f r y e ( S e c a l c e r e a l e ) t i s s u e s . P l a n t a n d S o i l 1 2 4 : 1 4 3 - 1 4 7 .
Young, C . C . , L.R. Zhue Thorne a n d G.R. W a l l e r , ( 1 9 8 9 ) . P h y t o t o x i c po t e n t i a l o f s o i l s a n d w h e a t s t r a w i n r i c e r o t a t i o n c r o p p i n g s y s t e m s o f s u b t r o p i c a l T a i w a n . P l a n t a n d S o i l 1 2 0 : 9 5 - 1 0 1 .
11 B i j l a g e 1 Nagekomen literatuur C a r b a l l e i r a , A. , ( 1 9 8 0 ) . " P h e n o l i c i n h i b i t o r s i n E r i c a a u s t r a l i s L . a n d i n a s s o c i a t e d s o i l " . J o u r n a l o f C h e m i c a l E c o l o g y 6 : 5 9 3 - 5 9 6 . L e a t h e r , G . R . a n d F . A . E i n h e l l i g ( 1 9 8 8 ) . B i o a s s a y o f n a t u a r a l l y o c c u r r i n g a l l e l o c h e m i c a l s f o r p h y t o t o x i c i t y . J o u r n a l o f C h e m i c a l E c o l o g y 1 4 : 1 8 2 1 - 1 8 2 8 . L u n d i n , P . , B. W i b r a n d t a n d K.O. J ö n s s o n ( 1 9 8 9 ? ) . B i o l o g i s k t e s t a v b e v a t t n i n g s v a t t e n . J o r d b r u k 4 9 : 1 9 1 - 1 9 7 . P u t n a m , A . R . e n C . S . T a n g ( 1 9 8 6 ) . T h e s c i e n c e o f a l l e l o p a t h y . J o h n Wi l e y & S o n s , N . Y . , 3 1 7 p p .
B i j l a g e 2
Testmethoden voor de remmende of stimulerende stoffen.
C a r b a l l e i r a ( 1 9 8 0 )
U i t L e a t h e r & E i n h e l l i g ( 1 9 8 8 ) U i t G r i m v a l l , e t a l . ( 1 9 9 0 )
Samples of plant material and the associated soil were repeatedly extracted with methanol. The extracts were concentrated to dryness under vacuum, redissQlved in distilled water, acidified to pH 2.5with^2_\_HCl. and extracted witli suliunc pther. lhe ether fraction was then reextracted using a saturated aquWas-sOÜium acetate solution. After acidification, organic ; compounds were separated with ether.
Chromogenic reactions on paper a n d thin-layer chromatography as well
as UV spectrophotometry were used to isolate and identify phenolic compounds, comparing the results obtained with those of standards of the pure samples.
Seed Germination Bioassaxs. The inhibition (or stimulation) of seed ger mination has been die most widely used bioassav for the determination of alle lopathic activity. However, the seed germination process is probably the least understoodjM all plant functions (Leather, 1987). Seed germination begins~with imbibition of water and ends w ith the protrusion of the radicle through the testa. Radicle elongation is by cell extension only and does not involve cell division. The biochemical events associated with germination are not well defined and may only be preparatory for the mobilization of reserves for seedling growth. Thus, definitive conclusions of allelopathic mechanisms in seed germination bioassays are limited but mav involve membrane alteration, resulting in loss of metabolites and the ability to establish the necessary osmotic potential for cell elongation (Koller and Hadas. 1982). Other processes, such as alteration of the phytochrome control of germination, may also be effected. We found that some naturally occurring volatile compounds stimulated the dark germination of Rumex sp. that normally require postimbibitional light (French and Leather. 1979). Other perturbations from allelochemicals of seed germination processes niay be involved, but we must await further knowledge of the biochemical events that occur during seed germination that are directly related to the germination process.
The greatest problem that affects the veracity of seed germination
bioas-says results from the manner in which the bioassav is conducted. Anderson and
Loucks (1966) emphasized the importance of the solution osmotic potential
when testing plant extracts; however, few reports on allelopathic effects con
f e r this precaution (Leather and Einhellig, 1986). Weidenhamer et al. (1987) reported that the number of seeds relative to the solution volume used in a seed germination bioassay was a factor in the results obtained. They found that the amount of ferulic acid available to each seed influenced the germination, rather than the concentration of chemical in the test solution. In conducting this research, care was taken to prevent anaerobic conditions by submersion of the seeds in water. We have found reports in the literature of allelopathic action where the investigators germinated the test species in volumes of solution that were 20 times the amount required for optimum germination without anaero-biosis.
13
Blum et al. (1984) offer recommendations for rhe standardization of
ger-mination bioassavs. However, their results are based on radicle growth subse
quent to germination and perturbations at any of the stages of germination and growth may have effects on any subsequent stage. Nonetheless, their observa tions regarding pH. microbial contamination, photolability of ailelochemical, and loss of test compound are very important when conducting germination bioassavs. Our recent review on this subject outlines additional precautions that should be observed when conducting seed germination bioassavs (Leather and Einhellig. 1986).
Radicle Elongation Bioassays. Radicle elongation is a more sensitive assay for allelochemicals than seed germination (Leather and Einhellig, 1985: Ein hellig. 1986). Like seed germination, radicle elongation is extremely sensitive to high (100 mosmol) osmotic potentials of solutions, and concentrations of purified extracts must be evaluated prior to assay (Bell. 1974). Generally, the radicle is completely dependent upon the seed (cotyledon) reserves for growth in the dark, and precautions must be taken to separate effects upon the seed and the mobilization of storage material by the ailelochemical during or immediately following germination. Blum et al. (1984) reported that surface sterilization of seeds with sodium hypochlonde modified radicle growth. Thus, as previously noted, care must be taken to minimize early effects of the ailelochemical upon the seed that may alter subsequent radicle growth.
The accuracy of results obtained from the measurement of radicles elon gating in Petri dishes is questionable. Few such radicles elongate on a straight course, and precise measurements are difficult. We have found that removing the radicle from seed germinated in Petri dishes and thoroughly drying to a
constant weight gives accurate results with small statistical e r r o r (Leather and
Hurtt, unpublished). Parker (1966) described a method to determine herbicide
uptake and effects on radicle elongation. We modified this method to use pre-germinated seed placed between chromatography sandwich plates that are main
tained at a 45° angle, thus having straight radicles for measurement
Additionally, this method allows only the radicle to be in contact with the test chemical solution (Leather and Einhellig, 1985).
Radicle elongation does afford greater possibilities for mechanism s t u d i e s
than seed germination. It is particularly suited for determining effects of alle lochemicals on hormones responsible for cell growth. Radicle elongation also
a l l o w s evaluation of ailelochemical effects on respiration and cell division.
Seedling Growth Bioassays. Seedling growth bioassays are extremely ver satile but require a greater quantity of chemical than is usually available during initial isolation and identification of allelochemicals. These bioassays usually have greater sensitivity and provide the basis for a variety of mechanism stud ies, such as nutrient uptake, water relations, and photosynthesis, but here again, it is difficult to determine the primary sites and mechanisms of action of the ailelochemical.
Blum and colleagues (Blum and Dalton, 1985; Blum et al. 1985a.b). used leaf expansion of cucumber seedlings grown in nutrient culture to determine the mechanism of action of ferulic acid and its microbial metabolic products. In these reports, they stress the importance of monitoring the loss of ailelochemical through absorption, microbial breakdow n, or other mechanisms, including dis sociation of the chemical in solutions of changing pH values.
Using a sorghum (Sorghum) seedling bioassay. we found that
[l 4C)salicyclic acid was rapidly taken up from the nutrient solution in which the
seedling was growing, and it was distributed throughout the seedling within 24 hr after treatment (unpublished results). Such rapid, widespread translocation severely limits the utility of seedling growth bioassays for pinpointing primary sites or mechanisms of ailelochemical action.
The Cucumber Root Bio-assay
Seeds of field cucumber (Weibull Favor) were placed in a Petri dish on a piece of
filter paper (Munktell 1410) moistened with the water to be tested. After 3 days in
darkness at 22°C and 70% humidity, 10 seedlings of average size were selected and rolled
into wet filter paper. The paper roll was put into a 300 ml beaker with 50 ml of the water
sample, and the seedlings were grown for 4 days at 24°C and 70% humidity. Photoperiod
(10,000 lux) was 10-12 h per day. After 2 days in the climate room, another 50 ml of the
water sample was added. At the end of the bioassay, the paper roll was unrolled and the
root length was measured for each seedling. Root discolouration,
gf rfM k*"™
and other visible root injuries were noted. Each water sample was tested in two of the
described seedling paper rolls.
U Î È A L L , £ T F I L .
