Germination and Growth Inhibitors as Allelochemicals - American

C h a p t e r 11

Germination and Growth Inhibitors as Allelochemicals W. Rüdiger and E. Lohaus

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Botanisches Institut der Universität München, Menzinger Str. 67, D-8000 München 19, Federal Republic of Germany Germination inhibitors have been discussed for a long time as one of the reasons for dormancy of seeds. Some observations, however, point to the possibility that such inhibitors may also act as allelochemicals. When we tried to germinate oat caryopses we observed a decreasing rate of germination with increasing density of caryopses. Inhibition of germination could also be achieved with aqueous extracts from the caryopses (1). Interestingly, the inhibitory activity of oat caryopses is by no means restricted to germination of oats. We found that the seeds of several other plants (e.g. of Raphanus sp. or of Amaranthus caudatus) are much more sensitive against inhibitors from oat caryopses than oats itself. This means that germination of such seeds is effectively inhibited i f they are in the immediate neighborhood of oat caryopses even i f germination of oats itself can take place. Oats are more active in allelopathy than other cereals (2). Such a l l e l o p a t h i c a c t i v i t y i s d e v e l o p e d d u r i n g m a t u r a t i o n o f o a t p l a n t s . Whereas o n l y l i t t l e a c t i v i t y i s found i n r o o t s , somewhat more

i s found i n stem and leaves. The highest a c t i v i t y , however, i s found i n the caryopses during maturation, e s p e c i a l l y i n the husks {3). In t h e f i r s t p a r t o f t h i s p a p e r , we s h a l l d e s c r i b e i s o l a t i o n and s t r u c t u r a l e l u c i d a t i o n o f t h e main i n h i b i t o r from t h e husks o f Avena s a t i v a , which can be c o n s i d e r e d as an a l l e l o c h e m i c a l a g a i n s t seeds o f s e v e r a l p l a n t s . The second p a r t o f t h e paper d e a l s w i t h a l l e l o c h e m i c a l s from r o s e seeds w h i c h , a l t h o u g h o f d i f f e r e n t chemical s t r u c t u r e , bear some r e l a t i o n s h i p t o t h e a l l e l o c h e m i c a l from o a t s . The b a s i s f o r t h i s t y p e o f i n v e s t i g a t i o n i s a q u a n t i t a t i v e b i o t e s t as d e s c r i b e d i n d e t a i l by Karl and Rudiger [V). The crude aqueous e x t r a c t from o a t husks i n h i b i t s g e r m i n a t i o n o f seeds from s e v e r a l p l a n t s , e . g . Avena s a t i v a , Sorghum s p . , P h a l l e r i s s p . , Raphanus s p . , Amaranthus c a u d a t u s , L e p i d i u m sativum L . . The i n h i b i t o r y a c t i v i t y increases with i n c r e a s i n g c o n c e n t r a t i o n of the e x t r a c t s . F i g u r e 1 shows t h e c o n c e n t r a t i o n dependency o f i n h i b i t i o n o f r o o t growth a f t e r f r a c t i o n a t i o n o f t h e e x t r a c t by e t h e r e x t r a c t i o n . Whereas l e s s a c t i v i t y i s found i n t h e n e u t r a l f r a c t i o n (pH 7 ) , at pH 1 about equal a c t i v i t y i s p r e s e n t i n t h e e t h e r l a y e r and t h e water phase. The l a t t e r c o n t a i n s b i o a c t i v e c o n j u g a t e s o f o r g a n i c 0097-6156/87/0330-0118$06.00/0 © 1987 A m e r i c a n C h e m i c a l Society

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

11.

RUDIGER AND LOHAUS

Germination

and Growth

119

Inhibitors

1

S

acids; after a l k a l i n e s a p o n i f i c a t i o n , the b i o a c t i v i t y i s also ext r a c t a b l e i n t o e t h e r a f t e r adjustment t o pH 1. F o r f u r t h e r f r a c t i o n a t i o n and s e a r c h f o r a c t i v e compounds, i t i s i m p o r t a n t t h a t t h e b i o t e s t be made q u a n t i t a t i v e as i n d i c a t e d i n F i g u r e 1. The main com pounds o f t h e a c i d f r a c t i o n a r e p h e n o l i c a c i d s . A f t e r m e t h y l a t i o n , t h e methyl e s t e r s were s e p a r a t e d by gas chromatography and i d e n t i f i e d by comparison w i t h a u t h e n t i c compounds, some o f which a r e shown here ( s t r u c t u r e s 1 - 4 ) . S i n c e t h e s e compounds (and t h e f o l l o w i n g ones) o c c u r n a t u r a l l y as f r e e c a r b o x y l i c a c i d s but a r e i n v e s t i g a t e d as t h e methyl e s t e r s , t h e l e t t e r "R" i s used i n t h e s t r u c t u r e s f o r e i t h e r " H " ( n a t u r a l compound) o r " C H " ( i n v e s t i g a t e d methyl e s t e r ) . 3

P h e n o l i c a c i d s have a l r e a d y been d i s c u s s e d as i n h i b i t o r y compounds. But i n h i b i t i o n o f g e r m i n a t i o n r e q u i r e s a c o n c e n t r a t i o n o f 1 0 ~ t o 10~ M p h e n o l i c a c i d s . (]_). Such high c o n c e n t r a t i o n may l o c a l l y be a c h i e v e d i f oat straw i s accumulated i n t h e f i e l d s ( 4 ) . T h i s c o u l d sometimes be a r e a s o n f o r poor p l a n t growth on f i e l d s a f t e r h a r v e s t o f o a t s w i t h o u t removal o f s t r a w . We d i d not f i n d such c o n c e n t r a t i o n s i n our husk e x t r a c t s , however; they were 100 - 1000 f o l d s m a l l e r than expected from b i o a c t i v i t y . P h e n o l i c a c i d s a r e t h e r e f o r e not t h e a c t i v e compounds i n our i n h i b i t o r y e x t r a c t s . C o n s e q u e n t l y , we undertook t o remove t h e p h e n o l i c compounds and r e t a i n t h e b i o a c t i v i t y as shown i n F i g u r e 2. The a c i d f r a c t i o n was a p p l i e d t o a Sephadex LH-20 column and e l u t e d w i t h w a t e r . The b i o a c t i v i t y was e l u t e d v e r y e a r l y ( F r a c t i o n A a n d A ) whereas most p h e n o l i c compounds were e l u t e d much l a t e r . The compounds o f f r a c tions k (and A , not shown here) were a g a i n i n v e s t i g a t e d by gas chromatography/mass s p e c t r o m e t r y . T h e i r s t r u c t u r e s ( § - 13) a r e g i v e n h e r e . The compounds a r e a l i p h a t i c c a r b o x y l i c a c i d s o f t h e t r i c a r b o x y l i c a c i d c y c l e and s t r u c t u r a l l y r e l a t e d compounds. T h e i r c o n c e n t r a t i o n i n t h e o r i g i n a l e x t r a c t i s at l e a s t one o r d e r o f magnitude s m a l l e r than t h a t o f t h e p h e n o l i c a c i d s . The a l i p h a t i c a c i d s § - 12 do not i n h i b i t g e r m i n a t i o n i η c o n c e n t r a t i o n s up t o 1 0 ~ - 1 0 ~ M . The t r u e i n h i b i t o r ( s ) must t h e r e f o r e be found amongst t h e many minor compounds o f f r a c t i o n A . In another c o n n e c t i o n , we 3

2

x

1

2

2

3

2

x


120

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

1

—ι

1

( Concentration )

1 2

_1

F i g u r e 1. I n h i b i t i o n o f r o o t growth o f Avena s a t i v a by t h e aqueous e x t r a c t from o a t husks a f t e r f r a c t i o n a t i o n . The aqueous e x t r a c t was e x t r a c t e d w i t h e t h e r f i r t a t pH 7 ( 0 - - - 0 ) and then a t pH 1 ( 0 - 0 ) ; ( · - · ) t h e r e m a i n i n g water phase. Lower p a r t : doubly r e c i p r o c a l p l o t . (Reproduced w i t h p e r m i s s i o n from r e f e r e n c e 1. C o p y r i g h t 1982 V e r l a g d e r Z e i t s c h r i f t f u r N a t u r f o r s c h u n g . )

F i g u r e 2 . Column chromatography o f t h e f r a c t i o n e t h e r pH 1 ( s e e F i g u r e 1) on Sephadex L H - 2 0 . F r a c t i o n s w i t h b i o a c t i v i t y (A, and A « ) a r e hatched. (Reproduced w i t h p e r m i s s i o n from r e f e r e n c e 6 . C o p y r i g h t 1982 V e r l a g d e r Z e i t s c h r i f t f u r N a t u r f o r s c h u n g . )


11.

Germination

RUDIGER AND LOHAUS

^COOR

H C 0

COOR

*C00R

H C

COOR

and Growth

0=C

H ^

COOR

HÇ

COOR

0

HC

H C

2

2

2

121

Inhibitors

H C

COOR

2

H C

COOR

2

7

H

3 v

^ /

C

C

H

8 HC 2

H-CO ο

C

COOR

CH

HO

9

COOR

HO

C

I

2

HC 2

2

I

C

I

HC 2

12

HC 0

2

COOR COOR

H0-

I

Q

3

HC

COOR

2

11

COOR

— C

COOR

H C

COOR

2

CH

I

COOR

io

H HO

COOR

I

C

ι HC

COOR

9

3

13

s t u d i e d minor compounds from t h e a c i d f r a c t i o n o f s e v e r a l cereals (5). We were a b l e t o i d e n t i f y 83 such compounds, but many remained u n i d e n t i f i e d . T h i s a n a l y s i s d i d not h e l p i n t h e i d e n t i f i c a t i o n o f t h e i n h i b i t o r . T o t a l a n a l y s i s i s not t h e b e s t way t o f i n d b i o a c t i v e com pounds. The i n h i b i t o r was i d e n t i f i e d by a c o m b i n a t i o n o f f u r t h e r s e p a r a t i o n by column chromatography and q u a n t i t a t i v e b i o t e s t (6). F i g u r e 3 shows t h e r e s u l t o f a n a l y s i s o f f r a c t i o n s 62-75 from t h e column chromatography by gas chromatography. The χ a x i s i s t h e r e t e n t i o n t i m e , t h e y a x i s i s t h e f r a c t i o n number o f column c h r o m a t o g r a p h y . The v e r t i c a l b a r s i n d i c a t e t h e o c c u r e n c e o f peaks w i t h a g i v e n r e t e n t i o n t i m e i n t h e s i n g l e f r a c t i o n s o f column chromatography. The i n h i b i t o r y a c t i v i t y was found o n l y i n f r a c t i o n s 6 8 - 7 2 . The o n l y peak t h a t o c c u r s e x c l u s i v e l y i n t h e s e f r a c t i o n s i s t h e one w i t h r e t e n t i o n t i m e 2 9 . 5 min ( i n d i c a t e d as b l a c k b a r ) . In F i g u r e 4 t h e r e l a t i o n s h i p between peak s i z e ( c r o s s e s ) and magnitude o f i n h i b i t o r y a c t i v i t y (open c i r c l e s ) i n t h e f r a c t i o n s 68 t o 72 i s g i v e n . The good c o r r e l a t i o n proved t h e i d e n t i t y o f t h e compound c a u s i n g t h i s peak w i t h t h e i n h i b i t o r even b e f o r e t h e i s o l a t i o n o f t h e compound. The approach i s d e s c r i b e d here i n some d e t a i l because i t may be r e l e v a n t f o r t h e s o l u t i o n o f s i m i l a r p r o b l e m s . S u b s e q u e n t l y , t h e compound was i s o l a t e d and i n v e s t i g a t e d by mass s p e c t r o m e t r y . The s t r u c t u r e o f t h e compound was d e r i v e d from t h e f r a g m e n t a t i o n p a t t e r n o f t h e p r o t o nated and t h e d e u t e r a t e d e s t e r ( 6 ) : i t i s p e n t a n e - 1 , 3 , 4 - t r i c a r b o x y l i c a c i d , o r d i h y d r o h e m a t i n i c a c i d ( T 4 h which had not been known b e f o r e as a n a t u r a l compound. I t had been o b t a i n e d by c h e m i c a l d e g r a d a t i o n of c h l o r o p h y l l s (_7, 8) and by t o t a l s y n t h e s i s ( 9 ) . T o t a l s y n t h e s i s


122


Gas Chromatography: RT [min] F i g u r e 3. A n a l y s i s by gas chromatography ( G O o f f r a c t i o n s 62-75 o f t h e l i q u i d chromatography. The o n l y GC peak which o c c u r s i n a l l i n h i b i t o r y f r a c t i o n s i s t h a t w i t h r e t e n t i o n time 2 9 . 5 m i n . (Reproduced w i t h p e r m i s s i o n from r e f e r e n c e 6. C o p y r i g h t 1982 V e r l a g der Z e i t s c h r i f t f u r N a t u r f o r s c h u n g . )

·

68

1

69

1

1

F 70r a c t i o71n

»

72

V -

73

F i g u r e 4. C o r r e l a t i o n o f seed g e r m i n a t i o n i n h i b i t i o n (x-x) w i t h GC peak s i z e o f compound RT = 2 9 . 5 min (o-o) o f f r a c t i o n s 68-73 of t h e l i q u i d chromatography (see F i g u r e 3 ) . (Reproduced w i t h p e r m i s s i o n from r e f e r e n c e 6. C o p y r i g h t 1982 V e r l a g der Z e i t s c h r i f t fur Naturforschung.)


Germination

11. RUDIGER AND LOHAUS

and Growth

Inhibitors

123

y i e l d s two p a i r s o f d i a s t e r e o m e r s (14§> 1 4 ^ · n a t u r a l compound from o a t s i s t h e t h r e o i s o m e r . We have not y e t e l u c i d a t e d t h e a b s o l u t e c o n f i g u r a t i o n o f t h e n a t u r a l compound. The s y n t h e t i c racemate has a somewhat s m a l l e r a c t i v i t y than t h e n a t u r a l compound. D u r i n g our s y n t h e t i c a p p r o a c h , we found t h a t s e v e r a l t r i c a r b o x y l i c a c i d s have b i o a c t i v i t y s i m i l a r t o t h a t o f d i h y d r o h e m a t i n i c a c i d . Some o f t h e s t r u c t u r e s ( ] J - 1Z) and t h e c o r r e s p o n d i n g a c t i v i t i e s ( T a b l e I) a r e g i v e n h e r e . E s s e n t i a l i s t h e p r e s e n c e o f t h r e e c a r b o x y l i c a c i d groups near each o t h e r and a c e r t a i n c h a i n l e n g t h o f t h e a l i p h a t i c r e s i d u e . Recent i n v e s t i g a t i o n s showed some i n h i b i t i o n o f r e s p i r a t i o n by t h e s e t r i c a r b o x y l i c a c i d s . G e r m i n a t i o n i s p o s s i b l y i n h i b i t e d v i a t h i s i n h i b i t i o n . A l t h o u g h t h e s e s y n t h e t i c compounds are not n a t u r a l a l l e l o c h e m i c a l s they c o u l d be used f o r t h e same p u r p o s e , namely f o r i n h i b i t i o n o f seed g e r m i n a t i o n . T

n

e

CH C00R 2

14 R

14§

146

1

1 1 1

1§:

R

1

=

(CH ) CH

3

1§:

R

1

=

(CH ) CH

3

1Z:

R

1

=

(CH )

2

3

CHCOOR 2

7

CHCOOR 2

1 5

CH

3

COOR

Table

I.

I n h i b i t i o n o f G e r m i n a t i o n o f Amaranthus caudatus Seeds by S y n t h e t i c T r i c a r b o x y l i c A c i d T r i m e t h y l E s t e r s

~ ~

% I n h i b i t i o n of Germination

Compound

1θ" Μ

ΊΟ M

14 a , b

100

54

15

100

20

16

100

50

17

5

0

4


124


The second example d e s c r i b e d here i s dormant seeds from Rosa c a n i n a . E x t r a c t s o f t h e s e seeds a l s o i n h i b i t g e r m i n a t i o n o f seeds o f s e v e r a l p l a n t s ( 1 0 ) . In F i g u r e 5 a scheme i s g i v e n f o r e x t r a c t i o n and s e p a r a t i o n oT~~three d i f f e r e n t i n h i b i t o r compounds. A l l t h e s e a r e p r e s e n t i n t h e a c i d f r a c t i o n . The f i r s t e s s e n t i a l s t e p i s chromatography on Sephadex LH-20, which s e p a r a t e s i n h i b i t o r I from i n h i b i t o r I I and I I I . I n h i b i t o r I was i d e n t i f i e d as a b s c i s i c a c i d . The o t h e r two i n h i b i t o r s were s e p a r a t e d by m e t h y l a t i o n w i t h diazomethane, f r a c t i o n a l d i s t i l l a t i o n , and column chromatography. The second i n h i b i t o r i s t h e α-pyrone 1§. R e a c t i o n w i t h diazomethane t r a n s f o r m s i t i n t o t h e b i c y c l i c compound 1§. T h i s b i c y c l i c compound i s even more a c t i v e than t h e p a r e n t α-pyrone 1§. S i n c e we sought s t r u c t u r a l r e q u i r e m e n t s f o r b i o a c t i v i t y here a l s o , w e t e s t e d s e v e r a l s y n t h e t i c α-pyrones (£0 - 2g) f o r b i o a c t i v i t y . These compounds had no i n h i b i t o r y a c t i v i t y . Be aTso t e s t e d t h e c y c l o p r o p a n e d e r i v a t i v e s £3 and 2 4 . In T a b l e I I , t h e b i o a c t i v i t y o f t h e b i c y c l i c compound T§ and two such d e r i v a t i v e s i s compared. The presence o f s e v e r a l c a r B o x y l i c a c i d groups seems t o be e s s e n t i a l ( o r a t l e a s t h e l p f u l ) f o r b i o a c t i v i t y i n t h i s case a l s o . COOR

COOR

11


11.

Germination

RUDIGER AND LOHAUS

and Growth

Inhibitors

125

Extraction with H 0 at 100'C 9

I crude extract Ether extract pH 7 Ether extract pH 1 Chromatography on Sephadex LH-20

y

\

RC-B

RC-A Inhibitor

methylation with diazomethane fractionation by distillation

/

RC-B 50 Lichroprep

1

Inhibitor III

\

RC-B 150 Lichroprep

I

Inhibitor II

F i g u r e 5. Scheme o f i s o l a t i o n o f g e r m i n a t i o n o f Rosa c a n i n a .

i n h i b i t o r s from


hips

126 Table

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY II.

I n h i b i t i o n o f G e r m i n a t i o n o f Amaranthus Seeds by C a r b o x y l i c A c i d Methyl E s t e r s

caudatus

% I n h i b i t i o n of Germination Compound

10~ M J

10

M

10 M

19

100

100

30

U

100

90

10

|4

90

40

10

Il

100

90

50

CH

3

2| (Product

o f r i n g opening o f 19)

u (unfavored

conformation

o f 14b)


11.

RUDIGER AND LOHAUS

Germination

and Growth

127

Inhibitors

The b i o a c t i v i t y i s s i m i l a r t o t h a t o f i s o h e m a t i n i c a c i d ( 2 § ) ( s e e T a b l e I I ) . We t h e r e f o r e r a i s e d t h e q u e s t i o n whether t h e r e c o u l d be some c l o s e r r e l a t i o n s h i p between t h e i n v e s t i g a t e d compounds from r o s e s and o a t s . Such r e l a t i o n s h i p i s i n d i c a t e d above. Opening o f t h e l a c t o n e r i n g o f 9 would l e a d t o t h e t r i c a r b o x y l i c a c i d derivative 2|. T h i s has a s t r u c t u r e l i k e a c e r t a i n c o n f o r m a t i o n o f d i h y d r o Rematinic a c i d [27). T h i s c o n f o r m a t i o n i s c e r t a i n l y d i s f a v o r e d b e cause o f s t e r i c h i n d r a n c e , but c o u l d e x i s t at a b i n d i n g s i t e o f an enzyme o r a r e c e p t o r . The p r e s e n c e o f a c y c l o p r o p a n e r i n g as i n 2§ c o u l d s t a b i l i z e such an o t h e r w i s e u n f a v o r a b l e c o n f o r m a t i o n t h a t Ts e v e n t u a l l y needed f o r high b i o a c t i v i t y . The l a s t i n h i b i t o r from r o s e seeds was i d e n t i f i e d as 2 , 5 - d i h y d r o f u r a n - 2 - c a r b o x y l i c a c i d ( 2 § ) . The s p e c i f i c a c t i v i t y o f t h i s compound i s not v e r y h i g h , but r o s e seeds c o n t a i n enough o f i t t o e x e r t measurable i n h i b i t i o n o f seed g e r m i n a t i o n . The b i o a c t i v i t y seems t o depend on t h i s p a r t i c u l a r s t r u c t u r e because a number o f s i m i l a r compounds (29 - 3Z) have no a c t i v i t y o r much lower a c t i v i t y than 2§.

28

32

29:

R

30:

R

31:

R

H

00H

33

R = H

34

R = Br

3Z


128

A L L E L O C H E M I C A L S : R O L E IN A G R I C U L T U R E A N D FORESTRY

The i d e n t i f i c a t i o n o f n a t u r a l g e r m i n a t i o n i n h i b i t o r s e n a b l e s us t o study t h e i r e f f e c t as a l l e l o c h e m i c a l s i n d e t a i l . Because most o f t h e s e compounds a r e n a t u r a l p r o d u c t s not p r e v i o u s l y known, they have t o be s y n t h e s i z e d at f i r s t i n o r d e r t o o b t a i n s u f f i c i e n t m a t e r i a l f o r such i n v e s t i g a t i o n s . T h i s work i s i n p r o g r e s s .

Acknowledgments The work was s u p p o r t e d by t h e BMFT, Bonn ( A z . : PTB 038519). We thank t h e BASF AG, Ludwigshafen, f o r s p e c i a l s u p p o r t and a of r e f e r e n c e compounds.

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Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Karl, R.; Rüdiger, W. Z. Naturforsch. 1982, 37c, 793. Putnam, A.R. and Weston, A.R. Abstracts of Papers, 190th National Meeting of the American Chemical Society, Chicago, IL, American Chemical Society: Washington DC, 1985; AGFD 201. Rüdiger, W.; Blos, I. unpublished results. Guenzi, W.D.; McCalla, T.M. Soil Sci. Soc. Am. Proc. 1966, 30, 214. Lohaus, E.; Blos, I.; Rüdiger, W. Z. Naturforsch. 1983, 38c, 524. Lohaus, E.; Blos, I.; Schäfer, W.; Rüdiger, W. Z. Naturforsch. 1982, 37c, 802. Ficken, G.E.; Jones, R.B.; Linstead, R.P. J. Chem. Soc. (London) 1956, 2272. Brockmann, H. Angew. Chemie 1968, 80, 233. Gray, C.H.; Nicholson, P.C. J. Chem. Soc. (London) 1958, 3085. Lohaus, E.; Zenger, C.; Rüdiger, W. Z. Naturforsch. 1985, 40c, 490.

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April 16,1986


Germination and Growth Inhibitors as Allelochemicals - American

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