Germination Regulation by Amaranthus palmeri and Ambrosia

19

G e r m i n a t i o n Regulation by A m a r a n t h u s palmeri and Ambrosia

artemisiifolia

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on January 1, 2016 | http://pubs.acs.org Publication Date: December 17, 1985 | doi: 10.1021/bk-1985-0268.ch019

JUDITH M. BRADOW Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, LA 70179 An apparent allelopathic interference having been observed in which residues of Palmer amaranth, Amaranthus palmeri S. Wats., inhibited the growth of onion, carrot and Palmer amaranth itself, aqueous and organic solvent extracts of amaranth plant parts were made and used to investigate the growth regulatory activity of the chemical constituents of this amaranth species. The resultant crude extracts, as well as isolated and identified chemical compounds, were assayed in vitro as germination regulators, by using both crop and weed seeds. Some crude organic solvent extracts proved inhibitory, but inhibitions of onion, Palmer amaranth, and carrot germination did not result from the same extracts or compounds. Four compounds have been isolated from Palmer amaranth: 2,6-dimethoxybenzoquinone, vanillin, phytol, and chondrillasterol. All showed biological activity, to varying degrees, in seed germination bioassays. Parallel studies of extracts and mixtures of compounds isolated from ragweed, Ambrosia artemisiifolia L., were also made. Water extracts of ragweed proved highly inhibitory of seed germination. Mixed sesquiterpenes isolated from ragweed were very inhibitory of onion, oat, ryegrass, and Palmer amaranth germination when applied in a dichloromethane solution pretreatment. Amaranthus Palmeri S. Wats., Palmer amaranth, is a coarse, weedy, drought-resistant, dioecious member of the Amaranthaceae and is related to A. retroflexus L., redroot pigweed, and the ornamentals _A. tricolor L., summer poinsettia, and /\. caudatus L., love-liesbleeding. Palmer amaranth was used by natives of the North American desert as a protein source and cereal to supplement maize and beans. Another member of the same family, k. spinosus L., spiny amaranth, has been reported to exhibit allelopathic activity toward coffee This chapter not subject to U.S. copyright. Published 1985, American Chemical Society

In The Chemistry of Allelopathy; Thompson, Alonzo C.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.


286

THE CHEMISTRY OF ALLELOPATHY

(_1); and D r . R. M. Menges, USDA, W e s l a c o , TX, has observed t h a t Palmer amaranth r e s i d u e s plowed i n t o f i e l d s l a t e r i n h i b i t the growth of o n i o n , c a r r o t , and Palmer amaranth i t s e l f (see Menges, t h i s p u b l i cation). The r e p o r t s from Texas prompted t h e f o r m a t i o n o f an i n t e r d i s c i p l i n a r y (and m u l t i - s i t e ) group t o i n v e s t i g a t e t h e apparent a l l e l o p a t h i c a c t i v i t y o f Palmer amaranth. D r . Ν. H. F i s c h e r , and h i s group at L o u i s i a n a S t a t e U n i v e r s i t y , Baton Rouge, LA, were a l r e a d y i n v e s t i g a t i n g t h e secondary m e t a b o l i t e s o f Ambrosia a r t e m i s i i f o l i a L . , common ragweed. A l t h o u g h t h e group i t s e l f had no f i e l d o b s e r v a t i o n s t o i n d i c a t e a l l e l o p a t h i c i n t e r f e r e n c e by A. a r t e m i s i i f o l i a , a t r o p i c a l ragweed s p e c i e s , A. cumanensis HBK, has been shown t o have a l l e l o p a t h i c p o t e n t i a l ( 2 j T and western ragweed, A. p s i l o s t a c h y a DC, has been found t o be i n h i b i t o r y i n l a b o r a t o r y seed g e r m i n a t i o n t e s t s (3) and appears t o have a r o l e i n o l d - f i e l d s succession ( 4 J . Common ragweed i t s e l f is r e p o r t e d t o e x e r t a l l e l o p a t h i c e f f e c t s i n t h e f i r s t s t e p s of secondary s u c c e s s i o n (j>). F i e l d o b s e r v a t i o n s i n d i c a t e d t h a t t h e i n h i b i t i o n s r e s u l t from Palmer amaranth r e s i d u e s , r a t h e r than t h e growing p l a n t i t s e l f , and i t was f e l t t h a t c o m p e t i t i v e i n t e r f e r e n c e a t the h i g h e r p l a n t l e v e l c o u l d be i g n o r e d i n f a v o r o f t h e study o f t h e apparent chemical interactions. Both " p h y t o i n h i b i t i n s " and " s a p r o i n h i b i t i n s " (j>) were t o be c o n s i d e r e d , b e g i n n i n g w i t h c h e m i c a l s o f p l a n t o r i g i n ( p h y t o i n hibitins). T h i s p a p e r , a s e c t i o n of a t h r e e - p a r t p r e s e n t a t i o n , d e s c r i b e s the i n v i t r o seed g e r m i n a t i o n a s s a y s performed w i t h crude and pure s u b s t a n c e s i s o l a t e d from the v a r i o u s p a r t s o f ragweed and Palmer amaranth. Materials

and Methods

The Palmer amaranth p l a n t s used as raw m a t e r i a l f o r crude e x t r a c t p r e p a r a t i o n and compound i s o l a t i o n were s u p p l i e d by D r . Menges or grown at Southern R e g i o n a l Research C e n t e r (SRRC) and h a r v e s t e d a f t e r seed d i s p e r s a l ( w a t e r - e x t r a c t e d samples o n l y ) . Ragweed p l a n t s were c o l l e c t e d i n Baton Rouge i n June o r i n Kenner, LA, j u s t b e f o r e flower anthesis. The d e t a i l s of o r g a n i c s o l v e n t e x t r a c t p r e p a r a t i o n and compound i s o l a t i o n and i d e n t i f i c a t i o n a r e d i s c u s s e d elsewhere (see F i s c h e r e t a l _ . , t h i s p u b l i c a t i o n ) . Water e x t r a c t s o f a i r - d r i e d l e a v e s o f ragweed ( 1 : 1 0 , w/w) and Palmer amaranth ( 1 : 1 6 , w/w) were made by b l e n d i n g t h e l e a v e s (and amaranth t h y r s e s ) i n d e i o n i z e d w a t e r , pH 6 . 8 , and a g i t a t i n g the s l u r r y at room t e m p e r a t u r e f o r 3 h b e f o r e f i l t r a t i o n through 4 l a y e r s of c h e e s e c l o t h . The f i l t r a t e was c e n t r i f u g e d 20 min a t 13,000 x g ( a v g . ) and the p e l l e t d i s c a r d e d . These crude water e x t r a c t s were used i n the seed g e r m i n a t i o n b i o a s s a y d e s c r i b e d below. The crude o r g a n i c s o l v e n t p r e p a r a t i o n s were produced by s e q u e n t i a l e x t r a c t i o n s o f the p l a n t m a t e r i a l w i t h p e t r o l e u m e t h e r ( P E ) , f o l l o w e d by d i c h l o r o m e t h a n e (DCM), and f i n a l l y methanol (MeOH). Some Palmer amaranth samples p r e p a r e d at SRRC were f i r s t e x t r a c t e d w i t h hexane (HX), r a t h e r than P E . The o r g a n i c s o l v e n t e x t r a c t s were s u p p l i e d f o r b i o a s s a y i n the form o f o i l s o r s o l i d s r e m a i n i n g a f t e r s o l v e n t removal i n v a c u o . A f t e r each e x t r a c t i o n s t e p a l i q u o t s o f the crude e x t r a c t s from the r o o t s , stems, or l e a v e s (and t h y r s e s ) were e v a l u a t e d f o r seed g e r m i n a t i o n r e g u l a t o r y a c t i v i t y . The g e r m i n a t i o n b i o a s s a y c o n s i s t e d o f g e r m i n a t i n g t h e seeds o f a number o f crop and weed s p e c i e s ( T a b l e I) f o r 72 h i n the dark at


19.

BRADOW

Germination Regulation by A . palmeri and A . artemisiifolia

287

T a b l e I . Seed S p e c i e s Used t o Determine In V i t r o G e r m i n a t i o n R e g u l a t o r y A c t i v i t y o f Palmer Amaranth and Ragweed E x t r a c t s


Seed s p e c i e s

No. seed/ replicate

A l l i u m cepa L . , 20 c v . Texas E a r l y Grano 25 Amaranthus p a l m e r i S . Wats. 25 Amaranthus r e t r o f l e x u s L . 15 Avena s a t i v a L. 20 Bromus s e c a l i n u s L . Cucumis s a t i v u s L . , 10 c v . Marketmore Daucus c a r o t a L . , 20 Danvers H a l f - l o n g Echinochloa c r u s - g a l l i ( L . ) Beauvois 20 Eraqrostis curvula ( S c h r a d e r ) Nees 20 Lactuca s a t i v a L. c v . Grand Rapids 20 20 c v . G r e a t Lakes Lepidium sativum L . , 20 C u r l y Cress Loi iurn s p p . 20 L y c o p e r s i c o n e s c u l e n t u m M i l l . ,» 20 c v . Homestead 20 Portulaca oleracea L. 15 Sorghum b i c o l o r ( L . ) Moench T r i f o l i urn i n c a r n a t u m L. 20 T r i t i c u m a e s t i v u m L. 15 *Not

used i n s t u d i e s o f Palmer amaranth

Class

Common name

Monocot Dicot Dicot Monocot Monocot

Onion Palmer Amaranth Redroot Pigweed Oat Cheatgrass*

Di c o t

Cucumber

Dicot

Carrot

Monocot

Barnyard

Monocot

Lovegrass*

Dicot

Lettuce

Dicot Monocot

Garden C r e s s Ryegrass

Dicot Dicot Monocot Dicot Monocot

Tomato Purslane* Sorghum Red C l o v e r Wheat

Grass*

extracts.

25 C i n 10 cm p l a s t i c p e t r i d i s h e s c o n t a i n i n g one 9 cm sheet o f Whatman #1 f i l t e r paper which had been s a t u r a t e d w i t h 3 ml o f a t e s t or c o n t r o l s o l u t i o n . The number o f seeds p e r r e p l i c a t e was chosen t o m i n i m i z e c o n t a c t between s e e d l i n g s a t 72 h and t o a v o i d w a t e r s t r e s s d u r i n g g e r m i n a t i o n i n the l a r g e r s e e d s . There were 8 r e p l i c a t e s o f each t r e a t m e n t and o f p a r a l l e l c o n t r o l s c o n s i s t i n g o f e i t h e r d e i o n i z e d w a t e r , pH 6 . 8 , o rO.1%( v / v ) d i m e t h y l s u l f o x i d e (DMS0) i n d e i o n i z e d w a t e r . The DMS0 was n e c e s s a r y t o a i d water s o l v a t i o n o f the e x t r a c t s made w i t h the l e s s p o l a r o r g a n i c s o l v e n t s . In a few i n s t a n c e s where DMS0 was i n e f f e c t i v e and t h e t e s t m a t e r i a l was e i t h e r a p u r e , i d e n t i f i e d compound o r more f u l l y c h a r a c t e r i z e d than a crude e x t r a c t , seeds were p r e t r e a t e d w i t h DCM s o l u t i o n s o f t h e s u b s t a n c e ( O . 1 mM where p o s s i b l e ) and germinated as above w i t h p a r a l l e l c o n t r o l s p r e t r e a t e d w i t h DCM a l o n e . This pretreatment t e c h n i q u e was u t i l i z e d i n a t i m e - c o u r s e g e r m i n a t i o n study o f t h e effects of chondrillasterol (5a-stigmasta-7,22-dien-3&-ol) isolated from Palmer amaranth, u s i n g t h e f o l l o w i n g f i v e amaranth s p e c i e s : Amaranthus caudatus L . , c v . L o v e - l i e s - b l e e d i n g ; A . p a l m e r i ; A. r e t r o f l e x u s ; A . t r i c o l o r L . , c v . Tampala; and A^ s a l i c i f o l i us L . ,


T H E C H E M I S T R Y OF A L L E L O P A T H Y


288

c v . Flaming F o u n t a i n . The seeds were soaked i n 3 ml O.1 mM c h o n d r i l lasterol i n DCM f o r 1.5 h; t h e s o l v e n t was a l l o w e d t o e v a p o r a t e , and the seeds were a i r - d r i e d f o r an a d d i t i o n a l 24 h b e f o r e u s e . The seeds were germinated under t h e c o n d i t i o n s d e s c r i b e d above, and g e r m i n a t i o n counts were made every 24 h f o r 7 d a y s . A l l seed manipul a t i o n s were c a r r i e d out under a green s a f e l i g h t . The c h o n d r i l l a s t e r o l was a l s o assayed f o r a c t i v i t y i n mung bean h y p o c o t y l and r a d i c l e e l o n g a t i o n t e s t s where mung b e a n s , V i g n a r a d i â t a L. W i l c z e k , c v . Golden Gram, were soaked 3 h i n 5 ml O.1 mM c h o n d r i l l a s t e r o l i n DCM. C o n t r o l s t r e a t e d w i t h DCM a l o n e were included. The s o l v e n t was a l l o w e d to e v a p o r a t e , and t h e seeds were p l a n t e d , 20 seeds per 10 cm p l a s t i c p e t r i d i s h , on a s i n g l e Whatman #1 f i l t e r paper d i s k s a t u r a t e d with 3 ml d e i o n i z e d w a t e r , pH 6 . 8 . U n t r e a t e d seeds were s i m i l a r l y p l a n t e d , u s i n g d e i o n i z e d water ( c o n t r o l ) , O.4% dimethylformamide (DMF c o n t r o l ) or O.05 mM c h o n d r i l l a s t e r o l i n O.4% DMF (4 r e p l i c a t e s e a c h ) . Incubation c o n d i t i o n s were t h e same as above and g e r m i n a t i o n and growth e v a l u a t i o n s were made a t 2 4 , 4 8 , 72, and 96 h. Some crude e x t r a c t s from ragweed l e a v e s and r o o t s were t e s t e d b e f o r e t h e m u l t i - s e e d s p e c i e s a s s a y s were r e g u l a r l y i n use at SRRC. These e x t r a c t s , P E , DCM, MeOH, and H 0 were t e s t e d , u s i n g two l e t t u c e seed c u l t i v a r s , Grand Rapids (GR) and Great Lakes ( G L ) , germinated at 28 C , a t h e r m a l - s t r e s s t e m p e r a t u r e , and r a d i s h (Raphanus s a t i v u s L . , c v . E a r l y S c a r l e t G l o b e ) germinated 64 h at 25 C, p l u s c a r r o t germinated 96 h a t 25 C . S i n c e t h e g e r m i n a t i o n d a t a o b t a i n e d f o r the c o n t r o l s , r e g a r d l e s s of s o l v e n t o r p r e t r e a t m e n t , f i t normal d i s t r i b u t i o n s w i t h d i f f e r e n t means and v a r i a n c e s a c c o r d i n g t o seed s p e c i e s , s t a t i s t i c a l a n a l y s e s were made u s i n g t h e s t a n d a r d normal d e v i a t e s f o r each species. To a l l o w comparisons between s p e c i e s the g e r m i n a t i o n data are shown as p e r c e n t of the g e r m i n a t i o n observed i n t h e a p p r o p r i a t e control. 2

R e s u l t s and

Discussion

In v i t r o g e r m i n a t i o n assays such as t h o s e used t o o b t a i n t h e r e s u l t s r e p o r t e d here are not i n t e n d e d as c l o s e a p p r o x i m a t i o n s of the f i e l d c o n d i t i o n s where t h e apparent a l l e l o p a t h i c i n t e r a c t i o n s o c c u r . The small q u a n t i t i e s of s u s p e c t e d a l l e l o c h e m i c a l s which a r e a v a i l a b l e f o r use i n b i o a s s a y s and t h e c o m p l e x i t y of the f i e l d s i t u a t i o n s d i c t a t e t h a t some s o r t of s c r e e n i n g assay be developed which is s i m p l e and r a p i d t o p e r f o r m , e a s i l y c o n t r o l l e d w i t h r e s p e c t t o e x p e r i m e n t a l c o n d i t i o n s , and r e p r o d u c i b l e . S i n c e we a n t i c i p a t e d t e s t i n g c r u d e e x t r a c t s , i s o l a t e d compounds, and m i x t u r e s of r e l a t e d c h e m i c a l s we chose a m u l t i - s p e c i e s assay where t o t a l g e r m i n a t i o n and pre-emergent s e e d l i n g development were t h e e x p e r i m e n t a l p a r a m e t e r s . The seeds v a r i e d c o n s i d e r a b l y i n s i z e and g e r m i n a t i o n r e q u i r e m e n t s . The mean c o n t r o l g e r m i n a t i o n v a l u e s f o r each s p e c i e s are a r e s u l t of more than 30 experiments c o n t a i n i n g 8 r e p l i c a t e s each and c o n t r o l germinat i o n data f o r each s p e c i e s f i t a normal d i s t r i b u t i o n . Using S t u d e n t ' s p a i r e d t - t e s t , the presence o f O.1% DMSO was found t o have no s i g n i f i c a n t e f f e c t (P >= O.05), except i n the case of wheat where DMSO d e c r e a s e d g e r m i n a t i o n 5.8%. C o n c e n t r a t i o n was a n o t h e r c o n f o u n d i n g f a c t o r . When u s i n g crude e x t r a c t s d e t e r m i n a t i o n o f t h e c o n c e n t r a t i o n s of i n d i v i d u a l components



19.

BRADOW

Germination Regulation by A. palmed and A. artemisiifolia

289

is i m p o s s i b l e . In d e a l i n g w i t h crude e x t r a c t s t h e most u s e f u l measure o f a p p l i c a t i o n r a t e proved t o be weight p e r c e n t (wt.%). The crude e x t r a c t c o n c e n t r a t i o n s used ranged from O.015 t oO.4wt.%. These l e v e l s a r e w i t h i n the c o n c e n t r a t i o n l i m i t s used i n e x o g e n o u s l y - a p p l i e d growth r e g u l a t o r s t u d i e s where m i x t u r e s o f g i b b e r e l l i c a c i d s ( O . 1 mM = O.035 wt.%) and a b s c i s i c a c i d isomers (O.05 mM = O.0003 wt.%) have been found e f f e c t i v e (_7). When t e s t e d w i t h s p e c i e s used i n o u r b i o a s s a y s , t h e a l l e l o c h e m i c a l , c o u m a r i n , i n h i b i t e d c a r r o t a t O.0005 wt.%, onion a t O.0019 wt.%, l e t t u c e a t O.0033 wt.%, and wheat a t O.0219-O.438 wt.% ( 8 ) . The wt.% c o n c e n t r a t i o n s o f t h e crude e x t r a c t s t e s t e d a r e i n d i c a t e d i n t h e f o l l o w i n g t a b l e s showing t h e e f f e c t s o f Palmer amaranth and ragweed on seed germination. Palmer Amaranth E x t r a c t s . The aqueous e x t r a c t s made from s e n e s c e n t Palmer amaranth l e a v e s and t h y r s e s a f t e r seed development and d i s p e r sal had l i t t l e e f f e c t on any o f t h e seeds t e s t e d ( T a b l e I I ) . The most c o n c e n t r a t e d ( 1 : 1 6 , w/w) e x t r a c t i n h i b i t e d o n l y l e t t u c e , t o m a t o , and r y e g r a s s . The o s m o l a r i t y o f t h e s o l u t i o n was 40.3 mOsm, i n s u f f i c i e n t t o a f f e c t g e r m i n a t i o n of most seeds (_9). The responses o f t h e t e s t seed t o i n c r e a s e d e x t e r n a l o s m o l a r i t y were determined by g e r m i n a t i n g t h e seeds i n a p o l y e t h y l e n e g l y c o l (PEG) s o l u t i o n o f 4 0 . 3 mOsm ( 1 0 ) . The o n l y t e s t seeds s i g n i f i c a n t l y a f f e c t e d by t h i s t r e a t m e n t were t h o s e o f tomato where P E G - t r e a t e d seed g e r m i n a t i o n was 69.3% o f t h a t o b s e r v e d i n d e i o n i z e d w a t e r . D i l u t i o n o f t h e aqueous e x t r a c t t o 1:32 (w/w) removed a l l i n h i b i t o r y a c t i o n , but t h e d i l u t e d s o l u t i o n i n c r e a s e d wheat g e r m i n a t i o n . Crude o r g a n i c s o l v e n t e x t r a c t s from t h e Palmer amaranth l e a v e s and t h y r s e s produced a m i x t u r e o f promotions and i n h i b i t i o n s o f g e r m i n a t i o n , depending on seed s p e c i e s and c o n c e n t r a t i o n o f t h e e x t r a c t . The two most n o n p o l a r s o l v e n t s u s e d , PE and HX, e x t r a c t e d m i x t u r e s i n h i b i t o r y o f c a r r o t (HX) and promotive o f sorghum ( P E ) . F u r t h e r e x t r a c t i o n s w i t h o t h e r o r g a n i c s o l v e n t s o f Palmer amaranth a e r i a l p a r t s , e x c l u d i n g stems, produced m i x t u r e s t h a t were a l l promotive o f cucumber g e r m i n a t i o n . The i n h i b i t i o n o f c a r r o t by both a DCM and a MeOH e x t r a c t prompted f u r t h e r , i n c o m p l e t e i n v e s t i g a t i o n s o f these extracts. No e x t r a c t s o f t h e l e a v e s and t h y r s e s a f f e c t e d o n i o n , and o n l y a second DCM e x t r a c t i o n o f l e a f t i s s u e and MeOH e x t r a c t i o n o f the r o o t s produced i n h i b i t o r s o f Palmer amaranth. Inhibitors of onion g e r m i n a t i o n were found i n MeOH e x t r a c t s o f both t h e r o o t s and the sterns. The stem e x t r a c t u s i n g MeOH a f t e r e x t r a c t i o n w i t h HX and DCM c o n t a i n e d an onion g e r m i n a t i o n i n h i b i t o r which was e f f e c t i v e at a very low c o n c e n t r a t i o n (O.025 wt.%). The c a r r o t i n h i b i t o r i n t h e DCM l e a f e x t r a c t f o l l o w i n g i n i t i a l HX e x t r a c t i o n was a l s o very active. Cucumber was promoted by r o o t and stem DCM e x t r a c t s , as was wheat by t h e HX/DCM e x t r a c t o f the stems. These b i o a s s a y s o f crude e x t r a c t s were i n t e n d e d o n l y as s c r e e n i n g t e s t s f o r t h e guidance f o r t h e n a t u r a l p r o d u c t s c h e m i s t s i n t h e i r i s o l a t i o n work, but a few g e n e r a l i t i e s were observed i n t h e results. Although a l l the seeds i n T a b l e I were used i n these a s s a y s , o n l y t h r e e s p e c i e s were promoted: wheat, sorghum, and p r i m a r i l y cucumber which was promoted by DCM e x t r a c t s from most o f the p l a n t p a r t s . F i v e s p e c i e s were i n h i b i t e d by t h e crude Palmer amaranth e x t r a c t s , i n c l u d i n g c a r r o t , o n i o n , and Palmer amaranth i t s e l f , as seen i n t h e f i e l d t e s t s . Ryegrass and sorghum were


290

T H E CHEMISTRY O F A L L E L O P A T H Y Table

Extract (Solvent

II.

E f f e c t s of Palmer Amaranth (Amaranthus E x t r a c t s on Seed G e r m i n a t i o n

Palmeri)

Concen.

Seed

% of Germination i n Control

Aqueous

1:16

PE HX PE/DCM

1:32 (w/w) O.380 wt.% O.015 O.391

HX/DCM PE/DCM/MeOH

O.330 O.016 O.423

HX/DCM/MeOH

O.031

Lettuce Tomato Ryegrass Wheat Sorghum Carrot Sorghum Cucumber Carrot Palmer amaranth Cucumber Cucumber Carrot Cucumber

70.7* 74.5* 86.0 120.5 114.4* 79.3 85.6* 107.4* 84.7 62.8* 114.8* 110.1* 74.7 113.8*

O.385 wt.% O.495

Cucumber Sorghum Ryegrass Palmer amaranth Onion

107.8* 86.7* 82.8* 83.7 93.1

HX/DCM

O.017

HX/DCM/MeOH

O.025

Wheat Cucumber Carrot Onion

117.5* 107.5* 84.2 83.8*

used)


Leaves and T h y r s e s (w/w)

Roots PE/DCM PE/DCM/MeOH

Stems

*P

Germination Regulation by Amaranthus palmeri and Ambrosia

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