Allelopathic Interference in a Wild Mustard (Brassica campestris L

C h a p t e r 24

Allelopathic Interference in a Wild Mustard (Brassica campestris L.) and Broccoli (Brassica oleracea L. var. italica) Intercrop Agroecosystem Juan J. Jiménez-Osornio and Stephen R. Gliessman 1

2

Instituto Nacional de Investigaciones sobre Recursos Bióticos, Retorno de Cerro Tuera No. 21. Col. Oxtopulco Universidad, 04310 Mexico, D. F. Mexico Agroecology Program, Environmental Studies, University of California, Santa Cruz, CA 95064

1

Downloaded by CORNELL UNIV on August 14, 2016 | http://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch024

2

Non-crop plants associated with the crop species offer possibilities for allelopathic weed control. In this study Brassica campestris (wild mustard), which is an important weed in Santa Cruz County, and broccoli, a common crop, were intercropped. The allelopathic potential of both species and the changes in this potent i a l throughout their l i f e cycle were demonstrated with experiments in the laboratory. Effects of different planting densities and sowing time of B. campestris on the crop yield are analyzed. Preliminary steps to separate the physiologically active compound(s) are described. The possibilities for the use of Brassica campestris in agroecosystem design as a non-crop plant that promotes pest control are described. From thé point of view of the evolution of agroecosystems, weeds are a good example of the capacity of organisms to adapt to the continued disturbances that humans produce in their environment. A very important component of the aggressive nature of weeds is allelopathic interference, the f u l l potential of which i s just being realized in the management of agroecosystems (1). Allelopathy refers to biochemical effects, both detrimental and beneficial of one plant (including microorganisms) on the germination, growth, or development of another plant Ç2). Studying the practices of peasants for cultivating land in Tabasco, Mexico, Chacon and Gliessman (_3) suggested the possibility that certain beneficial weeds (non-crop plants) might be able to repress harmful weeds through allelopathic interactions. An example of this could be weedy Brassica campestris. Tarahumara Indians in northern Mexico (Chihuahua) indicate that a plot of fertilized soil which was planted to mustard during the previous year will have a less dense stand of weeds. Such a statement suggests that residues or excretions of the mustard inhibit the germination and growth of other plants (4), suggesting its use as a weed controller. —' .pestris is a common weed in Santa Cruz County, CA. This cam

0097-6156/87/0330-0262$06.00/0 © 1987 American Chemical Society

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

24.

JIMÉNEZ-OSORNIO AND

GLIESSMAN

Allelopathic

Interference

263


S t a t e p r o d u c e s 95% o f the b r o c c o l i grown c o m m e r c i a l l y i n the U n i t e d S t a t e s (_5) and i t has been r e p o r t e d t h a t r e s i d u e s o f b r o c c o l i a r e p h y t o t o x i c and i n h i b i t the e s t a b l i s h m e n t o f o t h e r c r o p s (6^) . Both s p e c i e s , B. c a m p e s t r i s and 13. o l e r a c e a v a r . i t a l i c a , b e l o n g t o t h e B r a s s i c a c e a e , which c o n t a i n as a c h a r a c t e r i s t i c c h e m i c a l compounds the g l u c o s i n o l a t e s 8) . S p e c i e s from t h e B r a s s i c a c e a e f a m i l y have been i n f r e q u e n t l y r e p o r t e d as p l a n t s c o n t a i n i n g p h y t o t o x i n s t h a t can a f f e c t g e r m i n a t i o n , e s t a b l i s h m e n t , and growth o f o t h e r s p e c i e s i n n a t u r a l systems ( 9 ) , b u t f r e q u e n t l y r e p o r t e d i n agroecosystems (10, 16). In o r d e r t o d e s i g n more d i v e r s e agroecosystems w i t h p e s t c o n t r o l mechanisms b u i l t i n , t h e i n c o r p o r a t i o n o f some non-crop p l a n t s such as B. c a m p e s t r i s s h o u l d be c o n s i d e r e d and the a l l e l o p a t h i c p o t e n t i a l o f t h e s p e c i e s i n v o l v e d must be d e t e r m i n e d . The o b j e c t i v e s o f t h e study were: (a) t o d e t e r m i n e t h e p o s s i b l e a l l e l o p a t h i c p o t e n t i a l o f 13. c a m p e s t r i s and 13. o l e r a c e a v a r . i t a l i c a t h r o u g h o u t t h e i r l i f e c y c l e on d i f f e r e n t c r o p s p e c i e s and n o x i o u s weeds growing i n the a r e a ; (b) t o a s s e s s the d e n s i t y i n which B. c a m p e s t r i s c o u l d be p l a n t e d w i t h o u t l o w e r i n g b r o c c o l i p r o d u c t i o n ; (c) t o d e t e r m i n e what was t h e b e s t sowi n g time o f the non-crop p l a n t ; and (d) t o f i n d o u t i f r e s e a r c h on a l l e l o p a t h i c crop/weed i n t e r a c t i o n s can c o n t r i b u t e s i g n i f i c a n t l y on e s t a b l i s h i n g a l t e r n a t i v e weed c o n t r o l methods t h a t do n o t depend o n l y on p e t r o c h e m i c a l - b a s e d h e r b i c i d e s . M a t e r i a l s and

Methods

The e x p e r i m e n t s were done a t t h e Farm F a c i l i t y o f t h e A g r o e c o l o g y Program a t the U n i v e r s i t y o f C a l i f o r n i a a t Santa C r u z , where t h e r e a r e f a c i l i t i e s f o r l a b o r a t o r y , greenhouse, and f i e l d r e s e a r c h . The c l i m a t e i s M e d i t e r r a n e a n , and a v e r a g e s 40 i n c h e s o f r a i n f a l l a n n u a l l y . Laboratory

Experiments

To d e t e r m i n e 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 B. c a m p e s t r i s and 13. o l e r a c e a v a r . i t a l i c a , l e a c h a t e s from each s p e c i e s were made. E x t r a c t s were p r e p a r e d by s o a k i n g , f o r two h r , weighed amounts o f f r e s h o r d r i e d m a t e r i a l i n s u f f i c i e n t d o u b l y d i s t i l l e d water t o p r e p a r e a 10% e x t r a c t (of f r e s h m a t e r i a l ) o r 1.5% e x t r a c t (of d r y m a t e r i a l ) . Ext r a c t s were vacuum f i l t e r e d t h r o u g h p a p e r (Whatman #1). The o s m o t i c c o n c e n t r a t i o n was measured w i t h a f r e e z i n g - p o i n t osmometer (0 t o 500 mosm) and i n d i c a t o r seeds were soaked i n 10 mL o f the e x t r a c t f o r an hour p r i o r t o p l a n t i n g . E x t r a c t s (12 mL each) were added t o P e t r i d i s h e s 10 cm i n diame t e r c o n t a i n i n g 50 g of 30-mesh washed sand c o v e r e d w i t h f i l t e r paper c i r c l e (7-cm d i a m e t e r , Whatman #1). C o n t r o l s were m o i s t e n e d w i t h d o u b l y d i s t i l l e d w a t e r . Ten i n d i c a t o r seeds were p l a c e d on t h e f i l t e r p a p e r i n each d i s h w i t h t h e embryo down and the h y p o c o t y l p o i n t e d t o t h e c e n t e r o f t h e P e t r i d i s h . Each i n d i c a t o r / e x t r a c t c o m b i n a t i o n had 3 replicates. The P e t r i d i s h e s were k e p t i n a dark growth chamber f o r a p p r o x i m a t e l y 72 h r a t 25°C. The r a d i c l e l e n g t h o f each g e r m i n a t e d s e e d l i n g was measured a t 72 h r . T a b l e I shows the number o f e x p e r i m e n t s done, age o f p l a n t s , k i n d o f e x t r a c t , i t s c o n c e n t r a t i o n , and i n d i c a t o r s p e c i e s u t i l i z e d i n each e x p e r i m e n t . B i o a s s a y s w i t h d i f f e r e n t p l a n t p a r t s o f b o t h s p e c i e s were done



IV

X

18,20,24, 28,33,38, 43,46,51, 53,54,56, 58

X

X

X

10,20,30, 39,46,57

X

III

-

-

X

II

X

-

Broccoli fresh dry (10%) (1.5%)

18,31,45, 59

X

No. o f exp. I Age (days) 26,39,51, 27

45,51,55, 59,73,79, 80,82,84

43,45,51, 55,59,64, 69,72,73 77,79,80 82,84

34,43,53, 63,72,79, 90

Age (days)

Winter 1983

Season Fall 1982

Barley Vetch Ryegrass Radish

Fall 1983

Barley Summer Vetch 1983 Ryegrass Radish Wild r a d i s h Lettuce

Barley Broccoli Collards Ryegrass

Indicator species Barley Vetch

I . Number o r E x p e r i m e n t s , K i n d and C o n c e n t r a t i o n o f E x t r a c t s , Ages o f P l a n t s , and I n d i c a t o r S p e c i e s U t i l i z e d t o D e t e c t A l l e l o p a t h i c P o t e n t i a l o f B r a s s i c a c a m p e s t r i s L. and Brassica oleracea var. i t a l i c a

Mustard fresh dry (10%) (1.5%)

Table


H 70 >
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2

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Ο

70

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as

24.

JIMÉNEZ-OSORNIO AND GLIESSMAN

Allelopathic

Interference

265


t o d e t e c t which o f them c o n t a i n e d t h e i n h i b i t o r ( s ) . Extraction of t h e a l l e l o c h e m i c a l s from 13. c a m p e s t r i s l e a v e s was done w i t h s o l v e n t s o f d i f f e r e n t p o l a r i t y ( c h l o r o f o r m , a c e t o n e , methanol, and w a t e r ) . When a l l t h e f r a c t i o n s were r e a d y , 0.09 g were d i s s o l v e d i n 21 mL o f the s o l v e n t w i t h which t h e y were e x t r a c t e d and 3 mL used t o m o i s t e n a f i l t e r p a p e r c i r c l e (7-cm d i a m e t e r , Whatman #1). C o n t r o l s were moistened w i t h 3 mL o f t h e s o l v e n t a l o n e . The s o l v e n t s were a l l o w e d t o e v a p o r a t e and t h e f i l t e r p a p e r s then p l a c e d i n t h e c e n t e r o f P e t r i dishes c o n t a i n i n g 50 g o f 30-inesh washed sand. The, 12 mL o f d o u b l y d i s t i l l e d w a t e r were added t o each d i s h i n which 10 seeds were s e t . Some a s s a y s were done u s i n g s i n i g r i n ( S a r s y n t e x brand) and w i t h t h e c h l o r o f o r m - s o l u b l e f r a c t i o n o b t a i n e d from B. c a m p e s t r i s , a t t h e same c o n c e n t r a t i o n (0.013 g p e r P e t r i d i s h ) , u s i n g as i n d i c a t o r s p e c i e s : Hordeum v u l g a r e , L o l i u m m u l t i f l o r u m , Raphanus s a t i v u s , and Β. oleracea var. i t a l i c a . A l l t h e d a t a from t h e experiments were a n a l y z e d t h r o u g h an ANOVA program f o r a randomized complete b l o c k d e s i g n (17) and comparisons were made between t r e a t m e n t s . The p e r c e n t a g e s o f i n h i b i t i o n were c a l c u l a t e d by c o n s i d e r i n g t h e c o n t r o l as z e r o . Experiments

i n the F i e l d

Summer, 1983. I n o r d e r t o d e t e r m i n e t h e b e s t d e n s i t y a t which b r o c c o l i and B. c a m p e s t r i s c o u l d be p l a n t e d w i t h o u t a f f e c t i n g p r o d u c t i o n o f b r o c c o l i , an experiment was conducted i n a complete randomized b l o c k d e s i g n w i t h f i v e t r e a t m e n t s : 0, 2, 4, and 8 B. c a m p e s t r i s p l a n t s / n i i n t e r p l a n t e d w i t h b r o c c o l i a t a d e n s i t y o f 4.5 p l a n t / m and a c o n t r o l p l a n t i n g o f 13. c a m p e s t r i s a l o n e a t t h e same d e n s i t y . F i v e weeks a f t e r sowing, t h e b r o c c o l i was t r a n s p l a n t e d i n t o t h e f i e l d w h i l e w i l d mustard was p l a n t e d d i r e c t l y on t h e d a t e o f b r o c c o l i transplant. The a r e a was i r r i g a t e d e v e r y week w i t h overhead s p r i n k l e r s t h r o u g h o u t t h e experiment and f e r t i l i z e d 10.1 L/ha f i s h emul s i o n ("Grow F o r c e " brand) a t 30 and 57 days a f t e r s e t - u p o f t h e ex periment. The p l o t s were hand weeded s e l e c t i v e l y e v e r y 15 days, sam p l e s o f t h e v o l u n t e e r weeds were t a k e n t h r o u g h t h e p l o t method ( 1 8 ) , and t h e number o f d i f f e r e n t s p e c i e s , number o f i n d i v i d u a l s o f each s p e c i e s , and biomass (dry w e i g h t ) were r e c o r d e d f o r each p l o t . The dominance, f r e q u e n c y , d e n s i t y , and importance v a l u e were c a l c u l a t e d f o r each s p e c i e s i n each p l o t . Some 90 days a f t e r t h e s t a r t o f t h e experiment t h e b r o c c o l i was h a r v e s t e d and t h e mean d i a m e t e r o f each b r o c c o l i head and i t s mean w e i g h t were r e c o r d e d as w e l l as number o f i n d i v i d u a l s w i t h h a r v e s t a ble heads. 2

2

F a l l , 1983. T a k i n g i n t o a c c o u n t t h e r e s u l t s o b t a i n e d i n t h e f i r s t ex p e r i m e n t , a n o t h e r experiment w i t h f o u r t r e a t m e n t s i n a complete r a n domized b l o c k d e s i g n was done t o d e t e r m i n e t h e e f f e c t o f B. campes t r i s sowing time on b r o c c o l i p r o d u c t i o n . The t r e a t m e n t s were: (a) b r o c c o l i a l o n e , c l e a n weeded t h r o u g h o u t t h e experiment; (b) b r o c c o l i a l o n e , unweeded; (c) 13. c a m p e s t r i s p l a n t e d on October 7 and b r o c c o l i on October 15, c l e a n weeded (d) B. c a m p e s t r i s and b r o c c o l i p l a n t e d on October 15, c l e a n weeded. The same p r o c e d u r e s ( f e r t i l i z a t i o n , weedings, and h a r v e s t ) were f o l l o w e d as i n t h e summer experiment. P r o d u c t i o n d a t a from b o t h experiments were a n a l y z e d w i t h an ANOVA


266

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND

f o r a complete randomized b l o c k d e s i g n and means between t r e a t m e n t s were done.

FORESTRY

Duncan's comparisons

of

Results


B r o c c o l i p r o d u c t i o n . T a b l e I I shows r e s u l t s f o r the d i f f e r e n t t r e a t ments t e s t e d i n summer 1983. A l l the p a r a m e t e r s (mean number o f h a r v e s t a b l e heads, mean d i a m e t e r o f the i n f l o r e s c e n c e , and t o t a l biomass produced) i n d i c a t e t h a t growth o f b r o c c o l i was s t i m u l a t e d by 13. campestris. In the f a l l o f 1983, b r o c c o l i p r o d u c t i o n was v e r y d i f f e r e n t between t r e a t m e n t s (Table I I I ) . B. c a m p e s t r i s p l a n t e d on O c t o b e r 15 + b r o c c o l i produced a y i e l d l i k e t h a t o f weeded b r o c c o l i monocult u r e , but p r o d u c t i o n d i m i n i s h e d when J B . c a m p e s t r i s was p l a n t e d on Oct. 7 and b r o c c o l i on Oct. 15. A l l e l o p a t h i c a c t i v i t y o f B. c a m p e s t r i s and b r o c c o l i . T a b l e IV shows the a l l e l o p a t h i c a c t i v i t y o f e x t r a c t s o f f r e s h B. c a m p e s t r i s and b r o c c o l i on v a r i o u s i n d i c a t o r s p e c i e s . The o s m o t i c c o n c e n t r a t i o n o f t h e s e e x t r a c t s ranged from 0 t o 25 mosm/L; a c c o r d i n g t o B e l l (19) and Anaya and Rovalo (20) t h e s e c o n c e n t r a t i o n s a r e not l i k e l y t o a c c o u n t f o r i n h i b i t i o n o f g e r m i n a t i o n o r r a d i c l e growth. N e v e r t h e l e s s i n h i b i t i o n of such growth was sometimes o b s e r v e d , i n g e n e r a l more i n monoc o t s ( b a r l e y and r y e g r a s s ) t h a n i n d i c o t s . The e x t r a c t s from B. c a m p e s t r i s produced s t r o n g e r i n h i b i t i o n t h a n t h e b r o c c o l i e x t r a c t s , and i n s e v e r a l c a s e s the s p e c i e s t e s t e d were s t i m u l a t e d by b r o c c o l i r a t h e r than i n h i b i t e d . The g r e a t e s t a l l e l o p a t h i c p o t e n t i a l o f w i l d mustard was j u s t b e f o r e and d u r i n g the e a r l y p a r t o f the f l o w e r i n g stage. The r a d i c u l a r growth o f s p e c i e s from the same genus ( b r o c c o l i and c o l l a r d s ) was s t i m u l a t e d r a t h e r t h a n i n h i b i t e d by f r e s h extracts o f w i l d mustard. These r e s u l t s s u p p o r t the i d e a o f i n t e r p l a n t i n g B. c a m p e s t r i s w i t h a s p e c i e s from the same genus. T a b l e V summarizes the e f f e c t s o f e x t r a c t s o f dry t i s s u e s o f b r o c c o l i and w i l d mustard on the r a d i c u l a r growth o f i n d i c a t o r s p e c i e s (H. vu1gare, L. m u l t i f l o r u m , V. a t r o p u r p u r e a and R. s a t i v u s ) . The f i r s t g e n e r a l c h a r a c t e r i s t i c i s t h a t the i n h i b i t i o n produced by B. c a m p e s t r i s was s t r o n g e r than t h a t p r o d u c e d by the b r o c c o l i . The changes i n p e r c e n t a g e s o f i n h i b i t i o n depended on the age o f the p l a n t as w e l l as on the o c c u r r e n c e o f r a i n f a l l . When the m a t e r i a l was coll e c t e d a f t e r a r a i n y day and put t o d r y , the i n h i b i t i o n was not as s t r o n g as b e f o r e o r a t t h e b e g i n n i n g o f the r a i n . The d i f f e r e n c e s o f i n h i b i t i o n b e f o r e and a f t e r a r a i n a r e v e r y c l e a r i n the e x t r a c t s obt a i n e d from b r o c c o l i . As w i t h the f r e s h e x t r a c t s , monocots ( L ^ m u l t i f l o r u m and v u l g a r e ) were more i n h i b i t e d than d i c o t s . F i g u r e 1 shows the p e r c e n t a g e o f i n h i b i t i o n p r o d u c e d by extracts o f d i f f e r e n t p a r t s o f dry and f r e s h m a t e r i a l o f B. c a m p e s t r i s on the r a d i c u l a r growth o f H. v u l g a r e . A l l the e x t r a c t s o f d r y m a t e r i a l (1.5%) s i g n i f i c a n t l y i n h i b i t e d r a d i c u l a r growth, but o n l y e x t r a c t s (10%) o f f r e s h l e a v e s had t h i s e f f e c t , as can be seen i n F i g u r e 1. V. a t r o p u r p u r e a was l e s s a f f e c t e d than H. v u l g a r e . O n l y the e x t r a c t s o f d r y l e a v e s and f r e s h r o o t s i n h i b i t e d i t s r a d i c u l a r growth. Percentages of i n h i b i t i o n of r a d i c u l a r growth o f b o t h R. sativus and H. v u l g a r e produced by e x t r a c t s made w i t h nonaqueous s o l v e n t s are i n Table VI. Treatment m o i s t e n e d w i t h the s o l v e n t s a l o n e i n h i b i t e d such growth o f the i n d i c a t o r s p e c i e s , but not s i g n i f i c a n t l y as com-


24.



Table I I .

Broccoli

Production i n

Allelopathic

Interference

D i f f e r e n t Treatments,

267

Summer 1983

Total y i e l d Mean Mean Mean number o f per p l o t diameter weight individuals (tons/acre) per p l o t (cm) Treatment (g) 4.408 353.61 A 13.55 A 73 A 1 5.021 332.82 A 14.16 A 89 AB 2 6.254 358.35 A 14.62 A 3 101 Β 6.742 15.83 A 406.35 b 4 99 Β Means w i t h t h e same l e t t e r s a r e n o t s i g n i f i c a n t l y d i f f e r e n t , ρ = 0.05%. 1 = B r o c c o l i monoculture 2 = 2 _B. c a m p e s t r i s p l a n t s / m ^ + b r o c c o l i 3 = 4 B. c a m p e s t r i s p l a n t s / m ^ + b r o c c o l i 4 = 8 B. c a m p e s t r i s p l a n t s / m + broccoli 2

Table I I I . Harvestable B r o c c o l i Production i n D i f f e r e n t T r e a t m e n t s , F a l l 1983

Total y i e l d Mean Mean Mean number o f per p l o t weight diameter individuals (tons/acre) (cm) per p l o t Treatment (g) 0.840 68.81 A 70.33 A 1 7.37 A 0.585 48.64 Β 6.08 Β 2 69.33 A 0.721 59.58 AB 3 69.67 A 6.85 A 0.652 52.57 Β 4 6.18 Β 71.33 A Means w i t h t h e same l e t t e r s a r e n o t s i g n i f i c a n t l y d i f f e r e n t , ρ = 0.05% 1 = B r o c c o l i a l o n e , clean-weeded 2 = B r o c c o l i a l o n e , unweeded 3 = B r o c c o l i + B. c a m p e s t r i s p l a n t e d on October 15 4 = B r o c c o l i p l a n t e d on O c t o b e r 15 + B. c a m p e s t r i s p l a n t e d on O c t . 7.



F a l l 1982 F a l l 1982 W i n t e r 1983 W i n t e r 1983 Summer 1983 Summer 1983 Summer 1983 Summer 1983 Summer 1983 F a l l 1983 F a l l 1983

Season and y e a r

VG F VG F VG F F F F VG F

Phenological stage o f Brassica 19.00* -3.07 13.00* 38.00* 10.07 40.03* -6.15 DT 0 11.50* -10.40

Barley

DT DT DT DT 13.31 22.70 -10.42 -22.68 -35.65 DT DT

Lettuce

-13.08 DT DT -18.46 DT DT -11.67 DT DT -25.25* DT DT DT DT DT DT DT DT n o t t e s t ; VG = v e g e t a t i v e

DT DT -10.00 -11.66 DT DT DT DT DT DT DT

DT DT -1.38 10.59 DT DT DT DT DT DT DT DT DT DT DT 20.63 10.06 20.95* 0 25.65 -12.20 15.70*

9.28 14.36 0 20.65 0 9.7 did DT

Collards

Broccoli

Radish

Species

Ryegrass W i l d r a d i s h Vetch Brassica campestris DT DT 12.00* DT DT 17.00* DT 0 DT DT 19.36 DT 8.47 30.69* 0 -8.00 -7.83 -30.38* 22.26* -19.66 0 14.93 9 27.07* 7.65 16.06 11.85 DT -12.00 DT DT 6.1 -13.00

Indicator

italica B. o l e r a c e a v a r . -9.37 26.3* 17.16* 34 16.48* Summer 1983 VG -15.43 -9.66 0 43 VG -17.50* Summer 1983 35.48* 6.30 17.21 63 0 Summer 1983 VG 0 -19.60 F 9.85 17.02* 79 Summer 1983 DT -22.50* 0 43 VG -21.90* F a l l 1983 DT 8.00 -30.00* VG -20.50* F a l l 1983 45 • D i f f e r e n c e s among i n d i c a t o r s p e c i e s f o r t h i s age a r e s i g n i f i c a n t a t JD = 0.05%. growth; F = f l o w e r i n g .

51 57 45 59 20 30 39 46 57 18 47

Age o f p l a n t s (days)

T a b l e IV. Age a t Which B. c a m p e s t r i s o r B. o l e r a c e a v a r . i t a l i c a Showed A l l e l o p a t h i c A c t i v i t y ( f r e s h m a t e r i a l e x t r a c t s , 10g/100 mL) on R a d i c u l a r Growth o f Hordeum v u l g a r e , V i c i a a t r o p u r p u r e a , L o l i u m m u l t i f l o r u m , Raphanus r a p h a n i s t r u m , R. s a t i v u s , B. o l e r a c e a v a r . i t a l i c a , B. o l e r a c e a v a r . G e o r g i a , and Lactuca s a t i v a . Ν = 30. Numbers show p e r c e n t o f i n h i b i t i o n compared t o c o n t r o l .


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24.

JIMÉNEZ-OSORNIO A N D GLIESSMAN

Allelopathic

Interference

269

T a b l e V. Age a t Which B. c a m p e s t r i s o r B. o l e r a c e a v a r . i t a l i c a Showed A l l e l o p a t h i c A c t i v i t y (dry m a t e r i a l 1.5 g/100 mL) on R a d i c u l a r Growth o f Hordeum v u l g a r e , L o l i u m m u l t i f l o r u m , V i c i a a t r o p u r p u r e a and L a c t u c a s a t i v a . Ν - 30. Numbers show p e r c e n t o f i n h i b i t i o n as compared t o c o n t r o l s F a l l , 1983,


Indicator Age o f P l a n t s (days)

Pattern of r a i n f a l l

Barley

Ryegrass

B r a s s i c a campestris 1 day 28* 42* 56* r a i n e d 3 days 35* 41* 45* 45* 38* 44* r a i n e d 1 day r a i n e d 4 days 35* 50* 48* 40* 47*

18 20 24 28 33 38 43 46 47 51 53 54 56 58

rained

43 45 51 55 59 64 69 72 73 77 79 80 82 84 •Differences ρ = 0.05%.

Species Vetch

DT DT DT 57* 17 65* 60* 80* 48* 29* 44* 42* 48* DT

B. o l e r a c e a v a r . i t a l i c a 1 day DT 6 DT 36* DT 27 25 r a i n e d 3 days 11 6 41* 59* 32* 33* 50* 67* 50* 48* 28* r a i n e d 1 day 10 r a i n e d 4 days -5 28* 1 10 32 6 14 DT 30* among i n d i c a t o r s p e c i e s f o r t h i s age a r e rained

DT DT DT 18* 30* 24* 17* 30* 24* 26* 10 24* 17* 24*

Radish

25 24 43* 23* 52* 46* 90* 28* 29* 27* 31 13 28* 34*

8 DT 13 DT 21* DT 7 DT 32* -5 30* 33* 28* 24* 8 16* 9* 15 14* 15 12 7 4 5 13 -1 31* 12 significant at


270

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

pared t o a doubly d i s t i l l e d water c o n t r o l . Solvents with the ex t r a c t s were s i g n i f i c a n t l y d i f f e r e n t from t h e c o n t r o l . The f r a c t i o n t h a t produced t h e s t r o n g e r i n h i b i t i o n i n b o t h s p e c i e s was t h e one made w i t h c h l o r o f o r m . H. v u l g a r e was more s e n s i t i v e than R. s a t i v u s . Because o f t h e f r a c t i o n i n which the i n h i b i t o r ( s ) were p r e s e n t , t h e a l l e l o c h e m i c a l s i n B. c a m p e s t r i s a r e most l i k e l y i s o t h i o c y a n a t e d e r i v a t i v e s such a s a l l y l i s o t h i o c y a n a t e , a breakdown p r o d u c t o f t h e t h i o g l u c o s i d e , s i n i g r i n (21).

Table VI. Comparative E f f e c t s o f S o l v e n t s A l o n e and w i t h E x t r a c t e d M a t e r i a l from B. c a m p e s t r i s Leaves on t h e R a d i c u l a r Growth o f R. s a t i v u s and H. v u l g a r e


Indicator Treatment

H. v u l g a r e

Species R. s a t i v u s

0. 0 A 0. 0 A Control 3. 1 A 11. 0 A Control chloroform 13. 0 A 4. 0 A C o n t r o l acetone 19. 3 Β 7. 0 A C o n t r o l methanol 35. 9 C 53. 4 C C h l o r o f o r m f r a c t i o n (4.2 mg/mL) 25. 6 Β 15. 4 Β Acetone f r a c t i o n (4.2 mg/mL) 19. 3 Β 8. 0 A Methanol f r a c t i o n (4.2 mg/mL) 22. 4 Β 17. 3 Β Aqueous f r a c t i o n (4.2 mg/mL) 10. 7 A 14. 4 Β T o t a l e x t r a c t (4.2 mg/mL) Percentages o f i n h i b i t i o n with d i f f e r e n t l e t t e r s d i f f e r s i g n i f i c a n t l y a t ρ = 0.05% by Duncan's m u l t i p l e - r a n g e t e s t . P e r c e n t a g e s were com p a r e d o n l y between t h e same s p e c i e s . Ν = 30. 3 mL o f t h e s o l u b l e f r a c t i o n s were p u t i n each P e t r i d i s h and t h e s o l v e n t s w e r e a l l o w e d t o evaporate.

F i g u r e 2 shows t h e i n h i b i t i o n p r o d u c e d by s i n i g r i n (0.013 g p e r P e t r i d i s h ) compared w i t h t h a t produced by t h e c h l o r o f o r m f r a c t i o n o f B. c a m p e s t r i s . I t i s c l e a r t h a t t h e f r a c t i o n from IS. c a m p e s t r i s p r o duced more i n h i b i t i o n t h a n s i n i g r i n , and o n l y R. s a t i v u s was n o t i n h i b i t e d by e i t h e r s i n i g r i n o r the c h l o r o f o r m f r a c t i o n . E f f e c t s on weed s p e c i e s . I n t h e f i e l d experiments o f summer, 1983, t h e t o t a l weed biomass was n o t s i g n i f i c a n t l y d i f f e r e n t among t r e a t ments, and i n t h e f i r s t weeding (15 days a f t e r t h e b e g i n n i n g o f t h e experiment) a l l t h e t r e a t m e n t s agreed on t h e main weeds p r e s e n t , which were from t h e B r a s s i c a c e a e ( v o l u n t e e r B. c a m p e s t r i s and R. raphanistrum). I n subsequent weedings t h e r e were no p a r t i c u l a r p a t terns; t h e main weeds were: S p e r g u l a a r v e n s i s , Amaranthus r e t r o p l e x u s , C o n v o l v u l u s a r v e n s i s , Malva p a r v i f l o r a , Medicago sp., Chenopodium album, P l a n t a g o l a n c e o l a t a , Sonchus sp., Erodium sp., Anagal l i s a r v e n s i s , and C a p s e l l a b u r s a - p a s t o r i s . I n t h e f a l l o f 1983, weed samples were n o t t a k e n because weeds ( S p e r g u l a a r v e n s i s , Amaran thus r e t r o f l e x u s , and Chenopodium album) l e f t i n the f i e l d from p r e v i o u s experiments g e n e r a t e d c o n s i d e r a b l e h e t e r o g e n e i t y i n the* weed seed bank. Weed s p e c i e s were v e r y p a t c h y i n t h e d i f f e r e n t p l o t s , so the samples d i d n o t t r u l y r e p r e s e n t the t r e a t m e n t s .


24.

Allelopathic


271

Interference

60 Indicator Species 45

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I Vicia atropurpurea 30

1

15

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-15

F I ο w e r

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S

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e

m

F I ο w e r

L e a f

T

R ο ο t

S t e m

FRESH _ """EXTRACT

DRY —H EXTRACT"

F i g u r e 1. P e r c e n t a g e o f I n h i b i t i o n o f R a d i c u l a r Growth o f Hordeum v u l g a r e and V i c i a a t r o p u r p u r e a Produced by E x t r a c t s from Dry and F r e s h M a t e r i a l o f D i f f e r e n t P a r t s o f B r a s s i c a c a m p e s t r i s . Ν = 30. *p = 0.05%

Β

80 r-

• |

C o n t r o 1

Sinigrin

| !•£

π

4 0 \-

LI

Barley

Radish

Rye

Chloroform fraction

Broccoli

Indicator Species F i g u r e 2. P e r c e n t a g e o f I n h i b i t i o n o f R a d i c u l a r G r o w t h o f S e v e r a l S p e c i e s P r o d u c e d b y S i n i g r i n and t h e C h l o r o f o r m F r a c t i o n (0.013 g ) E x t r a c t e d f r o m B. c a m p e s t r i s . Ν = 30. Treatments w i t h different l e t t e r s d i f f e r s i g n i f i c a n t l y a t £ = 0.05% b y Duncan's m u l t i p l e range t e s t .


272

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND

FORESTRY


Discussion Other s t u d i e s have p r o v i d e d e v i d e n c e f o r 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 w i l d mustard (4) and b r o c c o l i (10, 14/ 22). The r e s u l t s from the l a b o r a t o r y experiments i n t h i s study demonstrate t h a t both s p e c i e s can produce a l l e l o c h e m i c a l s t h r o u g h o u t t h e i r l i f e c y c l e . I t i s v e r y l i k e l y t h a t t h e i n h i b i t o r s a r e breakdown p r o d u c t s o f g l u c o s i n o l a t e s , which are c o n s i d e r e d n o n t o x i c i n t h e m s e l v e s (23) but can y i e l d p h y s i o l o g i c a l l y a c t i v e p r o d u c t s upon h y d r o l y s i s by t h e enzyme m y r o s i n a s e . Through h y d r o l y s i s g l u c o s i n o l a t e s can yield isothiocyanates (24-28). Ju e t a l . (29) d e m o n s t r a t e d t h e c a p a c i t y o f t h i o c y a n a t e s as a l l e l o p a t h i c a g e n t s , and i s o t h i o c y a n a t e s a l s o have a l l e l o p a t h i c a c t i v i t y (30). The w a t e r - s o l u b l e e x t r a c t s o f w i l d mustard and b r o c c o l i p l a n t s were s p e c i e s s p e c i f i c , as shown by t h e r e s u l t s o b t a i n e d by o t h e r aut h o r s (.22, 31./ 32_) . The g r e a t e r i n s e n s i t i v i t y o f c r u c i f e r s i s a p p a r e n t l y r e l a t e d t o the p r e s e n c e o f s p e c i f i c m y r o s i n a s e s which a r e c a p a b l e o f t r a n s f o r m i n g t h e breakdown p r o d u c t s o f t h e g l u c o s i n o l a t e s (33). The s t r o n g e s t i n h i b i t i o n p r o d u c e d by e x t r a c t s o f f r e s h m a t e r i a l of w i l d mustard o c c u r r e d when i t was b o l t i n g o r j u s t a t the b e g i n n i n g o f f l o w e r i n g , w h i l e i n h i b i t i o n by f r e s h e x t r a c t s o r b r o c c o l i d i d not show any such c o r r e l a t i o n . N e v e r t h e l e s s , t h i s does n o t mean t h a t a l l e l o p a t h i c i n t e r f e r e n c e cannot t a k e p l a c e e a r l i e r i n the l i f e c y c l e o f the p l a n t . Jiménez-Osornio and G l i e s s m a n (34) have demonstrated t h a t seeds o f c r u c i f e r s can i n h i b i t t h e r a d i c u l a r growth o f some o t h e r s p e c i e s d u r i n g s i m u l t a n e o u s g e r m i n a t i o n as e a r l y as 60 m i n u t e s a f t e r sowing. Mustard e x t r a c t s p r o d u c e d s t r o n g e r i n h i b i t i o n than b r o c c o l i ext r a c t s (Tables IV and V ) . A l t h o u g h b o t h s p e c i e s b e l o n g t o the same genus, g l u c o s i n o l a t e c o n t e n t u n d o u b t e d l y d i f f e r s i n b o t h q u a n t i t y and q u a l i t y , between s p e c i e s , as w e l l as among v a r i e t i e s (_35 -40) . B r o c c o l i and w i l d mustard a r e o f d i f f e r e n t s p e c i e s ; i n a d d i t i o n , p e o p l e have been i s o l a t i n g c h e m i c a l c o n s t i t u e n t s o f b r o c c o l i f o r a l o n g time. On t h e o t h e r hand B. c a m p e s t r i s has been c o n s i d e r e d a weed and i t s c h e m i c a l c o n s t i t u e n t s have n o t been s t u d i e d as i n t h e c a s e o f b r o c coli. I n h i b i t i o n o f r a d i c u l a r growth was s t r o n g e r w i t h e x t r a c t s o f d r y m a t e r i a l : than t h o s e o f f r e s h m a t e r i a l and the o s m o t i c p r e s s u r e s o f d r y m a t e r i a l e x t r a c t s were h i g h e r t h a n from f r e s h m a t e r i a l , i n d i c a t i n g b e t t e r e x t r a c t i o n o f compounds. E f f e c t s o f e x t r a c t s w i t h d r y m a t e r i a l and w i t h f r e s h m a t e r i a l were not c o r r e l a t e d . T h i s may be because d r y i n g p l a n t m a t e r i a l causes h y d r o l y s i s of g l u c o s i n o l a t e s to isothiocyanates, while a u t o l y s i s of f r e s h c r u c i f e r s y i e l d s p r e d o m i n a n t l y n i t r i l e s (28). F i e l d s t u d i e s do not demonstrate c o n c l u s i v e a l l e l o p a t h i c i n h i b i t i o n o f weeds by w i l d mustard o r b r o c c o l i , but t h e r e a r e some i n d i c a t i o n s o f a l l e l o p a t h i c i n t e r f e r e n c e . F i r s t , t h e main weeds i n t h e f i r s t weeding were c r u c i f e r s i n a l l t r e a t m e n t s , b u t n o t i n t h e f o l l o w i n g weedings. They were s t i m u l a t e d t o g e r m i n a t e o n l y a t t h a t time. Second, b r o c c o l i p r o d u c t i o n was a f f e c t e d by B. c a m p e s t r i s : y i e l d s were i n c r e a s e d d u r i n g the summer. E a r l i e r p l a n t i n g o f mustard i n the f a l l i n h i b i t e d b r o c c o l i y i e l d s , but had no e f f e c t when mustard was p l a n t e d a t t h e same time b r o c c o l i was t r a n s p l a n t e d . In a d d i t i o n , s t i m u l a t o r y e f f e c t s o f c r u c i f e r s on o t h e r c r u c i f e r s o r o t h e r c r o p s has been o b s e r v e d b e f o r e (10, 16, 41).



24.


Allelopathic

Interference

273

In this study some assays showed stimulation by wild mustard extracts on broccoli radicular growth, and field experiments demonstrated that broccoli production can increase in the presence of the non-crop plant. Mean production of broccoli for Santa Cruz County varies from 4 to 6 tons/acre (42) . The yield obtained when 13» campestris was at a density of 8 plants/m2 was 6.74 tons/acre even with broccoli planted in lower density than in conventional monocrops. During the f a l l , broccoli production was reduced significantly, yet i t is important to mention that the variety (Premium Crop) is not the one most recommended for this season. Increasing diversity in agroecosystems is important not only in space but also in time, since cultivars may produce long-lasting effects on soils and soil fungi. The results obtained during the summer experiment suggest the use of B. campestris intercropped with broccoli during the summer but not during the f a l l . In order to manage a non-crop plant i t is very important to understand i t s allelopathic potential and the chemical structure of the active chemical(s) as well as specific effects, spatial dynamics, and active l i f e in the substrate. A l l this can give us a clear idea of the ecological significance of the allelochemicals and thus allow us to assess accurately the value of the chemical interference cf some plants with others in an agroecosystem. With such information we can begin to make suggestions for future management and research in those systems when plant/plant interactions are integral such as in intercropping and rotational plantings. It seems that progress towards a sustainable agriculture might benefit from a study of the naturally envolved chemical defenses of plants and their beneficial management through intercropping or planting crops with non-crop plants. It i s possible that humans, by selecting crops over centuries, have created cultivars that have lost many of their allelopathic substances (41). Utilization of non-crop plants in agroecosystems can incorporate these chemicals back into the system. High intraespecific and intraspecific chemical diversity are likely to have several beneficial consequences, including allelopathic interference with harmful weeds. According to the data presented by Josefsson (43) from 12 thioglucosides analyzed, a broccoli monocrop will have 6 of them, whereas the intercrop of wild mustard and broccoli permits the presence of 11 of them. The presence of a non-crop plant in a agroecosystem incorporates structural complexity and variety of secondary chemicals. Research of allelopathic interference is necessary in the design of agroecosystems in order to lessen dependence on petrochemical-based pesticides. The intercropping of non-crop species into crop systems offers alternatives for the design of more diverse and sustainable agroecosystems. Acknowledgments Support for this research i s most gratefully acknowledged from the Agroecology Program, University of California, Santa Cruz, the Columbia Foundation, and Universidad Nacional Autonoma de Mexico. Review of the manuscript by G.R. Waller and O.C. Dermer i s much appreciated. Literature Cited 1. 2. 3.

Gliessman, S.R. J. Chem. Ecol. 1983, 9, 991-9 Rice, E.L. "Allelopathy"; Academic Press: New York, 1984; p. 1. Chacon, J.C.; Gliessman, S.R. Agro Ecosystems 1982, 8, 1-11. Waller; Allelochemicals: Role in Agriculture and Forestry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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274

4. 5. 6. 7. 8.


9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

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Allelopathic Interference in a Wild Mustard (Brassica campestris L

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