A Rapid Seedling Bioassay for the Study of Allelopathy - ACS

C h a p t e r 31

A Rapid Seedling Bioassay for the Study of Allelopathy Donn G. Shilling and Fumio Yoshikawa 1

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on June 16, 2016 | http://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch031

Monsanto Agricultural Products Company, St. Louis, M O 63167

A bioassay was developed to evaluate the biological activity of pure allelopathic compounds qualitatively and quantitatively and to monitor activity of interest during the purification of chemical components in extracts. The effects of allelopathic compounds and analogs on various growth parameters (GP) were measured using the test species Echinochloa crusgalli (L.) Beauvois and Sesbania exaltata (Raf.) Cory. Root length and root fresh weight were the most sensitive GP, but these measurements were extremely time consuming. Therefore, a model was developed to ascertain predicted shoot-plus-root fresh weight (PSRFW) from total plant fresh weight, which is less sensitive due to seed weight, but rapidly measured. It was concluded that a seedling bioassay using PSRFW is an efficient method to evaluate the phytotoxicity of biological samples. The development of reliable bioassays is crucial to successful research in the rapidly growing field of allelopathy. The need for bioassays in the study of allelopathy is twofold. First, a bioassay is needed to determine i f a specific plant-plant interaction has a chemical basis. Often in these studies, both the potential donor species [i.e., the plant releasing compound(s)] and the bioassay species (i.e., receptor) are studied in situ (1). In other cases, donor species are extracted with various solvents and the extracts are bioassayed (2). Although time-consuming, these studies are essential i f an allelopathic interaction is to be demonstrated. Second, a bioassay is needed to help isolate and characterize the compound(s) causing the interaction. There are two considerations with these types of bioassays: the bioassay species and growth parameter(s) used to indicate biological activity. Nicollier et_ al. (3) used Lycopersicon esculentum (tomato) and Raphanus* sativus (radish) as bioassay species to help in characterization of dhurrin isolated from Sorghum halepense rhizomes. However, these two species were not involved in the actual allelopathic association. 1

Current address: Agronomy Department, University of Florida, Gainesville, FL 32611 0097-6156/87/0330-0334$06.00/0 © 1987 American Chemical Society

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


31.

SHILLING AND YOSHIKAWA

A Rapid Seedling

Bioassay

335

To i d e n t i f y f e r u l i c acid, L i e b l and Worsham (4) used weed species i n their bioassays that were actually growing i n association with wheat (Triticum aestivum L.) i n the environment. Thus, there i s d i v e r s i t y in terms of which plant species are used i n bioassays to indicate biological activity. In most studies (2-10), a whole plant system i s used to monitor the phytotoxic a c t i v i t y by measuring some aspect of growth (e.g., root length). Determining i n h i b i t i o n using a growth parameter such as root length can be tedious and time consuming. Time considerations become even more c r i t i c a l during the p u r i f i c a t i o n of chemical components i n extracts due to the large number of samples generated. Bioassays that monitor s p e c i f i c biochemical (11, 12) or physiological (13-15) processes have also been developed. Although these types of bioassays are generally more sensitive than growth bioassays, they are s p e c i f i c i n the types of b i o l o g i c a l a c t i v i t y detected. When extracts and/or compounds are unknown i n terms of structure and/or b i o l o g i c a l a c t i v i t y , a bioassay that detects s p e c i f i c a c t i v i t y may not be useful because interesting a c t i v i t y may be missed. The objective of this study was to develop an e f f i c i e n t whole plant bioassay that would i d e n t i f y diverse types of compounds that cause phytotoxic a c t i v i t y . Materials and Methods Light conditions and growth parameter evaluation. These experiments were conducted to determine which growth parameter(s) indicates the highest l e v e l of i n h i b i t i o n i n response to a selected chemical(s) under l i g h t and dark conditions. Two compounds (α-phenyllactic acid and p_-ethoxybenzoic acid) were i n d i v i d u a l l y dissolved i n acetone and the 2 mM solutions placed i n 60 ml glass jars with Whatman #1 f i l t e r paper. After the solvent was evaporated, either 0.22 g (60 seed) of Echinochloa c r u s g a l l i L. Beauvois (barnyardgrass) or 0.40 g (30 seed) of Sesbania exaltata (Raf). Cory (hemp sesbania) were placed in j a r s . Three ml (for barnyardgrass) and 3.5 ml (for hemp sesbania) of 15 mM MES buffer [2-morpholinoethanesulfonic acid] adjusted to pH 6.0 was added to each j a r . Seeds were germinated at 25°C either i n continuous darkness or using a 12-h photoperiod. Controls consisted of acetone-treated f i l t e r paper alone. After 72 h of incubation, the following factors were measured and used to determine percent growth i n h i b i t i o n : percent germination, root length, shoot length, root fresh weight, shoot fresh weight, seed fresh weight and t o t a l plant fresh weight. After drying plant material for 48 h at 80°C, weights of shoots, roots, seeds, and t o t a l weight were again determined. Growth parameters were compared on the basis of their s e n s i t i v i t y to the test compounds under l i g h t and dark conditions. The term shoot i s used for s i m p l i c i t y i n this paper. Actually, i n the case of barnyardgrass i t refers to coleoptile and leaf tissue, and with hemp sesbania, hypocotyl tissue. Development of model. Experiments were conducted to develop a model for predicting shoot-plus-root fresh weight (PSRFW) from t o t a l fresh weight (TFW). This seemed reasonable because of the apparent relationship between shoot fresh weight versus shoot length, and


336

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY


root fresh weight versus root length. Both barnyardgrass and hemp sesbania were grown as previously described, but no chemicals were used. Plants were incubated at 25°C using a 12-h photoperiod and harvested at the following times: 2, 4, 6, 8, 10, 15, 40, 51, 69, and 92 h. At these times, fresh and dry weights of shoots, roots, and seeds were determined. Regression analysis was used to determine the best model. (See S t a t i s t i c s section). Effects of a l l e l o p a t h i c compounds. To compare the s e n s i t i v i t y of shoot and root length to PSRFW, 25 additional compounds were tested at 2 mM as described i n the previous section. Hemp sesbania was harvested at 60 h and barnyardgrass at 72 h. These times were chosen i n an attempt to maintain s u f f i c i e n t solution volume during the experiment. Root and shoot lengths, fresh and dry t o t a l weights, and percent germination were then determined. TFW was used to determine PSRFW. S t a t i s t i c s . Data were subjected to analysis of variance and regression analysis by using the general l i n e a r model procedure of the S t a t i s t i c a l Analysis System (16). Correlation c o e f f i c i e n t s between growth parameters were determined with the same system. Equations were best f i t t e d to the cjata based on significance l e v e l of the terms of the equation and R values. Results and Discussion Both barnyardgrass and hemp sesbania gave consistently good germination (barnyardgrass, 93%; hemp sesbania, 89%) and produced an e a s i l y measured dominant root under both l i g h t and dark conditions. In addition, within each population ( i . e . , within each j a r ) of both species, growth was r e l a t i v e l y uniform. This was important, since i t was not feasible to measure every plant. Furthermore, both are weed species and, therefore, observed i n h i b i t i o n of growth could represent a c t i v i t y of economic significance. Using a whole plant versus a s p e c i f i c biochemical bioassay also allows f o r the i d e n t i f i c a t i o n of useful b i o l o g i c a l a c t i v i t y ( i . e . , a c t i v i t y that translates to the whole plant l e v e l ) . The use of both a monocot and dicot would also indicate any s p e c i f i c i t y of a c t i v i t y . Analogs of known natural products [α-phenyllactic acid (17) and £-ethoxybenzoic acid (18)] were used to determine d i f f e r e n t i a l response to l i g h t and to compare the s e n s i t i v i t y of various growth parameters (Table I ) . There would be an advantage to evaluating chemicals and/or extracts of unknown structure and/or a c t i v i t y i n the l i g h t with a nonspecific bioassay ( i . e . , whole plant). This type of bioassay would indicate not only growth i n h i b i t i o n , but also q u a l i t a t i v e changes i n plant pigmentation (e.g., bleaching and c h l o r o s i s ) . However, even with these stated advantages, there are differences i n s e n s i t i v i t y . A l l measures of shoot growth (length and fresh dry weights) of barnyardgrass were inhibited more i n the dark than under l i g h t conditions. This would be expected, as shoots are etiolated i n the dark and grow more rapidly. Perhaps the potential f o r the appearance of a d i f f e r e n t i a l response was greater. The same trend was true f o r hemp sesbania, although the differences i n s e n s i t i v i t y i n the l i g h t and dark were not as dramatic. Root



31. SHILLING AND YOSHIKAWA

A Rapid Seedling Bioassay

337

growth was similar or more sensitive under l i g h t conditions f o r both species. With these two compounds there would be an advantage to evaluating i n h i b i t i o n i n the dark; however, the a b i l i t y to i d e n t i f y a d i v e r s i t y of b i o l o g i c a l a c t i v i t y would be s a c r i f i c e d . Overall, root growth was the most sensitive growth parameter measured. In the l i g h t , root fresh weight of barnyardgrass and root length of hemp sesbania were the most sensitive indicators of a c t i v i t y . Although these growth parameters were s e n s i t i v e , their measurement was extremely time consuming. Total fresh weight (TFW) represents a growth parameter that i s rapidly determined but less sensitive. The decrease i n s e n s i t i v i t y was due partly to the fact that seed weight represented a tissue that was either nonresponsive or one that responded inversely to chemical i n h i b i t i o n ( i . e . , as the l e v e l of i n h i b i t i o n increased, the seed weight loss would p o t e n t i a l l y decrease). Note that f o r barnyardgrass grown i n the l i g h t , average shoot-plus-root fresh weight (SRFW) was almost twice as sensitive as TFW (48 versus 26% i n h i b i t i o n , respectively). To determine i f TFW could be used to obtain predicted shoot-plus-root fresh weight (PSRFW), a time course study was conducted to ascertain the relationship between TFW, SRFW, and seed fresh weight (SFW). Figures 1 and 2 i l l u s t r a t e relationships f o r barnyardgrass and hemp sesbania, respectively. As indicated, the percentage of TFW that was due to SFW changes with time. It was assumed that changes i n the relationship with time would simulate changes due to chemical i n h i b i t i o n . It was also apparent that the s e n s i t i v i t y of TFW ( i . e . , the need f o r PSRFW) decreases as chemical i n h i b i t i o n increases. This was caused by the fact that as the amount of seedling growth decreased, the amount of TFW represented by SFW increased. Thus, the need for a model to determine PSRFW was apparent i f the s e n s i t i v i t y of fresh weight was to be acceptable. With the aforementioned data, a l i n e a r regression was developed to determine the relationship between TFW and SRFW (Figures 3 and 4). With these equations, TFW could be used i n future experiments to determine PSRFW without actually measuring i t . The equations were tested for accuracy by solving the equations using TFW and correlating the results with actual shoot-plus-root fresh weight (ASRFW). Models were also determined f o r t o t a l dry weight and predicted shoot-plus-root dry weight, but the equations were not s i g n i f i c a n t . The PSRFW correlated well with ASRFW (Table II) f o r both barnyardgrass and hemp sesbania (0.97 and 0.91, respectively) for these two compounds. PSRFW correlated well with average shootplus-root length (barnyardgrass; 0.86; hemp sesbania; 0.88) and root length alone (barnyardgrass; 0.84, hemp sesbania; 0.87). Once the model was tested empirically, 25 additional compounds (19) were evaluated to compare the s e n s i t i v i t y of PSRFW to root length (Table I I I ) . These data indicated that PSRFW was more sensitive than TFW, but i t was not as sensitive as root length. Correlation c o e f f i c i e n t s also indicated the same trend (Table IV). There are two possible reasons f o r the lower s e n s i t i v i t y of PSRFW as compared to root length. F i r s t , PSRFW measured the growth of both shoot and root, and i n most cases, root growth was more sensitive to the tested compounds than shoot growth. A second weakness of using fresh weights to quantitate phytotoxicity was caused by compounds that induced r a d i a l expansion of roots (root swelling). Coumarin


338


Table I. The effect of α-phenyllactic acid and p_-ethoxybenzoic acid on various growth parameters of barnyardgrass and hemp sesbania.

Growth Parameters

Barnyardgrass Light Dark

— * *


%

Germination Shoot fresh weight Root fresh weight Average shoot-plusroot fresh weight Shoot dry weight Root dry weight Shoot length Root length Average shoot-plusroot length Total fresh weight Total dry weight

6 12

* *

10

48 35*

83

*

*

48* 9* 53*

42* 31* 54* 55* 73

15

Hemp sesbania Light Dark inhibition"

*

70

*

* 26 5

26 5

U

*

U

*

33

52 40 2

*

*

* 34* 14 12

18

*

34

*

*

30* 67

*

*

61

25 7

17 10

^Averaged across both chemicals at 2 mM. Values followed by asterisk are s i g n i f i c a n t l y d i f f e r e n t from the control at the 0.05 l e v e l according to the general l i n e a r model procedure.

10 20 30 4 0 50 60 70 80 90 100 TIME

(h)

* TFW = 0.24 + 0.007 (Time); F = 306.4, P>F 0.0001, R^ = 0.92. SFW = 0.30 + 0.002 (Time); F = 492.1, P>F 0.0001, R = 0.95 SRFW = -0.06 + 0.005 (Time); F = 183.9, P>F 0.0001, R = 0.87. D

c

Figure 1. The relationship between barnyardgrass t o t a l fresh weight (TFW), seed fresh weight (SFW), and shoot-plus-root fresh weight (SRFW) over time.





)\

Ο

. Sx

I

I

I

I

.

I

.

I

10 20 3 0 40 5 0 6 0 7 0 80 9 0 100 TIME (h)

? TFW = 0 . 4 9 + 0.02 ( T i m e ) ; F = 1 , 4 6 1 . 7 , P>F 0 . 0 0 0 1 , R = 0 . 9 8 . SFW = 0 . 5 + 0.02 (Time) - 0.000161 (Time) ; F = 9 2 . 1 , P> 0.0001, R = 0.87. SRFW = - 0 . 1 4 + 0.01 ( T i m e ) ; F = 4 6 9 . 8 , P>F 0 . 0 0 0 1 , R = 0 . 9 4 . C

F i g u r e 2. The r e l a t i o n s h i p between hemp s e s b a n i a t o t a l f r e s h weight (TFW), seed f r e s h weight (SFW) and s h o o t - p l u s - r o o t f r e s h weight (SRFW) o v e r t i m e .

» -Or

0.0 0.2 0.4 0.6 0.8 TOTAL

a

F = 2,532.8,

P>F 0 . 0 0 0 1 , R

1.0 1.2

1.4 1.6

FRESH WEIGHT (g)

2

= 0.99.

F i g u r e 3 . Model f o r t h e d e t e r m i n a t i o n o f p r e d i c t e d s h o o t - p l u s - r o o t f r e s h weight (PSRFW) from t o t a l f r e s h weight (TFW) f o r barnyardgrass.


339


340


caused t h i s t y p e o f r e s p o n s e i n hemp s e s b a n i a . Root l e n g t h was i n h i b i t e d by 82% b u t PSRFW o n l y 12%. I n p l a n t s t h a t were exposed t o t h e s e t y p e s o f compounds, t h e l o n g i t u d i n a l growth ( r o o t l e n g t h ) was a f f e c t e d more t h a n t h e o v e r a l l f r e s h weight o f t h e r o o t . Therefore, when a q u a l i t a t i v e assessment i n d i c a t e s t h i s t y p e o f a c t i v i t y , a more a p p r o p r i a t e growth measurement, such as r o o t l e n g t h , s h o u l d be used t o q u a n t i t a t e p h y t o t o x i c i t y . I t was e v i d e n t from t h i s s t u d y t h a t a whole p l a n t b i o a s s a y i s more a p p r o p r i a t e t h a n a s p e c i f i c b i o c h e m i c a l b i o a s s a y f o r t h e e v a l u a t i o n o f d i v e r s e c h e m i c a l t y p e s and/or e x t r a c t s c a u s i n g unknown l.8r

0.0

0.5

10

1.5

2.0

2.5

3.0

T O T A L F R E S H W E I G H T (g)

a

F = 3 6 8 . 7 , P>F 0 . 0 0 0 1 , R

2

= 0.93.

F i g u r e 4 . Model f o r t h e d e t e r m i n a t i o n o f p r e d i c t e d - r o o t f r e s h weight (PSRFW) from t o t a l f r e s h weight sesbania.

shoot-plus (TFW) f o r hemp

T a b l e I I . C o r r e l a t i o n c o e f f i c i e n t s between growth parameters a f f e c t e d by α - p h e n y l l a c t i c a c i d a n d ^ - e t h o x y b e n z o i c a c i d i n the l i g h t * a

Growth parameter Shoot f r e s h w e i g h t Root f r e s h weight Actual shoot-plusr o o t f r e s h weight Shoot l e n g t h Root l e n g t h Average s h o o t - p l u s root length T o t a l f r e s h weight

Barnyardgrass PSRFW TFW 0.82 0.69 0.92 0.95

Hemp s e s b a n i a TFW PSRFW 0.79 0.79 0.77 0.77

0.93 0.62 0.81

0.97 0.62 0.84

0.91 0.74 0.87

0.91 0.74 0.87

0.84

0.86 0.90

0.88

0.88 0.66

—

—

^Average f o r b o t h c h e m i c a l s a t 0 . 5 , 1.0 and 2 . 0 mM. A l l c o e f f i c i e n t s s i g n i f i c a n t at the 0.05 l e v e l . ^ T o t a l fresh weight. Predicted s h o o t - p l u s - r o o t fresh weight.


31.

341




b i o l o g i c a l a c t i v i t y . Numerous q u a l i t a t i v e observations were made which indicated varying modes of action for the compounds tested. Juglone caused blackening of the tips of barnyardgrass shoots and roots. Flavone caused severe bleaching i n barnyardgrass shoots and coumarin caused root swelling. Some of these observations would not have been made i f a s p e c i f i c biochemical bioassay were used. Although there are l i m i t a t i o n s , as indicated previously, the described bioassay appears to be an e f f i c i e n t method to evaluate the phytotoxicity of various samples, both q u a l i t a t i v e l y and quantitatively. Table I I I . The effect of 27 compounds on four growth parameters of barnyardgrass and hemp sesbania Chemical 2 mM

SL

Barnyardgrass RL PSRFW

a

*e

Benzoic acid p_-Ethoxybenzoic acid 18 £-Hydroxybenzoic acid 1 £-Aminobenzoic acid 0 o-Ethoxybenzoic acid 16 Vanillin 12 V a n i l l i c acid 4 G a l l i c acid 7* Shikimic acid * Protocatechuic acid 20 2,3-Dihydroxy* benzaldehyde 44 3-Ethoxy-4-hydroxybenzaldehyde _t-Cinnamic acid 37 3-Phenyllactic acid * Caffeic acid * F e r u l i c acid 27 o-Hydroxycinnamic * acid 27 Coumarin 100* Scopoletin * Umbelliferone * 4-Hydroxycoumarin 17 Flavanone * Flavone 55 2-Carbethoxy-5,7dihydroxy-4-methoxy* 2 isoflavone Quercetin Juglone 68 α-Phenyllactic acid 12

84

* 43

37 27 0 0 7 0 18

12 2 0 0 12 8

18 3 0 0 16 10

5 * 69* 22 66 12 0 0 * 30 81 0 0 0 0 3 0 15 10 12 0

51

25

27°* 0 0 8

*

2 0

1 3

* * * * * * * 51

*

19 5 6

* * * 30 2 6

1 6

*

2 0

9

°* *

*

* * 41 0 2

27 0

°*

°*

20 0 0 0 3 1

47 0 0 0 4 1

17

2

3

91°* 0

23°* 3 0 0

34°* 4 0 0

**

40 4* 67

39°*

82

7 9

* * 66

* * 74 1* 52 6 4 0

50°* 24 4 0

0 9 8 0 5

34

24

13

7.

12

*3*

3 2

4 0

2 0

!5* * * * 23 4 2

* 1 0 8 9

2

* 40

97 9 0 12

U

* 1 0

2 5

45

1 2

Hemp sesbania RL TFW PSRFW

* * 29 8

1 8

SL

*

2 9

5 7

9 6

* *

3 7

4 9

5 1

67

2 8

3 6

3 0

3 9

*

10 12 0 23 11 0 7

* 0

V "*

26 5 2

* 22

57

*

33

13*

46

7

°* 56

9. * * * 34 9

22

*

22

C

3 5

Shoot length. Root length. T o t a l fresh weight. Predicted shoot-plus-fresh weight. Values followed by an asterisk are s i g n i f i c a n t l y d i f f e r e n t from the control at the 0.05 l e v e l according to general l i n e a r model procedure. e


342

ALLELOCHEMICALS: ROLE INAGRICULTURE A N D

FORESTRY

Table IV. Correlation c o e f f i c i e n t s between growth parameters of barnyardgrass and hemp sesbania averaged for a l l 27 compounds Hemp sesbania

Barnyar•dgrass


Growth parameter Total fresh weight Root length Shoot length Average root-plusshoot length

TFW

PSRFW : 1

0.71 0.90

—

0.93 0.84 0.87

0.86

0.94

J

TFW

PSRFW

0.80 0.47

—

0.97 0.81 0.43

0.73

0.72

Compared a t 2 mM o n l y . ^ T o t a l f r e s h w e i g h t . °Predicted s h o o t - p l u s f r e s h weight. A l l c o e f f i c i e n t s s i g n i f i c a n t a t the 0.05 l e v e l . a

Literature Cited 1.

Putnam, A.R.; Duke, W.B. Ann. Rev. Phytopathol. 1978, 16, 431-51. 2. Stevens, K.L.; Merrill, G.B. J. Agric. Food Chem. 1980, 28, 644-46. 3. Nicollier, G.F.; Pope, D.F.; Thompson, A.C. J. Agric. Food Chem. 1983, 31, 744-48. 4. Liebl, R.A.; Worsham, A.D. J. Chem. Ecol. 1983, 9, 1027-43. 5. Lehle, F.R.; Putnam, A.R. Plant Physiol. 1982, 69, 1212-16. 6. Fay, P.K.; Duke, W.B. Weed Sci. 1977, 25, 224-28. 7. Williams, R.D.; Hoagland, R.E. Weed Sci. 1982, 30, 206-12. 8. Jankay, P.; Muller, W.H. Am. J. Bot. 1976, 63(1), 126-32. 9. Tang, C.C.; Young, C.C. Plant Physiol. 1982, 69, 155-60. 10. Muller, W.H.; Muller, C.H. Bull. Torrey Bot. Club 1964, 91, 327-30. 11. Leather, G.R.; Einhellig, F.A. In "The Chemistry of Allelopathy: Biochemical Interactions Among Plants"' American Chemical Society: Washington, D.C., 1985; pp. 197-218. 12. Kida, T; Takaro, S.; Ishikawa, T.; Shibai, H. Agric. Biol. Chem. 1985, 49, 1299-1303. 13. Esahi, Y.; Leopold, A.C. Plant Physiol. 1969, 44, 618-67. 14. Chrispeels, M.J.; Varner, J.E. Nature 1966, 212, 1066-67. 15. Jones, R.L.; Varner, J.E. Planta 1967, 72, 255-61. 16. Helwig, J.T.; Council, K.A. (Eds) "SAS User's Guide"; SAS Institute, Inc.: Cary, N.C., 1979. 17. Shilling, D.G.; Liebl, R.A.; Worsham, A.D. In "The Chemistry of Allelopathy: Biochemical Interactions Among Plant"; American Chemical Society: Washington, D.C., 1985; pp 243-71. 18. Shettel, N.L.; Balke, N.E. Weed Sci. 1983, 31, 293-98. 19. Rice, E.L. "Allelopathy"; Academic Press: New York, N.Y., 1974. RECEIVED December 23, 1985


A Rapid Seedling Bioassay for the Study of Allelopathy - ACS

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