Use of Bioassays for Allelochemicals in Aquatic Plants - ACS

23

Use of Bioassays for Allelochemicals in Aquatic Plants

Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 15, 2018 at 22:26:43 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

LARS W.J.ANDERSON Aquatic Weed Control Research Laboratory, Botany Department, Agricultural Research Service, U.S. Department of Agriculture, University of California, Davis, CA 95616 Lack of appropriate bioassays has hampered progress in detecting inhibitory or growth regulatory compounds in leachates or extracts of aquatic macrophytes. Assays relying on seed germination or callus growth in terrestrial plants are not necessarily relevant to the "target" aquatic plants. In these studies, sensitive bioassays were developed using explants of Hydrilla verticillata and vegetative propagules of Potamogeton nodosus and P. pectinatus. These systems respond to many growth regulators and herbicides much as do whole, intact aquatic plants. Responses to some potential allelochemicals have been demonstrated in small-volume exposures. Typical quantifiable responses include: frequency of new shoot production, elongation of new shoots, chlorosis, root production, necrosis of "parent" explant, and inhibition of sprouting. Explant systems should permit inexpensive large-scale evaluation of phytoactive compounds whether from natural sources or from synthetic, agrichemical production. The proliferation of various aquatic weeds in the United States causes losses and damages exceeding $300 million annually (1). In spite of this, there are very few herbicides available for use in aquatic sites. There are two main reasons for this dearth of effective products: potential sensitivity of the aquatic environment, and lack of sufficient research and development by agrichemical companies for what is perceived to be a "minor use" and "high risk" venture. Although there is some truth in this perception, i t has also resulted in the omission of economically important aquatic weeds in the primary chemical screening and identification programs of most companies. The general exception to this is use of duckweed (Lemna sp.) since i tiseasy and This chapter not subject to U.S. copyright. Published 1985, American Chemical Society

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

352

THE CHEMISTRY OF ALLELOPATHY

inexpensive to culture. Unfortunately, duckweed is also one of the l e a s t important a q u a t i c weeds and i t is not even i n the same f a m i l y as any o f the t e n most noxious a q u a t i c weeds: h y d r i l l a ( H y d r i l l a v e r t i c i l l a t a ) , w a t e r h y a c i n t h ( E i c h h o r n i a crassipes), E u r a s i a n w a t e r m i l f o i l (Myriophyllum spicatum), pondweed species (Potamogeton spp. - t h e r e are a t l e a s t f i v e important species), e l o d e a (Elodea canadensis), and coontail (Ceratophyllum demersum) (2). However, c u l t u r e and whole-plant testing using these more important weeds requires much more space and time and higher costs compared t o duckweed. These circumstances have p r o v i d e d impetus to investigate the p o t e n t i a l f o r using b e n e f i c i a l a q u a t i c p l a n t s which might out-compete more noxious weeds {3). One such plant, dwarf spikerush (Eleocharis coloradoensis) produces chemical(s) which exhibit i n h i b i t o r y e f f e c t s on o t h e r a q u a t i c p l a n t s ( J ) . The f u r t h e r c h a r a c t e r i z a t i o n and i d e n t i f i c a t i o n o f these a l l e l o c h e m i c a l s r e q u i r e s s e n s i t i v e b i o a s s a y s which are a l s o r e l e v a n t t o the "target" aquatic weed species. Although many convenient bioassays have been developed f o r plant growth regulators and a l l e l o p a t h i c compounds, they often bear no resemblance to the target plant. The objective of the research r e p o r t e d here was t o d e v e l o p s e n s i t i v e b i o a s s a y s which u t i l i z e near-whole p l a n t systems of appropriate target aquatic weeds and which r e q u i r e l i t t l e space and low volumes o f i n c u b a t i o n medium. Such bioassays could be used to help i d e n t i f y a c t i v e f r a c t i o n s o f c h r o m a t o g r a p h i c a l l y p a r t i t i o n e d a l l e l o c h e m i c a l s and c o u l d a l s o be used i n primary screening procedures f o r newly synthesized agrichemicals. Methods and Materials 1. P l a n t s o u r c e s . Two g e n e r a l types of plant bioassay systems were used: 1) v e g e t a t i v e propagules (winterbuds) o f American pondweed (Potamogeton nodosus) and sago pondweed (P. pectinatus; 2) e x p i a n t s o f h y d r i l l a ( H y d r i l l a v e r t i c i l l a t a r ( F i g u r e 1 ) . American pondweed "winterbuds" were c o l l e c t e d i n the f a l l i n de-watered i r r i g a t i o n canals i n Richvale, CA. and were maintained at ca. 6 C i n p l a s t i c bags. This treatment breaks f a l l dormancy i n 6 t o 8 weeks. Sago pondweeds were obtained from a commercial s u p p l i e r i n Oshkosh, WI., and were kept a t c a . 6 C to prevent sprouting. Vigorously-growing h y d r i l l a was c o l l e c t e d from t h e I m p e r i a l I r r i g a t i o n D i s t r i c t , CA., and shipped to Davis within 24 hours a f t e r harvesting. H y d r i l l a was kept i n 51 cm square χ 38 cm deep p l a s t i c tubs and kept thoroughly f l u s h e d with tap water. Intact shoots of the collected material were cut and planted into a standard soil "U.C. Mix" (.5 f t Delta peat, .5 f t ^ coarse sand, 45 g KNO3, 30g K2SO4, 713 g dolomite, 180 g gypsum, 320 g super phosphate). Potted cuttings were allowed to root 3 t o 5 weeks i n a l a r g e f i b e r g l a s s tank i n a greenhouse under 14-h l i g h t (natural l i g h t supplemented with cool-white fluorescent lamps = 250 μΕ m-2 s e c " ) . Only those p l a n t s w i t h h e a l thy-appearing, vigorous new growth were used i n subsequent bioassays. 2. P r e p a r a t i o n of vegetative propagules. Winterbuds of American pondweed were s u r f a c e - s t e r i l i z e d by rinsing them free o f soil and d e b r i s and soaking i n 1% hypochlorite (ca. 1:4 v/v d i l u t i o n of e

e

3

1


23.

ANDERSON

Bioassays for Allelochemicals in Aquatic Plants

353

Figure 1: L e f t : two-node expiants of h y d r i l l a showing new shoots (arrows) 2 weeks a f t e r e x c i s i o n ( e x p i a n t s 2 cm long); middle: ungerminated "winterbuds" of American pondweed (5 cm long); r i g h t : ungerminated tubers of sago pondweed (5 cm long).



354

Chlorox) f o r 20 min. Winterbuds were then rinsed 3 times with s t e r i l e d i s t i l l e d water to remove the hypochlorite. Sago pondweed " t u b e r s " were r i n s e d i n d i s t i l l e d water, but not surface s t e r i lized. 3. Preparation of h y d r i l l a explants. A stainless s t e e l razor was used t o remove the d i s t a l 4 t o 5 cm ( " a p i c a l e x p i a n t s " ) from e i t h e r r o o t e d h y d r i l l a , o r h y d r i l l a f r e s h l y r e c e i v e d from the f i e l d . Subtending sections containing two a d j a c e n t i n t a c t nodes ("2-node expiants") with whole whorls of leaves were removed, but 1 t o 2 intervening nodes were l e f t above the next cut ( F i g u r e 2 ) . No 2-node e x p i a n t s were taken below (proximal to) a subtending l a t e r a l branch. A l l expiants were kept i n tap water f o r 2 4 hours i n a growth chamber before use i n bioassays (25°C, 14-h day, ca. 200 μΕ m" s e c " ) . 4. Exposure to growth regulators, herbicides and allelochemicals. A l l t e s t compounds were dissolved i n s t e r i l e 1% Hoagland's medium (5) o r i n an organic solvent (methanol, acetone or ethanol) and subsequently d i l u t e d t o t e s t c o n c e n t r a t i o n w i t h s t e r i l e 1% Hoaglands medium. A l l experiments i n c l u d e d 1% Hoagland's medium c o n t r o l s , and c o n t r o l s containing appropriate concentrations of organic solvent. Generally, 5 winterbuds or tubers or 10 expiants were p l a c e d i n 500-ml foam-stoppered Erlenmyer flasks with 250 ml of medium. Treatments were run i n t r i p l i c a t e i n a growth chamber a t 25°C, 14-h day, 150-200 μΕ m" s e c " cool-white fluorescent l i g h t . Only expiants from a common batch of c o l l e c t e d o r t r a n s p l a n t e d h y d r i l l a were used i n an experiment. In some sets of experiments, l i g h t levels were varied with neutral density f i l t e r s o r temperatures were varied. Plant growth regulators used were: GA3 ( g i b b e r e l l i c a c i d ) , ABA ( a b s c i s i c acid) BA (benzyladenine), KT (Kinetin) ( A l l from Calbiochem, I n c . ) . A l l e l o c h e m i c a l s were DAD ( d i h y d r o a c t i n o d i o l i d e (6,7) ) and s o l s t i t i a l i n . Herbicides used were fluridone, chlorsulfuron, and sulfometuron. 5. Observation of e f f e c t s . Seven to 14 days a f t e r exposure t o t e s t compounds, length, number of leaves and presence of stomata i n sprouted winterbuds were determined. For sago pondweed tubers, l e n g t h , number o f new daughter plants and presence of roots were determined. H y d r i l l a e x p i a n t s were e v a l u a t e d f o r number o f and l e n g t h o f new shoots. In some experiments the c h l o r o p h y l l - a content of the a p i c a l 2 cm on a p i c a l expiants was determined by 3 s u c c e s s i v e extractions i n 90% acetone using a power-driven Teflon pestle. Absorbance of M i l l i p o r e - f i l t e r e d (.45μ ) acetone extracts was determined a t 6 30 , 6 45 , 6 65 nm on a spectrophotometer. C h l o r o p h y l l - a was c a l c u l a t e d by equations o f S t r i c k l a n d and Parsons (8). 2

1

2

1

Results American pondweed responded to GA3 and the herbicide sulfometuron within 7 days after exposure (Table I ) . As with t e r r e s t i a l plants, GA3 caused elongation of shoots (internodes) compared to controls, whereas sulfometuron almost c o m p l e t e l y blocked e l o n g a t i o n s i n c e no s i g n i f i c a n t lengthening o c c u r r e d between day 7 and 14. The presence of GA3 d i d not counteract the i n h i b i t i o n caused by sulfometuron. T h i s is r e a s o n a b l e s i n c e


23.

ANDERSON


- Apical

355

Meristems

- Expiant

#1

- Explant

#2

F i g u r e 2: Diagram of H y d r i l l a v e r t i c i l l a t a showing location from which apical meristens ("apical explants"_ and "two-node" expiants were excised.


356


Table

I

S h o o t l e n g t h o f A m e r i c a n P o n d w e e d 1 a n d 2 weeks a f t e r c o n t i n u o u s e x p o s u r e t o s u l f o m e t u r o n a l o n e and i n c o m b i n a t i o n w i t h d i f f e r e n t p l a n t g r o w t h r e g u l a t o r s (PGR)

Shoot Treatment

Control

3

b

10.3±O.8

Sulfometuron N-6

Benzyl Adenine

Gibberellic Kinetin Zeatin

Length

Acid

(KT) (ZT)

(BA)

(GA)

(cm)

2 weeks a f t e r treatment

1 week a f t e r treatment

16.6±O.6

C

7.2±O.6

7.6±O.3

11.6±1.1

15.7±O.8

16.3±O.4

28.4±1.5

1 1 . 1 ± 0 .7

17 . 2±1 .5

12.1+1.1

15 . 8±1 . 4

Sulfometuron

+ BA

6.3±O.8

7.4±1 .0

Sulfometuron

+ GA

6.5±O. 4

7.1±O.3

+ KT

6.4±O.5

7.9±O.7

6.9±O.7

7. 4±O. 4

Sulfometuron Sulfometuron

a

b c

+

GT

Concentration of sulfometuron PGRs was 1 0 - 5 ^ 1% H o a g l a n d s medium V a l u e r e p r e s e n t s X t S D ; n=20

was

1.0 p p b ( 2 . 7 X

9

10~ M)j


23.

ANDERSON


357

sulfometuron i n t e r f e r e s with c e l l d i v i s i o n v i a blockage of amino a c i d s y n t h e s i s (9) w h i l e G A 3 a f f e c t s c e l l expansion. In f a c t none of the growth regulators counteracted sulfometuron. Another type of response i n American pondweed is shown i n T a b l e I I . A b s c i s i c acid (ABA) causes a change i n l e a f development and morphology whereby exposed p l a n t s produce l e a v e s having stomata on t h e i r upper surfaces. These " f l o a t i n g " type leaves are more complex than the simple 2 - c e l l thick "submersed" l e a v e s t h a t are normally produced upon sprouting of winterbuds Q O ) . S o l s t i t i a l i n , a compound i s o l a t e d from Centaurea s o l s t i t i a l is, had no apparent e f f e c t on l e a f l e n g t h , number of leaves or ABA-induced f l o a t leaf formation i n American pondweed. In another b i o a s s a y ( T a b l e I I I ) , an a c t i v e compound (DAD) i s o l a t e d from whole dwarf s p i k e r u s h (Eleocharis coloradoensis) (6,7) appeared t o induce f l o a t i n g l e a f f o r m a t i o n i n American pondweed. The response to DAD was somewhat e r r a t i c and much less pronounced than t h a t observed f o r ABA a t even h i g h e r concen t r a t i o n s than ABA. ( P r e v i o u s work has shown t h a t high concen t r a t i o n s of ABA, e.g. 5-13 ppm, i n h i b i t sprouting of winterbuds). The response t o DAD is p a r t i c u l a r l y interesting since submersed "floating-type" leaves were observed on American pondweed when i t was grown with dwarf spikerush (3). G A 3 caused elongation i n sago pondweed, even i n the presence of s o l s t i t i a l i n (Table IV). At 1 t o 50 ppm, s o l s t i t i a l i n reduced the number of leaves per plant even i n the presence of G A 3 . At 50 ppm s o l s t i t i a l i n caused a r e d u c t i o n i n the number o f daughter p l a n t s , and seemed to have s l i g h t l y inhibited root production as well. The above r e s u l t s were obtained using volumes of 100-250 ml i n Erlenmyer f l a s k s . However, much s m a l l e r volumes can be used ( 2 - 5 ml) i f winterbuds are i n d i v i d u a l l y exposed i n g l a s s tubes. Table V shows the response of American pondweed winterbuds exposed to ABA f o r 72 h i n 8 mm (i.d) χ 14.5 cm tubes and then transferred to fresh 1% Hoaglands f o r 4 days. The e f f e c t on leaf development is the same as with larger volume exposures, but t h i s system would allow use of much smaller quantities of allelochemicals. H y d r i l l a e x p i a n t b i o a s s a y s . The g e n e r a l e f f e c t s o f temp e r a t u r e on p r o d u c t i o n and e l o n g a t i o n of new shoots on 2-node h y d r i l l a e x p i a n t s is shown i n Figure 3 . No shoots were produced at 15 C, but a t 36 C c a . 50% o f the e x p i a n t s had new shoots averaging 7 cm i n length within 7 days. In most cases, only one new shoot was produced per explant, but o c c a s i o n a l l y , two shoots were produced (either one at each node or two at one node). Most plants exhibit "apical dominance" which means t h a t the presence of a terminal (distal) meristem tends to suppress l a t e r a l shoot i n i t i a t i o n ( 1 1 ) . S i n c e l a t e r a l shoot production is an important c h a r a c t e r i s t i c to assess i n h y d r i l l a , the frequency of shoot production was determined i n s e q u e n t i a l l y c u t (distal to proximal) e x p i a n t s (Table V I ) . Even though the 4 cm a p i c a l meristem contained several nodes, almost none o f these produced new shoots. However, n e a r l y h a l f the 2-node expiants subtending the cut a p i c a l meristem produced new shoots. There was no apparent d i f f e r e n c e i n p e r c e n t o f new shoots produced once the apical meristem was removed. f



II

6

1 (ppm)

c

k

1.0±O.3 O.6±O.1

— —

3.6±O.2

11.7±0 .6 10.9±0 • 4

7.2±2.6

6.5±O.5

3.1±O.3

O.9±O.2

0

0

—

—

—

3.3±O.4

4.3±O. 4

6.1+O.2

c

1.1±0 .2

—

.01

0

0

2.2±O. 4

3.1±O. 3

3.9±1. 3

0

0

2.3±O. 4

0

leaves/plant 25-Day

—

Float 11-Day

10.2±0 .5

21 .9±1 . 4

17.0±0 .9

5.5±O.5

3.6±O.3

10.1±1 .8

19.5±1 .9

5.9±O.2

18.1±

Leaves/plant 11-Day 25 -Day

W i n t e r buds were e x p o s e d t o compounds i n 1% H o a g l a n d f o r 7 d a y s . V a l u e s a r e mean ± SE; 5 p l a n t s / r e p l i c a t e ; 3 r e p l i c a t e s / t r e a t m e n t , 11 Days p o s t t r e a t m e n t , p l a n t s were washed and t r a n s f e r r e d t o f r e s h 1% H o a g l a n d n o t c o n t a i n i n g t e s t compounds. Data a r e from p l a n t s kept 25 d a y s a f t e r t r a n s f e r s . One p l a n t had one l e a f w i t h s t o m a t a .

(ppm)

20

a

(ppm)

50

ABA 10" M + Solstitialin

6

6.2±O.5

14.3±O.9

1 (ppm) (3.6X10" M)

6

14.1±0·5

5

4

20 (ppm) (7.1X10" M)

6.3±O.5

14.5±1.4

10" M

16.5±4.3

D

a

o f S o l s t i t i a l i n and A b s c i s i c A c i d on A m e r i c a n Pondweed (Potamogeton n o d o s u s )

Length(cm) 11-day 25-day

Effect

Solstitialin 50 (ppm) (1 .8X10" M)

ABA

Control

Treatment

Table


a

1 4/27

12/23

5/28

0/30

3/31

6/29

7/29

6/27

E a c h t r e a t m e n t c o n s i s t e d o f two r e p l i c a t e s , f i v e p l a n t s p e r r e p l i c a t e . P l a n t s were s c o r e d 7 d a y s a f t e r s t a r t o f e x p o s u r e t o ABA o r DAD.

4

50 ppm DAD (1 .8X10~ M)

4

25 ppm DAD (O.9X10- M)

-

24.1

22.2

28.5 6/21 1/13

1/29

16/22

4/40

1Uppm DAD (3.5Χ10~ Μ)

5

48.0 12/25 7/26

21/33

9/16

16/37

5X10" M ABA (.131 ppm)

7

53.8 1 4/26

19/31

1 4/23

6

8/25

60.7

17/28

-

10.3

16/28

3/29

% Floating Leaves

18/28

0/29

2/29

7/30

Mean Exp.2,3,4

3

12/3 4

-

0/36

(72 H) No. 4

H) No. 3

(72

with Stomata/Total L e a v e s

H) No.2

{H

Leaves

( D i h y d r o a c t i n o d i o l i d e ) o n L e a f Development i n Potamogeton nodosus

(5 d a y s ) No.1

o f DAD

10" M ABA ( .26 3 ppm)

10" M ABA (2.63 ppm)

5

Control

(Exposure Period) Experiment

Treatment

TABLE I I I E f f e c t


4

(cm)

a

24.5±2.1 27.7±1.5 19.2±1.6

14.2±2.1 14.8±1.9 15.1±2.0

17.7±1 .9 18.9±1.9 18.9±1.8

13.7±1.9

13.8±1 .5

14.2±1.5

19.2±1 .8

— " ~ No. L e a v e s / p l a n t

7

66.7±13 66.7±13 66.7±18

100±0 93±7 80±0

66.7± 7

60.0±11

53.3±17

8 6 . 7 ±

n

u

t

% with daughter p l a n t

e

n

80 ±12 100 ±0 80 ±20

93± 7 100± 0 93± 7

66± 7

73± 7

73± 7

100± 0

% with roots

T u b e r s were e x p o s e d t o compounds f o r 7 d a y s i n 1% H o a g l a n d " t , V a l u e s a r e means ±SE.; 5 p l a n t s / r e p l i c a t e s , 3 r e p l i c a t e s p e r t r e a t m e n t

6

5

4

6

5

4

S o l s t i t i a l i n 20 ppm +Gibberellic Acid 10- M (35 ppm) 10" M (3.5ppm) 10- M (.35ppm)

15.1±1.1

14.5±1.0

14.8±1.0

14.9±1.3

Length

27 .0±2.2 26.3±1 .1 22.3±1 .3

—

G i b b e r e l l i c Acid 10" M (35 ppmw) 10" M (3.5 ppmw) 1 ϋ " Μ (.35 ppmw)

6

5

Solstitialin 50 ppmw (1.8X10- M) 20 ppmw (7.1X10" M) 1 ppmw (3.6X1û~ M)

Control

Treatment

5

E f f e c t o f S o l s t i t i a l i n and G i b b e r e l l i c A c i d o n Sago Pondweed ( P o t a m o g e t o n p e c t i n a t u s )

T a b l e IV


a

6

Μ ABA ppm)

7.4±.5

8.2±.4

10.2±.5

Length

(cm)

39.8±7

29.8±8

0

% Leaves with

in

Stomata

t o 2 ml ABA

F o r e a c h t r e a t m e n t 10 w i n t e r b u d s were i n d i v i d u a l l y s p r o u t e d i n t u b e s f o r 72 h, t h e n r i n s e d and t r a n s f e r r e d t o a 500 ml E r l e n m y e r f l a s k c o n t a i n i n g 200 ml f r e s h 1% H o a g l a n d ' s medium f o r 3 more d a y s . V a l u e s a r e mean ± S.E.

ΙΟ"" (.26

5X10 Μ ABA (.11 ppm)

- 7

Control

Treatment:

Plant

3

Response o f A m e r i c a n Pondweed t o 72 h e x p o s u r e 8 mm χ 14.5 cm B i o a s s a y T u b e s

TABLE V

362


7.0

LU CO

+1 IX

μ

6.0 5.0 4.0 3.0 i l

2.0 1.0

ί3|

5

15° 30° 36° 15° 30° 36°t36° 30° 15° 36° 30° I5°C A B C A Β C A Β C A Β C 7 days 14 days 21 days 28 days INCUBATION TIME

F i g u r e 3: E f f e c t of temperature on production and elongation of new shoots on h y d r i l l a two-node e x p i a n t s . Arrow i n d i c a t e s when e x p i a n t s o r i g i n a l l y maintained a t 15 o r 36 C were exxchanged. Values are means ± S.E.



VI

Expiants maintained

i n 1% H o a g l a n d

50

6

a

22

5

end)

30

4

(proximal

40

3

20

0

35

( 4 cm)

Expiants

Lateral

shoot

45

40

40

5

5-Nodes

Expiants

flasks.

Between

Expiants

production

S h o o t s on Two-Node

and new

Medium i n 500 ml E r l e n m e y e r

Consecutive

% New

position

2

end)

Meristem

No.

1 (distal

Apical

Expiant

R e l a t i o n s h i p between H y d r i l l a e x p l a n t

Table

3

364


R e s u l t s presented i n Table VII show that s u f f i c i e n t l i g h t is needed for new shoot formation on expiants and that the h e r b i c i d e f l u r i d o n e causes c h l o r o s i s i n new growth just as i n whole plants ( 12,13). These d a t a a l s o c o n f i r m that the apical expiant, which c o n t a i n s the t e r m i n a l meristem, is a poor system f o r assaying i n h i b i t o r s of new shoot production. T a b l e s V I I I and IX show responses of h y d r i l l a expiants to v a r i o u s p l a n t growth regulators and to s o l s t i t i a l i n . G A 3 , BA and solstitialin enhanced elongation at 10" or 10" M. Zeatin, on the other hand, d r a m a t i c a l l y i n c r e a s e d new shoot i n i t i a t i o n i n a p i c a l explants but had l i t t l e e f f e c t on 2-node explants. DAD at 50 ppm caused a s i g n i f i c a n t r e d u c t i o n i n new shoot l e n g t h on h y d r i l l a explants, but did not affect i n i t i a t i o n of new shoots (Table X). ABA was more e f f e c t i v e than DAD i n i n h i b i t i n g shoot elongation, but also d i d not block shoot i n i t i a t i o n . The h y d r i l l a 2-node e x p i a n t s were a l s o s e n s i t i v e t o the herbicides chlorsulfuron. One part per b i l l i o n chlorsulfuron (ca. 3 X 10" M reduced growth o f new shoots by almost 80% but had no e f f e c t on new shoot i n i t i a t i o n (Table XI). When the h e r b i c i d e was removed a f t e r 14 days, new shoots began to elongate. 4

5

9

Discussion A wide v a r i e t y o f p l a n t growth b i o a s s a y s are a v a i l a b l e f o r detecting growth regulatory and phytotoxic a c t i v i t y of chemicals i n t e r r e s t r i a l p l a n t s (14,15). These assays were developed primarily f o r the purpose of characterizing hormonal responses and for q u a n t i f i c a t i o n of endogenous hormones. However, when they a r e u t i l i z e d t o d e t e c t a l l e l o c h e m i c a l s the experimenter assumes a p h y s i o l o g i c a l and b i o c h e m i c a l s i m i l a r i t y i n mode o f e n t r y and mechanism o f a c t i o n which may o r may not e x i s t . T h i s is par t i c u l a r l y true f o r economically important a q u a t i c weeds, s i n c e t h e i r growth and development has been sparsely investigated. Given the range of secondary products i n aquatic plants (16) which might be a l l e l o p a t h i c , i t is important to t a i l o r b i o a s s a y s to f i t a given "target" plant and to use an assortment of q u a n t i f i a b l e ob servations. The r e s u l t s presented here demonstrate that these approaches can be a p p l i e d t o Potamogeton sp. and H y d r i l l a v e r t i c i l l a t a . In both assay systems, t y p i c a l responses to known plant growth regulators were observed i n most c a s e s . The unique response o f Ρ.nodosus t o ABA however, c l e a r l y shows the importance of not relying s o l e l y on " c l a s s i c a l " bioassays. Since aquatic plants i n general (and p a r t i c u l a r l y those which have "weedy" growth) are well adapted to conditions very d i f f e r e n t from t e r r e s t r i a l s i t e s , one might expect d i f f e r e n t growth r e sponses. Thus, the nature of a l l e l o p a t h i c interactions needs to be determined. In the case of dwarf spikerush for example, l e a c h a t e experiments showed t h a t production of daughter plants was i n h i b i t e d , not g e r m i n a t i o n of tubers (4). This implies that using a seed g e r m i n a t i o n (or tuber germination) assay alone would not be fruitful. H y d r i l l a e x p i a n t s responded t o h e r b i c i d e s e s s e n t i a l l y the same as whole plants: fluridone caused c h l o r o s i s i n new s h o o t s ,



1

2.5±1.9 7.5±2.5 35.02±2.8 25.0±6.4

Day 7

3

0 10.0 30.0 10.0

4.9±.2 6. 4±.4 5.7±.3 5.6±.2

5.2±.2 6.6±.5 5.6±.3 5.7±.2

4.6±.2 5.5±.3 5.8±.3 5.4±.2

Day 11

.386±.03 .542±.05 .409±.06 .337±.04

.382 .06 .867±.05 1.392±.38 .778±.07

Day 11

Values are mean + SE (n=15 for each

After 11 days, the terminal 2-cm were removed from 5 apices in each replicate. treatment).

r

0 3.3 16.7 10.0

5.8±.7 5.3±3.3 5.9±.3 5.2±.2

Day 7

3

Chlorophyll* (mg/g. f r . wt.)

b

5.0±2.8 17.5±2.5 22.5±8.5 35.0±1.8

3.3 3.3 16.7 19.4

Day 11

New Shoot Length (cm)

Various light levels were achieved using open mesh shade cloth. Expiants were exposed to fluridone in 1% Hoaglands medium for 11 days. Treatments consisted of 10 4-cm Apices/replicates, 3 replicates per treatment and 10 2-node explants/replicates 4 replicates per treatment. Values are mean ± SE.

0 7.5±4.8 10.0±7.1 30.0±14.7

0 6.7 10.0 16.1

Day 7

New Shoot (%)

4-cm Apical Tips

3

No light 10 60 135

3.0±2.4 15.0±2.9 37.5±2.5 27.8±8.4

Day 11

(New Shoot (%)

Two-Node Expiants

Fluridone (O.25 ppmw)

No light 1U 60 135

Treatment μΕ m~2 sec" ) Control

Treatment

VII

Effect of Fluridone on 2-node Expiants and Apical tips of Hydrilla v e r t i c i l lata exposed to different light l e v e l s

Table

366


TABLE

VIII

E f f e c t o f ABA and Z e a t i n o n New S h o o t P r o d u c t i o n f r o m A p i c a l S h o o t s and Two-node S e c t i o n s o f H y d r i l l a v e r t i c i l l a t a 5

Number o f new Source:

Apical

Control 10 ABA (.26 5 X 1 0 " ABA - 6

7

5X10" _ 66

8

ppm) O.5

ABA

1 I 0 ~ MM ZZ ee aa t i n (.26ppm) 10~ I 0 ~ MM ZZ ee aa t i n + 5 X 1 0 " M ABA 66

shoots

Expiants

per expiant

Two Node

.33*.2 1.0 ^.2 ±.31

.40*.1 .46*.1 .60*.1 1

O.10+·

.27+-

1.7 ± . 2 1.5 *.2

V a l u e s a r e means * S.E. f r o m t h r e e r e p l i c a t e s e x p i a n t s p e r r e p l i c a t e (15 e x p i a n t s ) . Light length = 24 h o u r d a y a t 2 B ° C

2

.27*.07 .47*.0

7

a

Expiants

of

five


23.

ANDERSON

367


T A B L E IX Effect

New

Treatment

4

- 5

Ga

6

Length

(cm)

on H y d r i l l a

% New S h o o t s

.8

36.7

4.20 + .9 5 . 6 7 ± .7 O.94± .1

40.0 50.0 30.0

M ( 2 6 . 0 ppm) M ( 2 . 6 ppm) M (.26 ppm)

1 .33± 2.0 ± 1.17±

30.0 16.7 20.0

M ( 3 5 . 0 ppm) M ( 3 . 5 ppm) M (.35 ppm)

7 . 8 7 ± 1 .6 2.7 ±1 .0 1.56± .5

50.0 43.3 26.7

a a b

M ( 2 3 . 0 ppm) M ( 2 . 3 ppm) M (.23 ppm)

4.7 ± . 5.10±2. 2.30±1,

50.0 33.3 23.3

a a b

itial ppmw ppmw ppmw

ABA 10" 10 10"

Shoot

2.32±

Control Solst 50 20 1

o f P l a n t Growth Regulator Two-node E x p i a n t s *

in (1.8X10~ M) (7.1X10~ M) (3.6X10 M) 4

5

_ 6

b

a

3

10" 10" 10~

4

5

6

BA 10~ 10" 10

4

5

- 6

a b

V a l u e s a r e means o f 3 r e p l i c a t e s , 10 e x p i a n t s p e r r e p l i c a t e 7 days posttreatment. V a l u e s w i t h t h e same l e t t e r 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 a t t h e 5% l e v e l u s i n g Duncan's M u l t i p l e Range T e s t .



368

TABLE X Response

of H y d r i l l a

Two-node

L e n g t h o f New

Expiants to Dihydroactinodiolide

Shoots

(cm)

% New

(DAD)

a

Shoots

Treatment : Experiment Control

1 Experiment

2

Experiment

1

Experiment

1.09

A

2. 42

A

60 A

30 A

50 ppm (1.8X10" M)

O. 42

C

1.2

Β

53

A

35 A

2 5 ppm ( 0 .9X1U~ M)

O.69

Β

1.5

Β

65 A

35 A

1 U ppm (3.5X10" M)

O.72

Β

1.83

AB

55 A

25 A

DAD

4

4

5

_ 5

ABA 10 M (2.6ppm)

-

O.16C

-

a V a l u e s a r e f o r 7 d a y s p o s t t r e a t m e n t , means o f 20 e x p i a n t s p e r treatment. V a l u e s w i t h same l e t t e r 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 a t 5% l e v e l u s i n q D u n c a n ' s M u l t i p l e Range T e s t .


25 A

2


30.0±10 43.4±12 34.0±17 43.3±18

30.0±10 43.3±5 33.3±3 36.6±9

30 .0±10 46 .6±15 36 .7±6 36 .7±9

1.65±O.02 O.98±O.14 O.75±O.11 O.72±O.10

1. 18±O.15 O. 2 4 ± O . 0 7 O. 21±O.09 O. 1 9 ± O . 0 8

O.21±0 .06

O.24±0 .09

O.19±1 .06

1 ppb

5 ppb

10 ppb

2 4°

O.91±0 .03

b

a

V a l u e s a r e mean ± S.E. f o r t h i r t y 2 -node e x p i a n t s , t e n p e r r e p l i c a t e . A l l a p i c e s were 4 cm l o n g a t s t a r t o f t r e a t m e n t , Between Days 14 and 2 4 e x p i a n t s were t r a n s f e r r e d t o f r e s h medium n o t c o n t a i n i n g Chlorosulfuron.

Treatment

Control

3

10

1 4

Shoots

Expiants

2 4°

% New

Two-Node

Posttreatment

verticillata

1 4

Shoots

Days

on G r o w t h o f H y d r i l l a

L e n g t h o f New

of Chlorosulfuron

10

Effect

TABLE XI

370


sulfometuron stopped shoot elongation. With the exception of subterranean turion ("tuber") production, one could view the explants as wholeplant "analogues" which are easily manipulated and which require much less space, time and resources to use. It may even be possible, with the correct plant growth regulator ratios, to induce tuber production on expiants. Furthermore, with sufficient care in sterilizing hydrilla tubers as a source, one could maintain axenic expiants. However, i t is important to be aware of variability which can affect the responses of expiants. These include external variables such as culture condition (temperature, light, day length, nutrient medium) and endogenous variables such as plant vigor, plant age, position on shoot (i.e. where the expiant was removed relative to the apical meristern) and internode length. By using "standard" (consistent) sets of external and endogenous conditions, reproducible responses are obtained. Ultimately, i t is important to develop explant bioassays which can help quantify allelochemicals. This will require greater availability of purified active products. However, at this time, the expiant systems can be used to shew qualitative effects whether in crude extracts, leachates or from HPLC fractionation (17). Once target species effects are characterized, other bioassays which may be more sensitive could be used as well. Acknowledgments I am indebted to Dr. Kenneth Stevens and Ms. Gloria Merril, USDA/ARS, Western Regional Research Center, for providing DAD and solstitialin. The technical assistance of Ms. Doreen Gee and Mr. Nathan Dechoretz is greatly appreciated. Literature Cited 1. Anderson, L.W.J. et al. USDA/ARS Aquatic Weed Research Planning Conference Report. 1982. 30pp. 2. Sculthorpe, C.D. The Biology of Aquatic Vescular Plants 1967, Edward Arnold, Ltd., London, p.16-20 3. Yeo, R.R. J. Aquat Plant Manage. 1984. 22, 4. Frank, P.Α., Dechoretz. N. Weed Sci. 1980 28, 499 5. Hoagland, D.R., Arnon, D.I. Calif. Agric. Exp. Stn. Circ. 347 1950 6. Stevens K.L.,Merrill, G.B. J. FoodChem.1980, 28, 644 7. Stevens K.L., Merrill, G.B. Experimentia 198137,1133 8. Strickland, J.D.H. Parsons, T.R. Handbook of Seawater Analysis, Fish. Res.Board Can. Bull.No.167 1968,311pp 9. Ray, T.B. Weed Sci. Soc. Amer. 1984 Abstract No. 223. 10. Anderson, L.W.J. Science 1978 201,1135 11. Phillips, I.D., Ann. Rev. Plant Physiol. 1975 26, 341 12. Anderson, L.W.J."Weed Sci: 1981 29 723 13. Bartels, P.G,. Watson, C.W. Weed Sci. 1979 26, 198 14. Evans, M.L. Ann. Rev. Plant Physiol. 197526,241 15. Mitchell, J.W., Livingston, G.A., Agriculture Handbk,NO.336U.S.D.A. 1968, 140 pp. 16. McClure, J.W. "Secondary Constituents of Aquatic Angiosperms"in Photochemical Phylogeny, J.B. Harborne (ed.) 1970 Academic Press, N.Y. 335 pp. 17. Martin, D.F.Misc.Report No.A-883-2 U.S. Army Corps of Engineers 1983, 37 pp. RECEIVED July 31, 1984


Use of Bioassays for Allelochemicals in Aquatic Plants - ACS

Recommend Documents