Fungicide Chemistry - American Chemical Society

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3 Uptake and Translocation of Carbon-14-Labeled Fungicides in Cereals Macro- and Microautoradiographic Studies

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Fritz Führ Institute of Radioagronomy, Nuclear Research Center Jülich GmbH, D-5170 Jülich, Federal Republic in Germany

Insecticides played the dominant role in plant protection until about 1960 when they were displaced in total application by herbicides. Herbicides were especially important in reducing wage-intensive work and resulted in a considerable increase in agricultural crop produc­ tion. As crops were planted more densely, infestation pressure from fungal diseases also increased and therefore fungicides have become increasingly important. New fungicides like azole compounds, with a wide spectrum of antimycotic and fungicidal activity (1-7) have been introduced and have replaced some of the fungicide standards such as the mercury seed dressing products. These new fungicides used as seed dressings are effective at low dosages and protect the plants from infection by certain seed- and soil-borne plant pathogens as well as against early season infections of powdery mildew and rust fungi (8, 9). Because of improved vigor, the small grain plant de­ velops additional earbearing stalks (10-13). In addition, treatment with these new fungicides during the last phase of grain filling protects the flag leaf and allows the plant to assimilate over a long period of time, so that the genetically determined productivity of the plant can be fully exploited.

0097-6156/86/0304-0053S06.00/0 © 1986 American Chemical Society

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

54

FUNGICIDE CHEMISTRY

U t i l i z a t i o n of Radiocarbon

To improve our use

C.

o f t h e s e new

f u n g i c i d e s , d e t a i l e d s t u d i e s need t o

be conducted t o determine the l e a f o r r o o t uptake and

transport

c e s s e s as w e l l as the d i s t r i b u t i o n mechanisms o f the new and/or

their

metabolic

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other plant b a r r i e r s . tion

i n the

phloem. largely

two

compounds through c e l l s ,

transport

systems of

I n the xylem ( l i g n i f i e d takes

place

i n the

from the r o o t t o the l e a f .

are

the

fungicides

membranes

and

on t r a n s l o c a ­

p l a n t , the

xylem and

the

c e l l s ) , w a t e r and n u t r i e n t t r a n s p o r t

d i r e c t i o n of

the

t r a n s p i r a t i o n stream,

I n the phloem ( l i v i n g

c e r t a i n plant n u t r i e n t s , sugars, pounds

cell

Recent s t u d i e s have c o n c e n t r a t e d

pro­

amino a c i d s and

d i s t r i b u t e d throughout

the

plant

transport

but

l e a v e s o r r o o t s i n t o the seeds o r o t h e r s t o r a g e

tissue),

p l a n t - s p e c i f i c com­ mainly

organs.

from The

the

distri­

b u t i o n and u l t i m a t e p l a n t a r e a p r o t e c t e d by a f u n g i c i d e i s d e t e r m i n e d by the e f f e c t i v e t r a n s p o r t a t i o n i n b o t h systems. and

The

t r a n s l o c a t i o n mechanisms have been d i s c u s s e d

Peterson

p o s s i b l e uptake

by

Edgington

( 1 4 ) , Crowdy (T5) and Buchenauer ( 1 6 ) .

Successful

a n a l y s i s of

small

q u a n t i t i e s of

the

chemical

t r a n s l o c a t e d r e q u i r e s the use of r a d i o a c t i v e i s o t o p e s The I n s t i t u t e of Radioagronomy has been c a r r y i n g out 14 C-labelled 18).

The

active plant

protection

substances

for

chemists,

phytopathologists,

i n the r a d i o i s o t o p e t e c h n i q u e s

being 14 such as C. studies with

13

r e s u l t s i n d i c a t e that intensive cooperation

protection cialists

years

cial

experimental include

facilities

practically

at

the

oriented

(17,

between p l a n t

phytophysiologists

and

spe­

i s necessary to f u l l y e x p l o i t

the a p p l i c a t i o n p o s s i b i l i t i e s and t o i n t e r p r e t the r e s u l t s .

which

and

Jillich

Nuclear

Research

spe­

Center

t e s t s supplemented by

de­

t a i l e d s t u d i e s under d e f i n e d c l i m a t i c c o n d i t i o n s enable p r a c t i c a l

and

r e l e v a n t r e s u l t s t o be

obtained

t i o n i s t o p r o v i d e new

i n s i g h t s and

t i v e n e s s and r e s i d u e b e h a v i o r

field

The

(17-19).

The

aim

of t h i s

contribu­

i n f o r m a t i o n on the system e f f e c ­

of azole f u n g i c i d e s .

The f o l l o w i n g t h r e e a c t i v e s u b s t a n c e s were used i n t h i s s t u d y (Figure 1): [benzene r i n g - U - ^ C ] t r i a d i m e n o l , [benzene r i n g - U - ^ C ] 14 t r i a d i m e f o n and [ C ] f l u o t r i m a z o l e . These s t u d i e s are i n c l u d e d as

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

Ο

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Triadimefon

N

Cl—( C I - ^ •* ^ )-- 0 --CÇH — C — C ( C H ) 3

3

u 1 -(4-Chlorphenoxy)-3,3-dimethyl-1 -(1,2,4-triazol-1 -yl)-2- butanon

OH Triadimenol

C

// l

^ Ο

- CH - CH - C(CH

3

1 -(4-Chlorphenoxy)-3,3-dimethyl-1 -(1H-1,2,4-triazoM -yl)-2- butanol

11 CF

3

Bis-phenyl-(3-Trif luormethylphenyl)-( 1,2,4,Triazolyl)-methan

F i g u r e 1. F u n g i c i d a l tions (*).

substances a p p l i e d

and ^ C - l a b e l i n g

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

posi­

FUNGICIDE CHEMISTRY

56

a c t i v e components i n t h e commercial p r e p a r a t i o n s B a y t a n , B a y l e t o n and Persulon, respectively.

C e r e a l Seed D r e s s i n g w i t h

Triadimenol 14

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The

uptake and d i s t r i b u t i o n

o f [benzene r i n g - U -

C ] t r i a d i m e n o l ap­

p l i e d as a seed d r e s s i n g i n s p r i n g b a r l e y and s p r i n g wheat was i n v e s ­ t i g a t e d under f i e l d a g r a r i a n ecosystems

c o n d i t i o n s (19). L y s i m e t e r experiments i n s m a l l 2 2 (0.25 - 1 m ) f i l l e d w i t h e i t h e r t o p s o i l (1 m

lysimeter) or w i t h undisturbed

s o i l c o r e s from a r a b l e l a n d were used

(20, 2 1 ) . The t e s t s o i l was a l o e s s loam (parabrown s o i l , w h i c h i s among t h e most f e r t i l e Germany ( 2 2 ) .

alfisol)

s o i l i n t h e whole F e d e r a l R e p u b l i c o f

C a l c u l a t i o n using the s p e c i f i c a c t i v i t y o f the l a b e l e d

f u n g i c i d e i n d i c a t e d t h a t a seed d r e s s i n g a p p l i c a t i o n o f 16 o r 12 yg a c t i v e substance/grain /100

(Table I ) corresponded t o 177 o r 160 g Baytan

kg seed (19).

T a b l e I : R a d i o a c t i v i t y and Q u a n t i t y o f A c t i v e Substance on t h e Seed G r a i n a f t e r D r e s s i n g w i t h 14 [benzene r i n g - U C]Triadimenol Crop Spring Barley

S p r i n g Wheat 30.0

36.2

Thousand g r a i n w e i g h t (g) R a d i o a c t i v i t y / g r a i n * (μ C i )

1.08

0.83

Active substance/grain*

0.016

0.012

(mg)

A c t i v e s u b s t a n c e / k g seed g r a i n (mg)

442

400

Baytan 25 DS/100 kg seed g r a i n (g)

176.8

160

*Mean v a l u e s o f 50 g r a i n s

The

distribution

u s i n g X-ray f i l m s causes b l a c k e n i n g s

of r a d i o a c t i v i t y i n

(macroautoradiographs). on t h e f i l m .

During

t h e p l a n t was determined The r a d i o a c t i v e r a d i a t i o n

t h e e a r l y development up t o

shoot e l o n g a t i o n , a maximum o f 7.5% o f t h e r a d i o a c t i v i t y a p p l i e d was t a k e n up and t r a n s l o c a t e d i n t o t h e wheat s t a l k s and l e a v e s w i t h t h e

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

3.

14

C-Labeled

FUHR

57

Fungicides in Cereals

m a j o r i t y o f the t r a n s l o c a t i o n o c c u r r i n g between the t e n t h and eighth

day a f t e r

p l a n t s show t h a t

sowing.

These m a c r o a u t o r a d i o g r a p h s

twenty-

o f t h e wheat

t h e r a d i o a c t i v e substance o f t h e m e t a b o l i t e s

were

t r a n s l o c a t e d i n t o the l e a f t i p s w i t h the t r a n s p i r a t i o n s t r e a m ( F i g u r e 2). By means o f s p e c i a l f i l m , the r a d i o c a r b o n Downloaded by UNIV OF CALIFORNIA SAN DIEGO on November 5, 2015 | http://pubs.acs.org Publication Date: April 22, 1986 | doi: 10.1021/bk-1986-0304.ch003

p l a n t c e l l s can a l s o be d e t e c t e d duced w i t h the the

o f t r i a d i m e n o l i n the

i n t h i n t i s s u e s e c t i o n s (8 ym) p r o ­

f r e e z i n g microtome.

Blackening

on the

film

reflects

p o s i t i o n o f the r a d i o a c t i v e substances i n the p l a n t c e l l s

24).

These

microautoradiographs

(Figure

3) show t h a t

s w e l l i n g phase o f the g r a i n s r a d i o a c t i v i t y m i g r a t e s t u d i n a l and c r o s s c e l l s o f the p e r i c a r p . through the t e s t a i n t o the a l e u r o n e t e i n s t o r a g e l a y e r i n the g r a i n .

during the

i n t o the l o n g i ­

Only a s m a l l amount moves

l a y e r , w h i c h i s l a r g e l y the

tein

from

the aleurone

ently

layer.

According

t o these

the f i r s t week o f g e r m i n a t i o n

an e f f e c t i v e

barrier

pro­

D u r i n g the f i r s t s i x days o f g e r m i ­

n a t i o n , s t a r c h i s broken down i n t o sugar and m o b i l i z e d w i t h the

graphs, during

(23,

against

microautoradio­

the p e r o c a r p

the penetration

pro­

i s appar­

o f the a c t i v e

substance i n t o the i n t e r i o r o f the g r a i n . Triadimenol tiles.

uptake

can occur

by c o n t a m i n a t i o n

The r a d i o a c t i v e seed d r e s s i n g was taken

t h r e e days o f g e r m i n a t i o n

o f the coleop-

up d u r i n g

the

first

o f the wheat s e e d l i n g ( F i g u r e 4a) and

could

then be found i n a l l the c e l l s o f the s e e d l i n g ( 1 9 ) .

A clear d i s t r i ­

b u t i o n g r a d i e n t o f r a d i o a c t i v i t y from the o u t e r t o the i n n e r c e l l s o f the s e e d l i n g i s thus v i s i b l e . i t y can be d e t e c t e d the

T h i r t y days a f t e r sowing, r a d i o a c t i v ­

i n c e l l s o f the l e a f t i p s ( F i g u r e 4b)

a c t i v e substance

and/or

i t s metabolites

indicating

has m i g r a t e d

with the

t r a n s p i r a t i o n f l o w a f t e r uptake i n the t r a n s p o r t t i s s u e (xylem) f o l ­ lowed by t r a n s l o c a t i o n i n t o n e i g h b o r i n g c e l l The

dressed

g r a i n s were sown i n the s o i l a t a depth o f 3-4 cm.

S w e l l i n g and g e r m i n a t i o n the ambient s o i l .

regions.

f o l l o w e d as a r e s u l t o f a b s o r b i n g water from

I n t e r i m r e s u l t s a t v a r i o u s p o i n t s i n the

ment showed t h a t d u r i n g

the

substance o n l y s u p e r f i c i a l l y a t t a c h e d ing,

develop­

s w e l l i n g phase up t o 80% o f the a c t i v e

t o the g r a i n d u r i n g the d r e s s 14 was r a p i d l y t r a n s f e r r e d i n t o the s o i l . Special C-analyses o f

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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FUNGICIDE CHEMISTRY

0.5—h 0.1

I .

' '

1,0-10.1 ,

.... ~ A 1

-1.0 /'

„._'-*· "•-22,1 0Λ —

0.3-

Developmental stages

A 10

2 5

5

V- · -0.7

Β 15

24.7 0.4

C 21

\

A —19.4

Τ D 28

0 4

— 0.1 »

/

E-F 38

11.5 M

days

F i g u r e 2. M a c r o a u t o r a d i o g r a p h s o f s p r i n g wheat p j a n t s i n v a r i o u s s t a g e s o f development a f t e r seed d r e s s i n g w i t h [ C]Baytan. (Reproduced w i t h p e r m i s s i o n from R e f . 19. C o p y r i g h t 1982 P f l a n z e n s c h u t z - N a c h r i c h t e n Bayer.)

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

FUHR

14

C-Labeled Fungicides in Cereals

59

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3.

F i g u r e 3. E n l a r g e d s e c t i o n s o f m i c r o a u t o r a d i o g r a p h s o f f r e e z i n g microtome c r o s s s e c t i o n s through wheat g r a i n s a f t e r seed d r e s s i n g w i t h [ CJBaytan ( i n t e r f e r e n c e phase c o n t r a s t p i c t u r e s ) , ( a ) Schematic (25) o f wheat g r a i n c e l l s t r u c t u r e s . Key: F, p e r i c a r p ; S, t e s t a ; AL, aleurone l a y e r ; and S t , s t a r c h y endosperm. ( b ) Wheat g r a i n s immediately a f t e r treatment w i t h f C ] B a y t a n . ( c ) Wheat g r a i n s a f t e r 6 days s w e l l i n g and g e r m i n a t i o n i n the s o i l , (d) Wheat g r a i n s a f t e r 6 days s w e l l i n g and g e r m i n a t i o n i n t h e s o i l (more g r e a t l y m a g n i f i e d ) . (Reproduced w i t h p e r m i s s i o n from Ref. 19. C o p y r i g h t 1982 P f l a n z e n s c h u t z - N a c h r i c h t e n Bayer.)

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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FUNGICIDE CHEMISTRY

F i g u r e 4. M i c r o a u t o r a d i o g r a p h s of f r e e z i n g microtome c r o s s s e c ­ t i o n s through a s p r i n g wheaj: embryo and the p r i m a r y l e a f t i p a f t e r seed d r e s s i n g w i t h [ C]Baytan ( d a r k - f i e l d m i c r o g r a p h ) , ( a ) Cross s e c t i o n through t h e embryo 3 days a f t e r s w e l l i n g , ( b ) Cross s e c t i o n through the p r i m a r y l e a f 30 days a f t e r sowing the d r e s s e d seed. (Reproduced w i t h p e r m i s s i o n from R e f . 19. Copy­ r i g h t 1982 P f l a n z e n s c h u t z - N a c h r i c h t e n Bayer.)

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

3.

14

C-Labeled

FUHR

soil

Fungicides in Cereals

segments c o n f i r m e d

61

t h a t , depending on the t e s t s o i l , a r a d i o a c ­

t i v i t y d i s t r i b u t i o n g r a d i e n t i s formed around the g r a i n up t o a d i s ­ tance o f 8 cm. A " d r e s s i n g a r e a centrations wardly

11

o f a c t i v e substance

r e s u l t s around the g r a i n w i t h c o n ­ and/or m e t a b o l i t e s

decreasing

out­

(26) .

In recent

s t u d i e s , when n o n - r a d i o a c t i v e l y d r e s s e d

winter

wheat

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g r a i n s were a l t e r n a t e l y p l a n t e d i n a s e r i e s w i t h r a d i o a c t i v e l y d r e s s ­ ed g r a i n s , r a d i o a c t i v i t y was d e t e c t e d i n the n o n - t r e a t e d

plants

indi-

C - l a b e l l e d compounds from the " d r e s s i n g

area"

14 e a t i n g an uptake o f via

the r o o t .

Uptake was about the same o r d e r o f magnitude as uptake

via

the g r a i n and s e e d l i n g ( 2 7 ) .

Only a c o m b i n a t i o n

of radioisotope

measurements and c h e m i c a l a n a l y s i s w i l l c o n f i r m whether t h i s i s s t i l l the a c t i v e s u b s t a n c e ( 2 8 ) .

I n o c u l a t i o n w i t h a p p r o p r i a t e pathogens i s

needed t o determine whether the c o n c e n t r a t i o n o f the a c t i v e substance in

the t i s s u e i s s u f f i c i e n t

t o provide

protection against

these i n ­

fections . A c o n c l u s i o n from these s t u d i e s i s t h a t c e r e a l d r e s s i n g may be a v e r y e c o n o m i c a l method t o p r e v e n t phase

o f development.

fungal plant diseases

New s y s t e m i c a l l y a c t i v e

t r i a z o l e type c o n t r o l the p a t h o g e n i c this surface.

i n the e a r l y

substances

of

Uptake o f the a c t i v e substance by the s e e d l i n g and i t s

t r a n s p o r t i n t o the e p i g e a l p a r t s o f the p l a n t r e s u l t s i n the t i o n o f t h e s e e d l i n g and the p r i m a r y borne f u n g a l pathogens. r e s u l t s o f chemical

leaf

from b o t h s o i l -

protec­ and a i r ­

T h i s d e t a i l e d i n f o r m a t i o n combined w i t h t h e

and r a d i o c h e m i c a l a n a l y s e s

o f t h i s type can

v i d e i n f o r m a t i o n t o a i d i n the improvement o f seed d r e s s i n g t i o n s and t e c h n i q u e s priate

the

f u n g i i n the seed as w e l l as on

uptake i s most

formula­

w i t h the o b j e c t i v e o f a p p l y i n g o n l y the

amount o f a c t i v e substance

pro­

and a t t h e l o c a t i o n where

appro­ plant

probable.

Uptake o f T r i a d i m e f o n v i a the Leaves

I f s p r a y i n g i s c a r r i e d out d u r i n g the p e r i o d o f i n t e n s i v e l e a f d e v e l ­ opment the

(e.g.

stalks

during

tillering

and e s p e c i a l l y d u r i n g

i n c e r e a l s ) , i t i s almost i m p o s s i b l e

development o f

t o achieve

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

uniform

FUNGICIDE CHEMISTRY

62 distribution

on

the

plant

surfaces.

i n t e r n a l t r a n s p o r t of a new is

important,

i n order

trum o f a c t i v i t y . fungal

diseases

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infection

f u n g i c i d e w i t h the a i d of

to f u l l y

the

the

plant

sowing. plant

on

as

w e l l as

spec­

e f f e c t can

eliminate

the

C-labelling

e x p l o i t t h e i r p o t e n t i a l broad

combat

pathogens a f t e r

a c t i v e substance must p e n e t r a t e

t i s s u e and be t r a n s p o r t e d t o n o n - t r e a t e d The

information 14

Only compounds w i t h a s y s t e m i c

on

since

Therefore,

i n t o the

plant

plant parts.

e f f e c t o f a seed d r e s s i n g l a s t s o n l y about 5-7

weeks a f t e r

Therefore,

protect

i n the

bear e a r s .

a

later

foliar

stages

of

A fungicidally

a p p l i c a t i o n i s needed

to

development e s p e c i a l l y as

the

the

tillers

a c t i v e substance used s u c c e s s f u l l y as

a

f o l i a r treatment i s t r i a d i m e f o n ( F i g u r e 1 ) , a n o t h e r compound o f the a z o l e group ( 1 , 3, 4 ) . To study the uptake and t r a n s p o r t o f [benzene 14 ring-U225

C]triadimefon

i n b a r l e y l e a v e s , 6 - 14 yg ΑΙ/leaf ( e q u a l

g ΑΙ/ha) were a p p l i e d

(29).

A p p l i c a t i o n was

f a c e when the

in strips

to

the

second

developing

to

leaf

made t o the b a s a l p a r t o f the upper l e a f s u r ­

l e a f was

almost

completely

developed.

The

upper l e a f p a r t s were s h i e l d e d w i t h a p l a s t i c s c r e e n take of r a d i o l a b e l l e d m a t e r i a l v i a the gas

untreated

to prevent

up­

phase.

M a c r o a u t o r a d i o g r a p h s ( F i g u r e 5) of the l e a v e s were t a k e n a t v a r i o u s times a f t e r t r i a d i m e f o n a p p l i c a t i o n and c l e a r l y showed t r a n s 14 l o c a t i o n of gions.

C - l a b e l l e d compounds i n t o the

untreated

the l e a f t i p s w i t h i n 12 days.

51%

t i p s as l a t e r confirmed

of p r e c i p i t a t i o n and

treated

the

by Buchenauer and

dew

formation

strip

on

l e a f s e c t i o n as w e l l as protected

siphe graminis var. h o r d e i ) . mildew was

observed i n the

although

Roehner ( 2 8 ) .

The

greenhouse r e s u l t s demonstrates the

against At

the

leaf

differ­ influence

the p e r s i s t e n c e o f the a c t i v e

l e a f as w e l l as i t s uptake and

remained c o m p l e t e l y

development) were

of the a c t i v e substance t r a n s l o c a t e d to the

ence between f i e l d and

substance on

found i n

However, i n the greenhouse where e n v i ­

r o n m e n t a l c o n d i t i o n s ( t e m p e r a t u r e , m o i s t u r e and dew more c o n s t a n t ,

The

leaf t i p re­

Under f i e l d c o n d i t i o n s , 28% of the r a d i o a c t i v i t y was

the

internal

untreated

transport.

upper l e a f

i n f e s t a t i o n w i t h mildew same t i m e ,

protection

part (Ery-

against

lower l e a f r e g i o n below the t r e a t e d l e a f

r e t r a n s l o c a t i o n i n t o t h i s p a r t amounted t o a maximum

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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3.

FUHR

14

C-Labeled

dayO

63

Fungicides in Cereals

day 1

day 3

day 6

day 12

F i g u r e 5. T r a n s l o c a t i o n o f r a d i o a c t i v i t y i n s p r i n g b a r l e y a f t e r t r e a t i n g the upper s i d e o f the lower h a l f o f the l e a f w i t h [ C]triadimefon (macroautoradiographic r a d i o a c t i v i t y d e t e c t i o n ) . (Reproduced w i t h p e r m i s s i o n from Ref. 19. C o p y r i g h t 1978 P f l a n z e n s c h u t z - N a c h r i c h t e n Bayer.)

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

FUNGICIDE CHEMISTRY

64 of o n l y 0.3%

of the a p p l i e d r a d i o a c t i v i t y .

e f f e c t or r e d u c t i o n i n i n f e s t a t i o n was

However, the p r o t e c t i v e

c o n s i d e r a b l y weaker i n

these

untreated basal regions. Microautoradiographs ing

showed

the

of the b a r l e y l e a f immediately

r a d i o a c t i v e substance

on

the

after

epidermis

(Figure

However, a s e q u e n t i a l time s e r i e s of m i c r o a u t o r a d i o g r a p h s Downloaded by UNIV OF CALIFORNIA SAN DIEGO on November 5, 2015 | http://pubs.acs.org Publication Date: April 22, 1986 | doi: 10.1021/bk-1986-0304.ch003

leaf are

sections

(8 ym)

r a p i d l y taken

shows t h a t

up

(Figure

almost e x c l u s i v e l y w i t h

the

spray­ 6).

of c r y o s t a t

t r i a d i m e f o n and/or i t s m e t a b o l i t e s

7).

Subsequent t r a n s p o r t

t r a n s p i r a t i o n flow

i n the

takes

place

xylem.

This

t r a n s p o r t system i s then r a p i d l y abandoned a g a i n so the a c t i v e sub­ stance

and/or m e t a b o l i t e s

move out of the t r a n s p o r t a t i o n stream

a r e found i n a l l l e a f c e l l s If

the

basal third the

triadimefon

spraying

of

i n c l u d i n g the

the

transverse

leaf

transport

reaches

the

leaf

t h r o u g h the

newly developed l e a v e s i s o b s e r v e d . 20%

leaf

upper

sheath

In t h i s way,

untreated

substance or i t s m e t a b o l i t e s

leaves.

The

surface

into up t o

the

of

the

leaf

can

the then

adjacent

approximately

can m i g r a t e

into

r e l a t i v e l y mobile a c t i v e

then are r a p i d l y t r a n s p o r t e d w i t h i n the

l e a f v i a the x y l e m , so t h a t b o t h the t r e a t e d as w e l l as the parts

of

(Figure 8),

sheath

of the a c t i v e substance and/or i t s m e t a b o l i t e s

the newly d e v e l o p i n g

and

(29).

become d e p l e t e d

with

an

untreated

accumulation

a c t i v e substance i n the l e a f t i p s and margins ( F i g u r e s 5 and

of

the

8)(29).

Residue S i t u a t i o n i n the C e r e a l G r a i n

This

one-directional transport

with

fungicide

transported

being

protected

behavior as

a

results whole.

substance must f i r s t

be

downward i n the

i t can be

e x c l u s i v e l y transported

treated

substance

is

l e a v e s ; how­

phloem of

leaf.

For f u n g i c i d a l com­

t r i a d i m e f o n , w h i c h are p r e d o m i n a n t l y i n the

the

t r a n s p o r t e d v i a the xylem to

the a p i c a l r e g i o n of the newly d e v e l o p i n g t r i a d i m e n o l and

leaf

l e a v e s i s l i m i t e d because the a c t i v e

transported

t r e a t e d developed l e a f b e f o r e

not

Active

i n t o the a p i c a l r e g i o n of newly d e v e l o p i n g

e v e r , r e d i s t r i b u t i o n i n t o these

pounds l i k e

in a

xylem,

this

type

of

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

if

transport

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FUHR

14

C-Labeled

Fungicides in Cereals

F i g u r e 6. M i c r o a u t o r a d i o g r a p h y d e t e c t i o n o f f C I t r i a d i m e f o n i m m e d i a t e l y a f t e r b e i n g sprayed onto the e p i d e r m a l c e l l s o f a b a r l e y ( l e a f c r o s s s e c t i o n u s i n g i n t e r f e r e n c e phase c o n t r a s t ) .

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

FUNGICIDE CHEMISTRY

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66

F i g u r e 7. R a d i o a c t i v i t y i n b a r l e y l e a f t i s s u e j£ter t r e a t i n g the upper s i d e of t h e lower h a l f of the l e a f w i t h [ C ] t r i a d i m e f o n (microautoradiography d a r k - f i e l d micrograph). (Reproduced w i t h p e r m i s s i o n from R e f . 19. C o p y r i g h t 1978 P f l a n z e n s c h u t z N a c h r i c h t e n Bayer.)

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

3.

14

C'Labeled

FUHR

behavior

67

Fungicides in Cereals

can be an advantage s i n c e t h e a c t i v e substance and/or meta­

b o l i t e s do n o t accumulate i n t h e d e v e l o p i n g This ^C-

i s illustrated

by d a t a

presented

i n Figure

9 (30).

The

l a b e l l e d f l u o t r i m a z o l e ( F i g u r e 1) was s p r a y e d onto s p r i n g b a r l e y 2

i n two l y s i m e t e r s (0.96 m 31). Downloaded by UNIV OF CALIFORNIA SAN DIEGO on November 5, 2015 | http://pubs.acs.org Publication Date: April 22, 1986 | doi: 10.1021/bk-1986-0304.ch003

seeds o r g r a i n .

each) u s i n g a c h r o m a t o g r a p h i c s p r a y e r ( 3 0 ,

S p r i n g b a r l e y a t t h e K-stage p r i o r t o e a r f o r m a t i o n was s p r a y e d

w i t h e i t h e r t h e a g r i c u l t u r a l use r a t e o f 125 g/ha o r a 2x r a t e (250 g/ha).

A t h a r v e s t , 57 days a f t e r t r e a t m e n t ,

about 1/3 o f t h e a p p l i e d

r a d i o a c t i v i t y was r e c o v e r e d

i n t h e s t r a w , c h a f f and g r a i n ( F i g u r e 9 ) .

A c t i v e substance e q u i v a l e n t

t o 5.3 mg/kg o f s t r a w r e s u l t e d from t h e

s i n g l e dose and about t w i c e as much a c t i v e substance accumulated i n the

residue

total

treated with

radioactive

located

t h e 2x c o n c e n t r a t i o n .

substances

i n the leaves

About

97% o f

( f l u o t r i m a z o l e and m e t a b o l i t e s )

o f the r i p e s p r i n g b a r l e y w i t h

little

the

were radio­

a c t i v i t y found i n t h e s t a l k s , c h a f f and g r a i n . The

nature

stances

and d i s t r i b u t i o n

and m e t a b o l i t e s

were

of the r a d i o l a b e l l e d active

determined

cochromatography i n c o m b i n a t i o n w i t h

using

liquid

thin

layer

scintillation

sub­

and

gas

counting.

About 76% o f t h e r a d i o a c t i v i t y found i n t h e s t r a w was f l u o t r i m a z o l e and

14% was c a r b i n o l , t h e major m e t a b o l i t e

grain,

on t h e o t h e r

hand, c o n t a i n e d

(31).

only

The awns, c h a f f and

traces

of r a d i o a c t i v i t y ,

l e s s than 0.1% o f t h e a p p l i e d r a d i o a c t i v i t y ( F i g u r e 9) w i t h t h e f l u o ­ trimazole

equivalents

mg/kg f o r b o t h spray locosystemic a very

i n the grain concentrations.

a c t i o n and i s p r o b a b l y

limited

computed

extent.

t o be l e s s

Fluotrimazole

than 0.01

displays only a

o n l y t r a n s l o c a t e d i n t h e xylem t o

The new growth, f o r example t h e e a r s ,

only

r e c e i v e s a s m a l l amount o f a c t i v e substance v i a t h e phloem. Conclusions

These examples c l e a r l y velop

fully

which migrate Transport plant.

systemic equally

show t h a t plant

i t i s n o t always d e s i r a b l e t o d e ­

protection

w e l l both

compounds,

i n t h e xylem

i . e . substances

and i n t h e phloem.

i n t h e xylem i s o f t e n s u f f i c i e n t t o e f f e c t i v e l y p r o t e c t t h e

Due t o t h e p h y s i o l o g y

o f the p l a n t , the storage

organs w h i c h

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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68

FUNGICIDE CHEMISTRY

F i g u r e 8. Uptake and t r a n s l o c a t i o n of r a d i o a c t i v i t y i n s p r i n g b a r l e y a f t e r t r e a t i n g the upper s i ^ g of the lower t h i r d of the l e a f , i n c l u d ­ i n g the l e a f s h e a t h , w i t h [ C ] t r i a d i m e f o n ( m a c r o a u t o r a d i o g r a p h i c r a d i o a c t i v i t y d e t e c t i o n ) . (Reproduced w i t h p e r m i s s i o n from Ref. 19. C o p y r i g h t 1978 P f l a n z e n s c h u t z - N a c h r i c h t e n Bayer.)

F i g u r e 9. D i s t r i b u t i o n of r a d i o a c t i v i t y and computed e c m i v a l e n t s i n r i p e s p r i n g b a r l e y 57 days a f t e r b e i n g [ C ] f l u o t r i m a z o l , sprayed r a d i o a c t i v i t y e q u a l s 100. p e r m i s s i o n from Ref. 30. C o p y r i g h t 1981 V e r l a g Eugen

a c t i v e substance treated with (Reproduced w i t h Ulmer.)

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

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3. FUHR

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C-Labeled Fungicides in Cereals

69

are used as fodder or for human nutrition are least contaminated. The translocation and accumulation of the fungicide could only be determined using the radiocarbon C active substance. The fluotrimazole results indicate that this is a very sensitive method. With the aid of radioactive labelling, it was computed that just 12.5 mg of active substance equivalents, i.e. compounds contain­ ing radiocarbon from fluotrimazole labelling, would be contained in the per hectare yield of 6,000 kg of spring barley (30, 31). Radio­ isotope labelling increases detection sensitivity to such an extent that even the slightest traces of residual carbon from an organic molecule can be quantified in the newly developing storage tissue. The active substance, with a correspondingly high specific radioac­ tivity even when applied at a very low quantity, can s t i l l be charac­ terized and identified by the combined methods of gas chromatography with mass spectrometry. For example, in an experiment where [3- C] metamitron, a triazine herbicide, had been sprayed preemergence to sugar beets (32), about 25% of the radiocarbon found in the sugar beets at harvest (188 days after spraying) had been utilized to form saccharose, probably derived from mineralized C0^ (33) . The recent studies with "C-labelled triazole fungicides attempt to gather information on residue analysis and biotests by applying macro- and microautoradiographic methods. Information about fungi­ cide uptake and transport behavior in plants, the orders of magnitude of the active fractions and indications concerning the residue situa­ tion in the plant can be evaluated. The use of specialized radioiso­ tope techniques in applied practical agriculture has been demon­ strated. These studies not only improve the application of these compounds but also assist the consumer by learning more about the residue in his food after treatment with a chemical plant protectant. Literature Cited 1.

Bűchel, K.H., Meiser, W., Kramer, W., Grewe, F., 8th Interna­ tional Congress of Plant Protection, Moscow, 1975, Section III, 111-118 (1975).

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

70

FUNGICIDE CHEMISTRY

2.

Grewe, F . , Bűchel, K.H., Mitt. Biol. Bundesanst. Land-Forstwirtsch. (Berlin-Dahlem), 59, 652-655 (1975). Kaspers, Η., Grewe, F . , Brandes, W., Scheinpflug, Η., Bűchel, K.H., 8th International Congress of Plant Protection, Moscow, 1975, Section III, 398-401 (1975). Bűchel, K.H., Kramer, W., Meiser, W., Brandes, W., Frohberger, P.E., Kaspers, H., 9th International Congress of Plant Protec­ tion, Washington, DC, 1979, Abstract of Paper No. 475 (1979). Frohberger, P.E., Pflanzenschutz-Nachr. Bayer (Ger. Ed.) 31, 11-24 (1978). Brandes, W., Kaspers, Η., Kramer, W., Pflanzenschutz-Nachr. Bayer (Ger. Ed.) 32, 1-16 (1979). Bűchel, K.H., Plempel, Μ., In: J.S. Bindra, D. Lednicer (Eds.); Chronicles of Drug Discovery, Vol. 2, 235-269, John Wiley & Sons Inc. (1983). Kuck, K.H., Scheinpflug, H., Tiburzy, R., Reisener, H.J., Pflanzenschutz-Nachr. Bayer (Ger. Ed.) 35, 209-228 (1982). Smolka, S., Wolf, G. Pflanzenschutz-Nachr. Bayer (Ger. Ed.) 36, 97-128 (1983). Kolbe, W., Pflanzenschutz-Nachr. Bayer (Ger. Ed.) 31, 38-59 (1978). Főrster, Η., Buchenauer, Η., Grossmann, F . , Z. PflKrankh. PflSchutz 87, 473-492 (1980). Kolbe, W., Pflanzenschutz-Nachr. Bayer (Ger. Ed.) 35, 72-103 (1982). Rawlinson, C . J . , Muthyalu, G., Cayley, G.R., Plant Pathol. 31, 143-155 (1982). Edgington, L.V., Peterson, C.A., In: M.R. Siegel, H.O. Sisler (Eds.): Antifungal Compounds, Vol. 2, 51-89, Marcel Dekker Inc., New York-Basel (1977). Crowdy, S.H., In: R.W. Marsh (Ed.): Systemic Fungicides, 92-114, Longman, London-New York (1977). Buchenauer, H., Untersuchungen zur Wirkungsweise und zum Verhalten verschiedener Fungizide in Pilzen und Kulturpflanzen. HabilSchrift Univ. Bonn (1979).

3.

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on November 5, 2015 | http://pubs.acs.org Publication Date: April 22, 1986 | doi: 10.1021/bk-1986-0304.ch003

4.

5. 6. 7.

8. 9. 10. 11. 12. 13. 14.

15. 16.

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

3.

17.

18.

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on November 5, 2015 | http://pubs.acs.org Publication Date: April 22, 1986 | doi: 10.1021/bk-1986-0304.ch003

19.

FUHR

14

C-Labeled Fungicides in Cereals

71

Fűhr, F., In M. L'Annuziata, J.O. Legg (Eds.); Isotopes and Radiation in Agricultural Sciences Vol. 2, 239-270, Academic Press London (1984). Fűhr, F., Rhein.-Westfael. Akademie der Wissenschaften, Vortraege Ν 326, 7-48, Westdeutscher Verlag Opladen (1984). Steffens, W., Fűhr, F., Kraus, P., Scheinpflug, Η., Pflanzenschutz-Nachr. Bayer (Ger. Ed.) 35, 171-188 (1982).

20.

Fűhr, F., In: Agrochemicals: Fate in Food and the Environment, IAEA Vienna, 99-111 (1982).

21.

Fűhr, F., Cheng, H.H., Mittelstaedt, W., Landw. Forsch. SH 32, 272-278 (1976). Műckenhausen, Ε., Entstehung, Eigenschaften und Systematik der Bőden der Bundesrepublik Deutschland, DLG-Verlag Frankfurt (1977).

22.

23. 24.

25. 26. 27. 28. 29. 30. 31. 32. 33.

Fűhr, F., Wieneke, J., Angew. Bot. 47, 97-106 (1973). Wieneke, J., Fűhr, F., In: W.P. Duncan, A.B. Susan (Eds.), Synthesis and Applications of Isotopically Labelled Compounds. Proc. Int. Symp. Kansas City, MO, USA, 6-11 June 1982, Elsevier Scientific Publishing Co., Amsterdam, 373-374 (1983). Biebl, R., Germ. Η., Praktikum der Pflanzenanatomie, Springer, Wien-New York (1967). Thielert, W., Steffens, W., Fűhr, F., Kuck, K.H. Pflanzenschutz-Nachr. Bayer (Ger. Ed.), 1985 (in preparation). Thielert, W., Steffens, W., Fűhr, F., Scheinpflug, H. Pflanzenschutz-Nachr. Bayer (Ger. Ed.), 1985 (in preparation). Buchenauer, Η., Roehner, Ε., Z. PflKrankh. PflSchutz 89, 385-398 (1982). Fűhr, F., Paul, V., Steffens, W., Scheinpflug, Η., Pflanzenschutz-Nachr. Bayer (Ger. Ed.) 31, 116-131 (1978). Steffens, W., Wieneke, J., Z. PflKrankh. PflSchutz 88, 343-354 (1981). Wieneke, J., Steffens, W., Z. PflKrankh. PflSchutz 88, 385-399 (1981). Mittelstaedt, W., Fűhr, F., Landw. Forsch. SH 37, 666-676 1981. Műller, L . , Mittelstaedt, W., Pfitzner, J., Fűhr, F., Jarczyk, H.J., Pestic. Biochem. Physiol. 19, 254-261 (1983).

RECEIVED October 1, 1985

In Fungicide Chemistry; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.