7th international congress of pesticide chemistry

Pestic. Sci. 1990, 30, 321-366

7th International Congress of Pesticide Chemistry

The following are extended summaries of papers and posters presented at the 7th International Congress of Pesticide Chemistry ( IUPAC) held on 5-10 August 1990 at Hamburg, Germany. The papers published here are entirely the responsibility of the authors and do not reflect the uiews of the Editorial Board of Pesticide Science.

Phosphonous peptides in the Regulation of Acetyl-CoA Formation

Alexander I. Biryukov, Irina G. Vasilyeva, Yurii N. Zhukov, Elena N. Khurs & Radii M. Khomutov

Engelhardt Institute of Molecular Biology of the USSR Academy of Sciences, Moscow, USSR

The dipeptide alaphosphalin (L-alanyl-L-1 -aminoethylphosphonic acid; Ala-Ala-P) is known to act by interfering with the biosynthesis of bacterial cell walls (as with penicillin, Dcycloserine and some other antibiotics), the true active agent being L-1- aminoethylphosphonic acid (Ala-P).' This summary reports an investigation of the influence of Ala-Ala-P and the related dipeptides L-alanyl-L- 1 -aminoethylphosphonous acid (Ala-Ala-Ph,), ~-alanyl-~-l-amino-2-hydroxy-ethylphosphonic acid (Ala-Ser-P) and ~-alanyl-~-l-amino-2-hydroxyethylphosphonous acid (Ala- Ser-P,), on other metabolically important processes in bacteria and in fungi. No record was found in the literature of Ala-Ala-P action other than inhibition of D- Ala and D-Ala-D-Ala biosynthesis in bacteria.'

The synthetic dipeptide Ala-Ala-P, also possesses antibacterial activity; protein biosynthesis is inhibited by I-aminoethylphosphonous acid (Ala-PHI produced from Ala-Ala-P, as a result of hydrolysis (aminopeptidase activity) within the cell.' No substantial antibacterial activity was found for Ala-P in uiuo, probably because of low rates of uptake into and transport within the cell, although Ala-P, acts similarly to Ala-Ala-P, .'

It has been shown recently that Ala-P, is a new, strong inhibitor of melanin synthesis in the phytopathogenic fungus Pyricularia oryzae and that it inhibits conidial and mycelial g r ~ w t h . ~ Further investigations4 suggested that Ala-P, undergoes intracellular transamination to the phosphonous analogue of pyruvate (Pyr) acetylphosphinate (Ac-PH) which inhibited oxidative decarboxylation. A

32 1

Pestic. Sci. 0031-613X/90/$03.50 0 SCI, 1990 SCI. Printed in Great Britain

322 7th International Congress of Pesticide Chemistry

TABLE 1 Antimicrobial and Enzyme Inhibitory Activities of Phosphoanalogues of Pyruvate, Alanine,

Serine and Their Dipeptides

Compound Target

Ac-PH Ac-P Ala-PH Ala-P Ser-P Ser-P,

Ala-Ala-P Ala-Ser-P Ala-Ser-P,

Ala-Ala-P,

Inhibition of pyruvate

dehydrogenase 1 5 0 a

(PSml-')

3 x 10-7 (0.05) 4 x (0.5)

Inactive Inactive Inactive Inactive Inactive Inactive Inactive Inactive

Inhibition of pyruvate

dehydrogenase afer incubation

with E. coli ISO0

(pg mir ' 1

3 x 10-7 (0.07) 4~ (0.6) 6 x (6)d 5 x (6)d

Inactive 5 x 10-5 (5)' s x 10-5 (10)s 5 x 10-5 (ioy

5 x 10-5 Inactive

Pyricularia oryzae

Mycelial Melanine growth synthesis M I C ~ M I C ~

(w mlr 1 (pg ml- '1

Inactive Inactive

Inactive Inactive

1/1 OOO'J

lO/loOok 5 1 9

50/50k

10175 100/750

Inactive Inactive 0.1/10

Inactive Inactive 10/100 1/1

Inactive' 100/1000 1011 0

Inactive Inactive

0.4' Inactive Inactive

Not tested 0.1" 0.5"

Inactive Not tested

a Figures in parentheses are in terms of molarity. Minimum inhibitory concentration. In minimal medium; Ref. 2.

d , e ~ f , ~ , h After incubation for 3, 4, 5, 6 or 10 h respectively. ' In minimal and complete medium respectively. 'Conidial growth inhibited at 5/100 pg m1-I.

Conidial growth inhibited at 1.111.5 pg m1-l. Conidiophores decolorised in minimal medium at 50 pg m1-I. " In minimal medium; Ref. 1.

similar mode of Ala-P, action was suggested for the inhibition of anthocyanin synthesis.'

Extracts of the substrate from incubation of dipeptides with Eschericia coli, except for that from Ala-Ser-P, effectively inhibited oxidative decarboxylation of Pyr and thus influenced the Acetyl-CoA level (Table 1). However, the dipeptides themselves did not inhibit the purified pyruvate dehydrogenase (PDG) extracted from E . coli. It seemed most probable that the inhibition of PDG was due, in the first instance, to hydrolysis of the dipeptides by aminopeptidase, yielding the corresponding amino acids. The enzyme activity was inhibited only slightly by the alanine and serine analogues at 1 mM but the inhibitory activity of the E . coli extract was much greater when Ala-P,, Ala-P and Ser-P, were incubated for a suitable length of time (Table 1). The obvious explanation of this effect is the enzymatic transformation of the amino analogues into keto derivatives, similar to alanine and serine transformations (leading to Pyr) in the same system. It is probable that Ac-PH and Ac-P were formed, either from Ala or Ser phosphoanalogues in the course of their transamination or deamination as suggested by (1) the existence of

7th International Congress of Pesticide Chemistry 323

transaminase and deaminase activity in E . coli, (2) the absence of activity in the substrates containing alanine and serine phosphoanalogues in the alanine and serine dehydrogenase reactions and (3) the existence of antagonism between the alanine analogues and amino-oxyacetic acid.

In the case of E . coli, it seems improbable that Ala-P, can inhibit protein biosynthesis at the stage of alanine activation by alanyl-tRNA synthetase, as was proposed in Ref. 2, because this analogue has been shown in this laboratory to be a poor competitive inhibitor of this enzyme (Ki for Ala-P,= 2.5 x M and for Ala- P,=g-Ox 10-3 M).

Thus the dipeptides were transformed into the Pyr phosphoanalogues, the important inhibitors of oxidative decarb~xylation.~-'.

References 1 . Allen, J . G., Atherton, F. R., Hall, M. J. , Hassall, C. H., Holmes, S. W., Lambert, R. W.,

2. Dingwall, J. G., Abstr. 3rd International Conference on Chemistry and Biotechnology of

3. Khomutov, R. M., Khurs, E. N., Dzhavia, V. G., Voinova, T. M. & Ermolinsky, B. S .

4. Biryukov, A. I., Vasilyeva, I. G., Zhukov, Yu. N., Khurs, E. N. & Khomutov, R. N.,

5 . Laber, B. & Amrhein, N., Biochem. J . , 248 (1987) 351-8. 6. Kluger, R . & Pike, D. C., J . Amer. Chem. SOC., 99 (1977) 4504-6. 7. Birukov, A. I., Vasilyeva, I. G., Zhukov, Yu. N., Khurs, E. N. & Khomutov, R. M. In

Proceedings Symposium 'Prospects for Amino Acid Biosynthesis Inhibitors in Crop Protection and Pharmaceutical Chemistry', ed. L. G. Copping, J. Dalziel & A. D. Dodge, British Crop Protection Council, Farnham, 1989, pp. 213-15.

8. Baillie, A. C., Wright, K., Wright, B. J. & Earnshaw, C . G., Pestic. Biochem. Physiol., 30

Nisbet, L. J. & Ringrose, P. S., Nature (London), 272 (1978) 56-8.

Biologically Active Natural Products, Sofia, Bulgaria, 2 (1985) 87-103.

Bioogr. Khimiyai, 13 (1987) 14224.

Abstr. Proceedings 19th Meeting FEBS 89, Rome, Italy, 1989, Th 264.

(1988) 103-12.

Andoprim? -a Novel Fungicide with an Unconventional Mode of Action and Special Selectivity for Phytophthoru Species

Gisela Grunwaldt,' Horst Lyr," Reinhold Wollgiehnb & Manfred Klepel'

"Biologische Zentralanstalt Berlin, Stahnsdorfer Damm 81, 1532 Kleinmachnow, Germany, bInstitut fur Biochemie der Pflanzen, Weinberg 3, 4050 Halle, Germany, 'Chemische und Pharmazeutische Werke Fahlberg-List GmbH, Alt Salbke 6 M 3 , 301 1 Magdeburg, Germany

Andoprim 4-methoxy-N-(4,6-dimethylpyrimidin-2-yl)aniline; Fig. 1 ; I) is a rather selective antifungal compound designed by Fahlberg-List, GDR. It exerts its activity almost exclusively against Phytophthoru spp. In contrast to phenylamides, Pythium spp. are remarkably less sensitive, while fungi of other taxa are insensitive. t Andoprim is not a BSI-approved Common name. (Tech. Ed.)

324

Fig. 1. Structure of andoprim.


1c

e n D x 6

e 4 0 4

2

15 36 45 60 75 ! i l i a

l01 b

8-

6-

Time ( m i d

Fig. 2. Effect of andoprim on (a) uptake and (b) incorporation of ['Hluridine by Phytophthora nicotianae, (0) control, (U) lornglitre-' andoprim, (A) 20mglitre-' andoprim.

The high degree of selectivity of andoprim, and the lack of cross-resistance to phenylamides, stimulated an investigation of the mode of action, despite the poor performance of this compound under field conditions.

Cultures of Phytophthora infestans (P.i.) and P. nicotianae (P.n.) were grown on Sakai agar medium and subcultivated on pea juice. The concentrations of andoprim necessary for a mycelial growth inhibition of c. 50% were 2 and 5 mg litre-' for P.i. and P.n. respectively.

Measurements of antimycin-sensitive respiration and of potassium efflux were performed using the Warburg technique and flame photometry. No influence of andoprim on this stage of metabolism or on cellular integrity could be observed. This agrees with other work which showed that only prolonged exposure to andoprim changes the fungistatic into a fungicidal effect. This behaviour resembles the response to dimethomorph [(E,Z)-4-(3-(4-~hloropheny1)-3-(3,4- dimethoxyphenyl)acryloyl)morpholine] but is different from that with phenylamides.'

Lipid metabolism in P i , studied by uptake and incorporation of [14C]acetate, was not influenced significantly. The overall lipid synthesis and the qualitative phospholipid composition were nearly unchanged as compared with untreated controls. Thus, the relative shifts between the various lipid classes were considered to be of a secondary nature, possibly reflecting an altered physiological state of the mycelia.

Profound effects were found on uptake and incorporation of labelled amino acids (['4C]leucine) and nucleosides (C3H]uridine, C3H]thymidine) by P.n. (Fig. 2). Since the values for inhibition of incorporation into macromolecules were almost equal to


TABLE 1 Effect of Andoprim on Uptake and Incorporation of Substrates by

Phytophthora nicotianae and P . infestans ~~ ~ ~

Fungus, substrate Andoprim conc. Uptake Incorporation (mg litre- ') ( % of control afer 100 min)

P . nicotianae ['4C]leucine

[3H]uridine

[ 3H]thymidine [32P]Na-phosphate [ 14C]Na-acetate P . infestans [ 14C]Na-acetat e [ 4C]D-glucose [ 3H]thymidine

10 20 10 20 20 20 20

15 15 15

58 23 49 18 32 21 34

105 75 55

55 25 45 21 36 18 38

84 -

those of uptake of the respective precursors, it is suggested that the reduced synthesis of nucleic acids and proteins is merely the reflection of a decreased uptake of the precursors and that there is no direct effect on synthetic processes.

Therefore the uptake of various substrates by P.n. and P.i. were tested (Table 1). The data suggest that all actively driven uptake processes are inhibited to

remarkable, although varying, extents. Differences between the fungi were found regarding acetate uptake, which is nearly unchanged in P.i. and clearly reduced in P.n. Uptake of glucose, which can occur partly as facilitated diffusion, is only slightly i nfIuenced .

Thus, andoprim is considered to be an agent affecting, preferentially, active transport processes across the cellular membrane. The lack of a severe inhibition of glucose uptake can explain the maintenance of a normal respiration rate, even over longer periods of time. The same is true for acetate uptake and lipid synthesis in P.i. Therefore, a rather unconventional mode of action is proposed for andoprim: an interference in a still-unknown manner with active cellular transport systems. This may bring about a chronic deficiency in most extracellular substrates essential for growth and development. However, fundamental systems like energy production and compartmentation are unimpaired. This provides an explanation for most of the experimental data but does not yet explain the selectivity of the andoprim action to Phytophthora spp without the assumption that active transport in Phytophthora differs in a structural or functional detail from that in other fungi.

Reference 1 Lyr, H. & Muller, H. M., Reinhardsbrunn Symposium on Systemic Fungicides and

Antifungal Compounds, Tagungsberichte der Akademie der Landwirtshaftswissen- schaften der DDR, in press.

326 7th lnternational Congress of Pesticide Chemistry

(-Carotene Accumulation and Bleaching by New Pyrimidine Compounds

Reynold Chollet," Gerhard Sandmanqb Regina Diethelm," Hansruedi Felix," Karlheinz Milzner" & Peter Bogerb

"SANDOZ-Agroresearch, 4002 Basel, Switzerland bLehrstuhl f u r Physiologie und Biochemie der Pflanzen, Universitat Konstanz, 7750 Konstanz, Germany

A series of pyrimidine derivatives has been synthesized' (Table 1). Their mode of action as bleaching compounds and their herbicidal activities against several plants have been investigated. Information concerning these chemicals can be found in Patents EP 0055692 and DOS 3101426.

In extracts of Scenedesmus treated with KM 143-958 several acyclic carotenes and oxygenated derivatives could be detected in addition to the xanthophylls and a- and p-carotenes that are typically found in green algae (Fig. 1). The dominating carotenes were three isomers of (carotene (Fig. 1; 8-10) and 1 5 4 phytoene (16), together with traces of all-trans phytoene (17). The ratio of (carotenes to phytoene was 5:l . The accumulation of these carotenes indicates that K M 143-958 preferentially inhibits (carotene desaturase, and also phytoene desaturase to a certain extent. Phytoene-desaturase inhibitors also caused the accumulation of phytofluene; its 1 5 4 s (14) and all-trans (15) isomers could be identified. Compounds that are known to block the carotene pathway at the <carotene desaturase step are hydroxypyridines,' aminotriazole,' other pyrimidine^,^ a tetrazole4 and dihydropyrones.' Details of these compounds are given in a recent review.6 Because phytoene desaturase catalyzes the desaturation of phytoene and phytofluene,6 it can be assumed that (carotene desaturase converts tcarotene and also neurosporene. Neurosporene was not detectable, but p-zeacarotene was found (11). This carotene is the cyclization product of neurosporene indicating that the latter had been formed in substantial amounts and was immediately converted to p- zeacarotene by lycopene cyclase. p-zeacarotene has also been found in the fungus Phycomyces in which carotene interconversion had been inhibited.7 In addition to phytoene and <carotene, their epoxides and hydroxylation products have been identified. The oxygenated phytoene derivatives have also been detected in heterotrophic Scenedesmus that had been treated with herbicidal inhibitors of phytoene desaturase.8 In parallel, accumulation of (carotene by KM 143-958 was accompanied by the formation of its corresponding oxygenated derivatives (Fig. 1 ;

The carotene pattern observed in Scenedesmus treated with K M 143-958 resembles more or less that of Aphanocapsa treated with LS 80707.' Because in-vivo studies with this inhibitor have directly demonstrated inhibitory interaction with <- carotene desaturation (and also with phytoene desaturation), it is concluded that KM 143-958 has the same inhibitory properties. This finding is supported by in-vitro inhibition of these desaturation steps by other substituted

The pyrimidine compounds had similar effects in all three assay systems (grasses/ dicots, Lemna and Scenedesmus) presented in Table 1. Higher concentrations were

4, 5).

TAB

LE 1

G

ener

al S

truc

ture

of

Pyrim

idin

es a

nd T

heir

Effe

cts

on G

row

th a

nd P

igm

ent C

onte

nt o

f D

iffer

ent P

lant

s ,R

2 R

2:

I =a

lly1

I11

= 2-

CI-a

lly1

--C

H, F

I1 =

2-Br

-ally

1 B

:

IV =

B(R

=H

) V

=B

(R=

CH

,)

R

Rl 'N

H

A

=O,S

,NH

,NC

H,,

R

, =i

-C,H

,,sec

-C,H

,,n-C

,H,,

~ ~

~_

_

~~

i?'

Com

poun

d Su

bstit

uent

s lo

g P

15

0 01M)

E,,

(kg

a.i.

ha-'

) E50 h')

& Sc

ened

esm

us

Gra

sses

and

dic

ots

Lem

na

rl z

Aut

otro

phic

H

eter

o-

- tro

phic

G

row

th

Gro

wth

Bl

each

ing

-2

R,

R2

;. A

Gro

wth

C

ar

Chl

Car

Bl

each

ing

Pre-

em

Post

-em

KM

146

-428

K

M 1

46-4

32

KM

145

-155

K

M 1

43-9

58

KM

142

-712

K

M 1

43-9

61

KM

304

-187

K

M 3

05-1

32

KM

147

-606

K

M 3

05-1

46

KM

304

-185

-0-

-0-

-0-

-0-

-0-

-0-

-NH

- -N

H-

-S-

-N(C

H3)

- -N

(CH

3)-

sec-

C,H

, i-

C3H

7 i-

C3H

7 i-

C3H

7 n-

C3H

7 n-

C&

,3

i-C

3H7

i-C

3H7

i-C3H

7 i-

C3H

7 i-C

3H7

IV

5.15

1 0.

1 0.

1 0.

1 ++

0.

4 0.

4 1

v 5.

34

3 0.

1 0.

1 0.

1 ++

0.

5 0.4

3

I1

4.71

1

0.1

0.1

00

1

++

0.4

0.6

3 II

I 4.

22

3 0.8

0.8

0.

1 ++

0.

6 0.8

3

III

4.23

1 0.

2 0.

1 0

1

++

1 .o

1 .o

20

III

5.77

10

7-0

8-0

n.d.

+

3.0

0-3

100

IV

n.d.

5

0.4

0.3

1.5

0 4

3.0

8 II

I n.

d.

20

1.0

1.0

2.0

0 4

3.0

30

IV

4.39

20

10

10

2.

5 0

4 2.

0 10

0 II

I n.

d.

40

30

30

50

0 4

0.8

50

I n.

d.

n.d.

10

0 n.

d.

50

0 4

0.8

100

0.2

0.6

0.6

0.6

2.0

5.0

6.0

20

100 50

100

~ ~

~_

_

~ ~~

_

__

__

_~

Sets

of f

our g

rass

es an

d fo

ur d

icot

wee

ds w

ere

used

for a

pplic

atio

n on

who

le p

lant

s. B

leac

hing

and

grow

th w

ere

rate

d vi

sual

ly th

ree

wee

ks

afte

r app

licat

ion

(+ +

=com

plet

ely

blea

ched

, O=f

ull g

reen

pig

men

tatio

n). L

emna

was

gro

wn

in m

iner

al n

utri

ent s

olut

ion

cont

aini

ng th

e in

hibi

tors

. Vis

ual e

stim

atio

n of

ble

achi

ng a

nd co

untin

g of

fro

nds (

grow

th) w

as d

one a

fter s

ix d

ays.

For

test

con

ditio

ns w

ith S

cene

desm

us s

ee

Ref

. 13

. 4

n.d.

, not

det

erm

ined

.


Retention time (min)

Fig. 1. HPLC separation of carotenoids from a Scenedesmus culture grown heterotropically in the dark for six days in the presence of 10 PM KM 143-958. Detection was at 425,350 and 285 nm (inset of traces). Optical spectra of selected carotenes were recorded on-line with a Waters 994 diode-array detector (compounds 8,11,14 and 16, right part, with wavelengths given in nm). Carotenoids were identified from their spectra and by cochromatography with reference compounds'." as: 1 =neoxanthin + violaxanthin, Z=antheraxanthin, 3=lutein fzeaxanthin, 4= hydroxy-(carotene, 5 = (carotene epoxide, 6= hydroxyphytoene, 7 = phytoene epoxide, 8-10 = t-carotene isomers, 11 =b-zeacarotene, 12 = ctcarotene, 13 =pcarotene, 14= 154s phytofluene, 15= all-trans phytofluene, 16= 15-cis phytoene, 17 = all-trans phytoene. Separation was carried out on a 25 cm Spherisorb ODs-1.5 pm column with acetonitrile+methanol+ 2-propanol (85 + 10+5 by volume) as eluent at a flow of

1 mlmin-'.

necessary for growth inhibition than for a decrease in pigment content with both Lemna and autotrophic Scenedesmus. With Scenedesmus, decreased carotenoid (Car) as well as chlorophyll (Chl) content was seen under autotrophic and heterotrophic growth conditions, as is known from typical inhibitors of carotenogenesis.6 Pre-emergence and post-emergence activity goes in parallel with growth inhibition of autotrophic Scenedesmus, indicating that this microalga is a reliable species for screening for bleaching activity. No peroxidative activity was detected with Scenedesmus (assayed by light-induced eethane formation data not shown).

Bleaching of new tissue as well as growth inhibition after pre- and post-emergence application depend strongly upon whether or not the A-bridge of the compound is an oxygen atom (Table 1). Of seemingly least importance is the R, end of the structure. However, no reliable SAR consideration can be made at the moment. Lipophilicity (log P , determined according to Ellgehausen et ~ 1 . " ) of the analogs tested is generally high but the present data are not conclusive as to how far lipophilicity determines the activity of a specific compound.


References

1. European Patent 0055692, DOS 3101426. 2. Burns, E. R., Buchanan, G. A. & Carter, M. C., Plant Physiol., 47 (1971) 1448. 3. Ridley, S. M., In: Carotenoid Chemistry and Biochemistry, ed. G. Britton & T. W.

Goodwin. Pergamon Press, London, 1982, pp. 35348. 4. Kerr, M. W. & Whitaker, D. P., Proc. Brit. Crop Prot. Conf., Weeds, vol 3, Brit. Crop

Protection Council, Thornton Heath, UK, 1987, pp. 1005-13. 5. Vial, J. & Borrod, G., Z . Naturforsch., 3% (1984) 459. 6. Sandmann, G. & Boger, P. In: Target Sites of Herbicide Action, ed. P. Boger &

G. Sandmann. CRC Press, Boca Raton, FL, 1989, pp. 2544. 7. Williams, R. J., Davies, B. H. & Goodwin, T. W., Phytochernistry, 4 (1965) 75940. 8. Sandmann, G. & Albrecht, M., Z . Naturforsch., 45c (1990) 487-91. 9. Sandmann, G., Bramley, P. M. & Boger, P., J . Pestic. Sci., 10 (1985) 19-24.

10. Mayer, M. P., Bartlett, D. L., Beyer, P. & Kleinig, H., Pestic. Biochem. Physiol., 34

11. Ernst, S. & Sandmann, G., Arch. Microbiol., 150 (1988) 5 9 W . 12. Ellgehausen, H., D'Hondt, Ch. & Fuerer, R., Pestic. Sci., 12 (1981) 219-27. 13. Sandmann, G. & Boger, P., Z . Naturforsch., 41c (1986) 729-32.

(1989) 111-17. 1

Enzyme Kinetic Studies on the Interaction of Structurally Unrelated Herbicides with Acetolactate Synthase

Klaus-P. Gerbling & Clemens Kotter

Schering AG, Agrochemical Research, PO Box 65 03 11, Miillerstrasse 170-178, D-1000 Berlin 65, Germany

Acetolactate synthase (ALS; EC 4.1.3.18) catalyses an important step in the biosynthesis of branched amino acids. This enzyme is the target for herbicides which belong to different chemical classes (Table 1). It is not clear whether these herbicides interact with one common or with different binding sites of acetolactate synthase. To answer this question, enzyme kinetic studies were performed with ALS isolated from maize.

A general feature of all the herbicides shown in Fig. I is the time-dependent increase in inhibitory potency. Therefore the kinetic parameters of the various compounds were determined at different incubation times. Initial inhibition parameters were measured at 0-5 min and transitional parameters at 5-10 min after onset of the reaction. Experimental data for all compounds were computer fitted to the equations for different inhibition mechanisms. This fitting procedure allows inhibition mechanisms to be determined from the kinetic values.'

The results of the computer fits are summarized in Table 1. Obviously the inhibition type varies with the analysed time period and is different for the compounds examined.

This study indicates the existence of specific areas on ALS from maize for the


TABLE 1 Inhibition Mechanisms and Constants for Analysed Herbicides as obtained by Computer Fitting. In the Case of Mixed Type Inhibition ki represents the Dissociation Constant

corresponding with the Free and k, , with the Substrate-Bound Enzyme

Compound Mechanism of Mechanism of inhibition at inhibition at

0-5 min 5-10 min

MixedjCompetitive MixedjNon-competitive k , = 6 x lo-' M k , = 4 . 3 x M k,,= 1.3 x lo-' M k, ,=3.1 X M

Sulfon ylurea 0 Noncompetitive Uncompetitive II k , = 4 x lo-' M k , = 1.5 x 10-5 M

'CH,

Imidazolinone Mixed/noncompetitive Noncompetitive

k , = l x M

Triazolopyrimidine

O H - - E Pyrimidin yl

Competitive Uncompetitive k , = 2 - 3 ~ 1 0 - ~ M k,= 1 X M

binding of the herbicides analysed. The reason for the variation of the inhibition type with time is not clear.

Reference 1. Segel, I. H., Enzyme Kinetics. John Wiley & Sons, New York, 1975.


PyriculariaSpecific Targets: Phospholipid Biosynthesis versus Melanisation

Dieter Berg, Gerd Haenssler & Wolfgang Kramer

Bayer AG, Business Sector Agrochemicals, D-5090 Leverkusen-Bayerwerk, Germany

It has been reported earlier that Pyricularia oryzae appressoria need a high physical stability to invade the host tissue of rice plants.' Principally, three partners contribute to the stability of fungal membranes viz. melanines, phospholipids and sterols.* Melanines from Pyricularia oryzae are derived from pentaacetate and are characterised chemically as cross-linked polymers of 1 ,8-dihydroxynaphthalone. Inhibition of 1 &dihydroxynaphthalene biosynthesis by e.g. tricyclazole or pyroquilone results in apathogenic pigment ( - )-isolates3 with weak appressoria that cannot penetrate the rice plant tissue. Inhibition of lecithin synthesis by e.g. edifenphos, on the other hand, proved to be a strategy for combating rice blast disease as well, and, finally, blocking sterol synthesis should represent a mechanism that should also lead to active compounds. The first two mechanisms seem to be Pyricularia-specific, whereas inhibition of sterol biosynthesis is known to lead to broad-spectrum fungicides (for review see Ref. 4).

A test set was established that incorporated these three mechanisms with the aim of designing or screening rice blast fungicides.

Various strategies specifically to prove inhibition of melanin biosynthesis have been described in the literature. Penetration of artificial membranes by Pyriculariu uppressoriu5 is still more or less a biological test system, even though it delivers quantitative data in addition to mechanistic clues.

Isolation of melanin precursors and their quantification by HPLC analysis6 represents a highly accurate test system which, however, is limited in test capacity. Therefore it was decided to use a simple agar diffusion test where, of course,

TABLE 1 Inhibition of Melanisation of Pyricularia oryzae by Tricyclazole

and Reversion with 1,8-Dihydroxynaphthalene (1,8-DHN)

k&TN Tricyclazole

Diameter of inhibition zone" (mm)

A. i . (pg nil- ') 20 10 5 2.5

Tricyclazole 85 65 50 20 Tricyclazole 44 23 + IOOpgrnl-' 18-DHN

- -

Inhibition of pigmentation, no growth inhibition.

332

Control


Edifenphos (10 pgml-')

PG IL

['4C]Acetate incorporation into lipids of P . oryzae. TLC-separation on silica gel (CHCI, + CH,OH + CH,CO,H + H,O (85 + 12.5 + 12.5 + 3 by volume). PC= phosphatidyl-choline-, P G = phosphatidyl- glycerol-, PE = phosphatidylethanolamine, NL = neutral lipid-fractions.

Fig. 1. Effect of edifenphos on in-vitro phosphatidylcholine (PC)-biosynthesis of P . oryzae.

TABLE 2 Effect of KWG 2168 on Sterol Synthesis in P. oryzae

Sterol (%) Control K WG 2168"

OH CH,F

KWG 2168b

CH,F

N

OH CH,F

KWG 2168b

Ergosterol 82.8 10.5 A'-Ergostenol 3.6 7.2 A'**' -Stigmastadienol 4.5 5.2 As-Stigmastenol 9.1 20.5 Obtusifoliol - 9.0 24-Methylenedihydrolanosterol - 47.6

a lopgrnl- ' Inhibits sterol-14-demethylase.

fungitoxic effects are not observed after application of e.g. tricyclazole. After 3 4 days, however, melanisation leads to a grey mycelium and the remaining white diffusion zones can be used to calculate a minimum melanisation inhibition concentration (MMIC-value). The advantage of this test system is that reversion experiments by addition of melanin precursors like 1 &dihydroxynaphthalene can be performed to localise the site of inhibition (Table 1). This test arrangement allows a large number of compounds to be tested and additionally provides rapid information on mode of action.


TABLE 3 Inhibition of Growth and Melanisation, respectively, of P y r icu lar ia oryzae by Edifenphos , KWG 2168, and Tricyclazole in Agar

Diffusion Test ~

Compound p g m l - '

Edifenphos KWG 2168 Tricyclazole

13.4" 0.8" 0.16b

Minimum inhibition concentration. Minimum melanisation inhibition

concentration.

The second Pyricularia-specific mechanism is represented by a number of phosphorous esters like 'Kitazin'B, 'Conen'a and edifenphos and has previously been described in terms of inhibition of the S-adenosylmethionine (SAM)- dependent methylation of phosphatidylethanolamines (PE) to the corresponding lecithins.6 A test system was established using edifenphos as a standard compound. A homogenate of Pyricularia oryzae shake culture was incubated with ['4C]acetate and the labelled lipids were separated by TLC and scanned for radioactivity (Fig. 1).

After administration of edifenphos, the radioactivity in the phosphatidylcholine (PC) fraction decreases whereas the PE fraction accumulates. Again this is a test system which gives a clue on mode of action and additionally delivers quantitative data with respect to inhibition of PC synthesis.

Biosynthesis of sterols, as the third membrane component causing physical stability, can be followed in a traditional way by fermentation of Pyricularia oryzae in the presence of sub-lethal concentrations of test compound, isolation of sterols and their quantification by GC and G G M S . 7

Table 2 shows data for the Pyricularia-active triazole KWG 2168. The sterol distribution in Table 2 clearly indicates that KWG 2168 is a classical inhibitor of sterol-14-demethylase. Although inhibitors of sterol synthesis have been studied inten~ively,~ no commercial products against rice blast disease on this basis are available as yet.

Table 3 shows a comparison of the in-vitro activities of edifenphos, KWG 2168 and tricyclazole in which tricyclazole appears to be the most active compound against Pyricularia oryzae, followed by KWG 2168 and then the much less active edifenphos.

In general, the three different approaches to combating P. oryzae in vitro proved to be successful. In-vivo experiments (data not discussed) correlated well with the in-vitro efficacies. Whereas melanisation and methylation of phosphatidylethanolamines obviously exhibit Pyricularia-specific targets, sterol synthesis inhibition leads to broad-spectrum fungicides which may control rice blast disease besides other fungi.


References 1. Sisler, H. D., Woloshuk, C. P., Wolkow, P. M . Tag.-Ber., Akud. Landwirtsch.-Wiss.,

D D R , Berlin, 222 (1984) 17-28. 2. Akatsuka, T. & Kodama, O., Nihon Noyaku Grrkkaishi ( J . Pestic. Sci.), 9 (1984) 375-81. 3. Inoue, S . , Maeda, K., Uematsu, T . , Kato, T . Nihon Noyaku Gakkaishi (J . Pestic. Sci.),

4. Berg, D. & Plempel, M. (eds). Sterol Biosynthesis Inhibitors: Pharmaceutical and

5. Chida, T., Uekita, T. & Hirano, K., Ann. Phytopathol. Soc. Japan, 48 (1982) 58-63. 6. Greenblatt, G. A. & Wheeler, M. H., J . Liq. Chromatog., 9 (1986) 971-81. 7. Berg, D. & Plempel, M . , J . Enzyme Inhibition, 3 (1989) 1-11.

9 (1984) 731-6.

Agrochemical Aspects. VCh/Ellis Horwood Ltd, 1988.

Modes of Action of Sterol-Biosynthesis-Inhibiting Fungicidal Amines

Dieter Berg, Wolfgang Kramer & Joachim Weissmuller

Bayer AG, Business Sector Agrochemicals, D-5090 Leverkusen-Bayerwerk, Germany

In the past decade, fungicidal amines have become economically important, especially for the treatment of cereal diseases.’ They are applied either alone or in combination with sterol C,,-demethylation inhibitors (DMIs).’ The amines exhibited a more pronounced variation in mode of action than the so-called az01es.~ A study has been made of this variability of mode of action, i.e. inhibition of sterol synthesis, substituent dependence of intrinsic activity, and stereochemical implications using aryl N-alkyl-substituted heterocyclic-aliphatic amines of the basic structure I:

I CH3 R , =substituted phenyl or cyclohexyl X=O, S, CH, I

I Y O Y =0, S, CH, R,, R , =alkyl, cycloalkyl, etc n=O, 1

R I-X-(CH2),-C-K-CH3

/Rz CH,-N CH3 q

‘R 3

Further variations led to spiro-ketal-amines of the general structure 11:

R 2 ,N<

CH, R3

R ’ = alkyl, cycloalkyl, aryl, etc.

R2, R 3 = alkyl, cycloalkyl, etc. X = O(S, CH,)

The compounds were tested with respect to their potency towards sterol

The amines studied showed a remarkable variability with respect to their mode of

Aryl-substituted 4-methylene-piperidino and morpholino dioxolanes caused an

biosynthesis in fungi.

action.


accumulation of non-sidechain-alkylated sterols, indicating that the S-adenosyl methionine (SAM)-dependent alkylation of lanosterol is the site of inhibition of sterol synthesis. Comparison of 24-methylene-dihydrolanosterol with KWG 2562 (111) in its most active cis-dioxolane form and with cis-dimethylmorpholine by molecular modelling indicates a possible analogous inhibition of this reaction by these products.

CH

111 KWG 2562 'CH,

CH3 P H ,-CH -K-CH 3gcH qH CHz-C---x-CH,(" I I 0 0

I 0 0 CH3 S N CH3 S N

H3C

IV KWC 2693 V KWC 3070

The cyclohexyl-substituted 4-methylene-piperidino dioxolanes again exhibited a broad variability with respect to their mode of action, e.g. KWG 2693 (IV) accumulating 14-cl-methyl-A8~24-cholestadienol as well as A',s,22-ergostatrienol and oxidosqualene in Pyricularia oryzae. A',8-22-ergostatrienol accumulation indicates interaction with A' +A7-isomerisation and the occurrence of oxidosqualene could be explained by inhibition of the cyclisation reaction or by damming up after blocking side chain alkylation. In yeast, however, 2-alkyl-cyclohexyl-analogues in particular (e.g. KWG 3070; V) led to accumulation of A8-14-ergostadienol which indicates a block of A14-reduction. Analogous considerations are valid for the tetrahydrofurans and isoxazolidines.

The oxa- and dioxa-spiro-decanes (11) represent inhibitors of A14-reduction with the A/S-isomer being the most active.

Figure 1 summarises the observed modes of actions of fungicidal amines on sterol biosynthesis in fungi. Four prominent steps could be inhibited by various compounds:

(1) Inhibition of squalene epoxidase could be observed with alkylamines4 and the subsequent cyclisation of oxidosqualene to lanosterol was prevented by cyclohexyl-dioxolanes.

(2) The SAM-dependent methylation ofthe sterol side chain was inhibited by the SAM-antagonist sinefungine (Berg, D., unpublished results) as model compound, as well as by arylalkyldioxolanes, e.g. KWG 2693.

(3) The reduction of the A14-double bond, occurring as an intermediate during C,,-demethylation, is inhibited by phenylpropylamines such as fenpropimorph and fenpropidin' and by 1,4-dioxaspirodecanamines (e.g. KWG 3850).

HO-CO-CH,

HO-CO--CH,

Squalene Squalene epoxide

Lanosterin

CH, I I

CH 1 I I

C-CH, CO- S -COA HMG--CoA

OH I HMG -CoA-Rcductdsc

C-CH, CHI- OH Mevalonic dcid

OH

1

24-Methylenedi- hydrolanusterin ( 3 )

HO HO

A8.?4,(28)- Ergwtadien-ol

H O

Ergosterin

Inhibition of Squalene epoxidase by e.g. allylamines Inhibition of SAM-dependent methylation by e.g. sinefungine, azasterols Inhibition of C ,,-Reductase by e.g. phenylpropylamines Inhibition of As-A' Isomerase by e.g. tridemorph or phenylpropylamines

Fig. 1. Modes of actions of fungicidal amines on sterol biosynthesis in fungi.


(4) A8+A7-isomerase is a well described target for tridemorph as well as, at least

These studies confirm that the mode of action of sterol-biosynthesis-inhibiting fungicidal amines is much more variable than that of the socalled azoles.

partially, for the phenylpropylamines.6

References

1. Kuck, K.-H. & Scheinpflug, H., In Chemistry of Plant Protection, ed/ H. Hoffmann 8~ G. Haug. Springer-Verlag, Berlin, 1986, pp. 65-96.

2. Schulz, U. & Scheinpflug, H. In Sterol Biosynthesis Inhibitors-Pharmaceutical and Agrochemical Aspects, ed. D. Berg & M. Plempel. Ellis Horwood Ltd, Chichester, 1988,

3. Mercer, E. I., In Sterol Biosynthesis Inhibitors-Pharmaceutical and Agrochemical Aspects, ed. D. Berg & M. Plempel. Ellis Horwood Ltd, Chichester, 1988, pp. 12&50.

4. Ryder, N. S. In Sterol Biosynthesis Inhibitors- Pharmaceutical and Agrochemical Aspects, ed. D. Berg & M. Plempel. Ellis Horwood Ltd, Chichester, 1988, pp. 15147.

5. Kerkenaar, A., van Rossum, J. M., Versluis, G. G. & Marsmann, J. W., Pestic. Sci., 15 (1984) 177-87.

6. Baloch, R. I., Mercer, E. I., Wiggins, T. E. & Baldwin, B. C., Proc. Brit. C r o p Prot. Conf.-Pests and Diseases, 1984, Vol. 3 , pp. 893-8.

pp. 211-61.

S-Adenosylmethionine (SAM)-Antagonists: Insecticidal Inhibitors of Juvenile Hormone Synthesis

Martin G. Peter,O Hans-Jorg Ferenz,b Dieter Berg' & Benedikt Beckerc

"Institute of Organic Chemistry and Biochemistry, University of Bonn, Gerhard Domagkstr. 1, D-5300 Bonn 1, Germany *Insect Physiology Group, University of Oldenburg, PO-Box 2503, D-2900 Oldenburg, Germany 'Bayer AC, Business Sector Agrochemicals, D-5090 Leverkusen-Bayerwerk, Germany

The development of insects can be disturbed by various factors, e.g. juvenile hormone (JH) agonists or antagonists.' Sinefungiq2 and S-adenosylmethionine (SAM) antagonist, was chosen as a model inhibitor of JH biosynthesis, as methylation of farnesoic acid is a late step in such biosynthesis, being followed by epoxidation in the o-position. In uitro, the inhibition of the methylation of farnesoic acid was followed by using a crude methyl-transferasecontaining enzyme preparation from female locust corpora allata.3 Methylation of farnesoate was strongly inhibited by sinefungin concentrations below 0.1 mM and completely inhibited by concentrations of c. 0.5 mM.

The production of juvenile hormone JH I11 by intact corpora allata, stimulated by farnesoic acid, was estimated in organ culture. TLC analysis of extracts of the culture medium revealed that the radioactivity was incorporated almost exclusively


in JH 111. At sinefungin concentrations of 0.3 mM, incorporation into JH I11 was already markedly depressed and the hormone production was inhibited to background level by concentrations > 3 mM.

The farnesoate-0-methyl-transferase exhibited classical Michaelis-Menten kinetic^.^ It showed an apparent K , for SAM of 1.2 x M and a V,,, of 14.3 x M. With [14C]SAM as substrate, the locust 0-methyltransferase was competitively inhibited by sinefungin with a Ki of 0.5-1.0 x M.

It was therefore decided to look for in-vivo effects of sinefungin. With female locusts, an increase of mortality was found only at rather high concentrations. The cuticle had an unusual reddish-brown colour and a paper-like consistency.

Dose-dependent reductions in ovary weight, oocyte development, and total haemolymph protein content were caused by increased amounts of sinefungin. Higher amounts suppressed any development of ovaries and fat body, and both tissues remained in the premature stage of development. Injection of smaller amounts of sinefungin caused the locust ovaries to show retarded growth and often resorption of partly matured oocytes occurred. Attempts to restore the process of sexual maturation in sinefungin-injected female locusts by topical application of high doses of the juvenile hormone agonist ZR-515 (methoprene)’ were not successful. However, there were signs of enhancement of fat body development and increase of haemolymph protein concentration.

Finally, sinefungin was tested in uivo for insect growth-retardant effects and mortality. Surprisingly, it proved to be effective against some species. e.g. Phaedon cochliariae and Ceratitis capitata when applied to larval stages. With a delay of 2-3 weeks, mortality was observed with up to 0.004 % active ingredient. This is quantitatively comparable to the efficacy of furadan which, of course, is a fast-acting insecticide.

In summary, sinefungin exhibited the expected competitive inhibition of farnesoic acid 0-methyl transferase with a K , of 0.5-1.0 x lop6 M. Thus the JH biosynthesis was strongly inhibited by the SAM-antagonist. An in-vivo reversion by methoprene (ZR-515) was not convincingly successful, however. Sinefungin showed remarkable insecticidal properties after 2-3 weeks, but the expected immediate effect could not be detected. The compound proved to be a slow-acting inhibitor of insect development. Its efficacy in uivo is surprisingly high since it is a relatively polar compound. In general, the concept of inhibition of JH-synthesis for new insecticides proved to be relevant.

mole min- ’. The K , for farnesoic acid was 29 x

References 1. Rembold, H., In Physiologische Schliisselprozesse in Pfranse und Insekt, ed. P. Bayer.

Universitatsverlag Konstanz, 1985, pp. 191-212. 2. Butler, T . F., J . Antibiot., 26 (1973) 464. 3. Ferenz, H.-J., Peter, M. G. & Berg, D., Agric. B i d . Chem., 50 (1986) 1003-8. 4. Ferenz, H.-J. & Peter, M. G., Insect Biochem., 17 (1987) 1119-22. 5 . Edwards, J. P. & Meran, J. J., In Chrmie der Pflanzenschutz und Schiidlings-

bekampfunysmitzel, ed. R . R . Wegler. Vol. 6, Springer-Verlag, 1981, pp. 185-214.


Substituted Tetrahydropyrimidinones. A New Class of Herbicidal Compounds Inducing Chlorosis by Inhibition of Phytoene Desaturation

Peter Babczinski,” Martin Blunck,b Gerhard Sandmann; Robert R. Schmidt,“ Kozo Shiokawad & Kazuomi Yasuid

“Agrochemicals Division, Bayer AG, Monheim, 5090 Leverkusen, Germany *Pharmaceutical Division, Bayer AG, 5600 Wuppertal, Germany ‘University of Konstanz, 7750 Konstanz, Germany dNihon Tokushu Noyaku Seizo K. K., Agricult. Chem. Ind., 3-1-1, Toyoda, Hino-shi, Tokyo, Japan

Impairment of plant pigment biosynthesis is one of the most prominent and attractive principles of herbicidal mode of action. Many biochemical steps in both chlorophyll and carotenoid biosynthesis are plant-specific and therefore not of mammalian toxicological importance, and some of the respective enzymes are targets for important commercial herbicides like protoporphyrinogen oxidase (e.g. nitrodiphenylethers, cyclic imides)’ or phytoene desaturase (PD; e.g. fluometuron, diflufenican2).

A considerable number of bleaching herbicides have been discovered over the past 15 years (Fig. l), and for many of them their biochemical target has been located at the PD step. Several classes of compound with PD inhibitory activity exhibit remarkable structural similarities, i.e. meta-CF, phenyl ring substituents, a carbonyl-containing heterocyclic ring system, or methylated amino functions. By contrast, profoundly different chemical structures show a common biochemical mode of action. In this respect, PD inhibitors resemble herbicidal inhibitors of photosystem I1 whose manifold representatives all fit into a common protein binding niche the understanding of whose molecular interaction is emerging.3

Compounds of Type I (Table 1) (1,3-diaryl-2-pyrimidinones; ‘cyclic urea~’)~-’ are new bleaching herbicides. Chemically, these compounds combine several structural elements and principles of well known chlorotic substances, e.g. the meta-CF, phenyl substituent, which is typical of rather a large group of compounds including fluridone, fluometuron and norflurazon. A carbonylcontaining heterocyclic system is a second common feature. Clearly, fluridone and fluometuron are parent structures for the new compounds.

The biological activity of the new compounds in pre-emergence upland tests is comparable to that of fluridone but with better selectivity in cotton. Both dicot weeds and grasses are controlled completely at 1000 g ha- ’. Under paddy-field conditions, the efficacy of weed control is acceptable, but selectivity to transplanted rice is inferior to that of pyrazolate.

The physiological mode of action of the cyclic ureas is a bleaching of newly grown green plant material. In a pre-emergence application, leaves of e.g. cress plantlets (the main test system used in the present investigations) grow completely white without any early visual damage. Cytologically, cells deteriorate and the vacuole looks very disorientated. Accumulation of mitochondria around chloroplasts is


I l - P v T H C H 3 \ / I \ /

CF3 I CF3 0 CF3 NHCH, CH,

Fluridone Norflurazon Flurtamone

Flurochloridone Diflufenican WL 110 547

I N (CH, 12 Dichlormate Amitrole

Difunon" (EMD-IT 5914)

OH

Pyrazolinate Pyriclor

Fig. 1. 'Bleaching Herbicides' which act at the Phytoene Desaturation Stage

"Difunon is not a BSI/ISO approved Common Name.


observed, and the latter lack thylakoid stacks. Necrosis of leaves is only observed after prolonged illumination for several days.

The biochemical mode of action of the new compounds has been investigated by looking for the accumulation of metabolic intermediates in treated leaves. As revealed by HPLC of leaf extracts, a material which cannot be observed in controls is drastically increased. It has been identified by UV and mass spectroscopy as the carotenoid precursor, phytoene. Therefore, it has to be assumed that chlorosis is caused by inhibition of carotenoid biosynthesis. Loss of chlorophyll therefore should be a secondary phenomenon.'

To exclude the possibility that phytoene desaturase might be inhibited by indirect or regulatory mechanisms, in-vitro studies with isolated enzyme have been carried out (Table 1). Conversion of phytoene into B-carotene was investigated with thylakoids from the unicellular cyanobacterium Anacystis. [ '4C]Geranylgeranyl pyrophosphate was converted into phytoene and further on into B-carotene. NTN 28621 (0.1 p ~ ) prevented about half of the carotene formation by inhibition of phytoene conversion. Inhibition was complete with 1 p~ inhibitor. The other derivatives are weaker inhibitors of phytoene desaturase.

Proof of secondary effects on chlorophyll bleaching has been obtained from dim- light experiments (150 lux). Treated plants do contain chlorophyll (c. one-third of that in dim-light control plants), and also phytoene, but no carotenoids. Those plants look artificially green because 'warm' coloured pigments are lacking. Illumination (2 h; 20 000 lux) brings the chlorophyll content in untreated plants to about double the dim-light value; in treated plants it is reduced to zero, and complete bleaching is observed. Obviously, chlorophyll biosynthesis is not a target of cyclic urea herbicides.

TABLE 1 In-vitro Phytoene Desaturation by Anacystis Thylakoids in Presence of Substituted

Tetrah ydropyrimidones

0 X=(CH,), n=2,3,4

I pANQ or -CH,-CH-CH,- I 'R,' ' X j R, CH,

Condition Radioactivity (dpm) in R , R, X

Phytoene B-Carotene"

Control 176 2304 NTN 28621 (0.1 pM) 963 1233 3-CF3 H -CH,-CH-CH,-

I NTN 28621 (1 p ~ ) 2784 0 CH3 NTN 26105 ( 1 p ~ ) 1194 914 3-CF3 2-C1 (CH,), NTN 28594 (1 pM) 344 2154 3-C1 H (CH,),

[ ''C]Carotenes were formed from [ 14C]geranylgeranyl pyrophosphate by Anacystis thylakoids equivalent to 120pg chlorophyll for 2 h.


The new compounds can be easily prepared with good yield by cyclization of alkylated anilines. They are all stable, soluble in many organic solvents, and practically insoluble in water.

From structure-activity relationships, using greenhouse experiments under paddy conditions, it can be concluded that two phenyl substituents, a substituent (preferably-CF, in the meta position), and a six-membered cyclic urea ring system are required for biological activity. If both phenyl rings are substituted, the second substituent must not be in the para position. Mono-methyl substitution of the pyrimidone system is allowed in the 5-position.

Using more than 150 compounds synthesized, correlation between cress pigment biosynthesis inhibition parameters and pre-emergence greenhouse data yields a correlation factor of r = 0.933. Not unexpectedly, out of seven compounds tested, the correlation between in uitro and in uiuo is much worse and shows deviations of at least three positions in a rank order. Obviously, parameters like uptake, partition, transport, and metabolism have to be considered. This is not the case in experiments with green plant cell cultures (Catharanthus roseus), where excellent correlation with in-vitro data is achieved.

Selectivity of cyclic urea herbicides can be demonstrated in cotton as well as in in- vivo greenhouse experiments, and also by the determination of plant pigment biosynthetic activity in this plant (in-vivo experiment). Clearly, tolerance is well correlated with less well-pronounced inhibition of pigment accumulation. The molecular reason for the selectivity certainly remains unknown.

As expected for urea derivatives, high persistence in soil is not a problem with this class of herbicide. In bio-availability experiments (using cress plants grown in unsterile soil) metabolism of cyclic ureas starts within two weeks of herbicide maintained in wet soil. In our hands, fluridone (which is known to be per~is tent)~ needs almost double that time for a decrease in bio-availability. Decreased persistence, as compared to fluridone, has also been observed in greenhouse experiments.

Acknowledgements The authors thank Prof. M. Zenk for providing the plant cell culture. The contributions of Th. Rother (EM pictures), Th. Grol3, A. Hacklander, and H.-J. Steckel are gratefully acknowledged.

References 1 . Matringe, M., Camadro, J.-M., Labbe, P. & Scalla, R., Biochem. J . , 260 (1989) 231-5. 2. Wightman, P. & Haynes, C., Proc. Brit. Crop Prot. ConJ Weeds (1985) 171-8. 3. Tietjen, K., Kluth, J. , Andree, R., Haug, M., Lindig, M., Muller, K. H. , Wroblowsky, H .

& Trebst, A., Abstr. 7th Internat. Congr. Pesticide Chem. ( I U P A C ) , Hamburg (1990) Poster No. 161, Code 04B-05.

4. Aya, M., Jap. Pat. Applic. 81/23,122 (1981). 5. Aya, M., Europ. Pat. Applic. 58,868 (1982). 6. Aya, M., Jap. Pat. Applic. 82/22,109 (1982). 7. Aya, M., Europ. Pat. Applic. 86,411 (1983). 8. Mayfield, S. P. & Taylor, W. C., Mol. Gen. Genet., 208 (1987) 309-13. 9. Banks, P. A., Ketchersid, M. L. & Merkle, M. G., Weed Science, 27 (1979) 631-3.


Allosamidin Enhances Chitin Biosynthesis in Artemia salina Microsomes

Martin G. Peter, Fritz Schweikart & Herbert Wiederstein

Institut fur Organische Chemie und Biochemie der Universitat, Gerhard-Domagk-Str. 1, D-5300 Bonn 1, Germany

The polysaccharide, chitin, is an essential component of the cell walls of fungi and yeasts as well as of the integument of arthropods. Cell division in micro-organisms or moulting in arthropods requires chitin biosynthesis and degradation. Inhibitors of these biochemical processes are useful tools for the study of chitin metabolism and for the control of insects as well as of fungal infections. The mechanism of chitin biosynthesis in arthropods is largely unknown (for a recent review, see Ref. 1). Solubilization of membrane-bound chitin synthase has not been reported so far, though cell-free systems have been established from insect2 and crustacean3 sources. In this report, results on chitin biosynthesis in membrane preparations from the crustacean species Artemia salina are summarized. Full details are published el~ewhere.~.’

Chitin synthesis: The assay was adapted from the A . salina microsomal chitin synthase assay, described by H ~ r s t . ~ When 1OOOOg pellets of homogenates of nauplii (harvested 72 h after hatching) were incubated with 0.6 p~ UDP-[U- ‘‘C]GlcNAc, incorporation of radioactivity into chitin accounted for 0.084 (& 0.036) pmol GlcNAc within 2 h.’ This ‘basic synthesis rate’ was enhanced by a factor of 5.2 in the presence of the established activators trypsin and GlcNAc. However, C3H]acetyl chitin, added to the assay mixture, was degraded considerably to C3H]GlcNAc (identified by HPLC). Thus, the membrane fraction contained not only chitin synthase, but also endo- and exochitinase. The hydrolytic activity was not removed by repeated washing of the membranes. Consequently, the ‘true’ chitin synthase activity may have been masked by endogenous chitinase.

Inhibition of chitinase by allosamidin: Allosamidin (Fig. 1) is a natural product, isolated recently from Streptomyces sp. No. 1713 by Sakuda et ~ 1 . ~ 7 ’ Allosamidin is a highly potent and specific inhibitor of insect chitinases (Bombyx mori) but not of chitinase from plants (yam); also of lysozyme or B-N-acetyl-D-glucosaminidase.* When a membrane fraction from Artemia was incubated with [3H]acetyl chitin and 100 p~ allosamidin, the formation of soluble radiolabelled products was only 7 % of the ‘basic synthesis rate’.4 HPLC analysis of these soluble products revealed a composition of 66 % GlcNAc and 33 % (GlcNAc),. Thus, the small amount of chitin degraded in the presence of the inhibitor is hydrolysed primarily to the monomer.

OH

I HO NH HO Ac HO Ac CH3

Fig. 1. Structure of Allosamidin.


However, when an inhibitor of N-acetyl-D-glucosaminidase was included (i.e. 20 mM 2-acetamido-2deoxygluconolactone), the product mixture consisted mostly of (GlcNAc), in addition to higher oligomers. Thus, allosamidin is an inhibitor of the endo-chitinase of A . salina. Kinetic analysis showed a K , of 50 p~ at pH optimum (pH=4.5). At pH=7, the inhibition of chitinase by allosamidin is even more effective, probably because of tight ionic interaction with a carboxylate group at the active site of the enzyme (cf. Ref. 9).

Chitin synthesis in the presence of allosamidin: The inhibition of endochitinase allows observation of the 'true' chitin biosynthesis rate. The experiments with UDP-[U-'4C]GlcNAc were repeated in the presence of 5-20 p~ allosamidin.' A c. nine-fold enhancement over the 'basic synthesis rate' was found. When lOpg trypsin and 20 p~ allosamidin were present, incorporation of GlcNAc into chitin was 4.89 ( k 091) pmol. Likewise, an incorporation of 4.63 ( f 0.23) pmol GlcNAc was found in the presence of 5 pg trypsin, 1 mM GlcNAc and 10 PM allosamidin. Thus, the apparent synthesis rate was enhanced up to 58-fold in the presence of allosamidin.

Synthesis of a photo-labile substrate analogue: One promising approach towards the isolation of labile enzymes makes use of photoaffinity labelling. N-diazo- acetylglucosamine was synthesised in high yield,' as described first by Burkhardt et al.' Acylation of 2-amino-l,3,4,6-tetra-0-acetyI-2-deoxy-~-glucose with Z - glycine in the presence of DCCI was followed by removal of the Z-residue by catalytic hydrogenation, diazotization and cleavage of the 0-acetyl groups by means of sodium methanolate in methanol. The compound shows absorption at A,,, = 253 nm (log E = 4.10) and is photolyzed rapidly at 254 nm. It does not compete with the binding of UDP-[U-'4C]GlcNAc to the membranes. However, a 1.2-fold stimulation of chitin biosynthesis was observed in the presence of diazo-GlcNAc which, in this respect, is similar to the effect of GlcNAc. Probably the activator analogue binds to the same regulatory sub-unit as the natural activator.

Conclusions: Chitin synthase assays should be checked for the presence of endogenous chitinase activity. The results of this study suggest that chitin synthase assays may be greatly improved by the use of an endochitinase inhibitor which will drastically enhance the apparent synthase activity. Synthesis of N-diazoacetyl- glucosamine a-phosphate was completed recently.

References 1. Cabib, E., Ado. Enzymol., 59 (1987) 59-101. 2. Cohen, E. & Casida, J. E., Pestic. Biochem. Physiol., 17 (1982) 301-6. 3. Horst, M. N., J . B id . Chem., 256 (1981) 1412-19. 4. Schweikart, F., Isogai, A,, Suzuki, A. & Peter, M. G. In Chitin and Chitosan, ed. G.

SkjPk-Briek, T. Anthonsen & P. Sandford. Elsevier, London, 1989, pp. 269-78. 5. Peter, M. G. & Schweikart, F., Biol. Chem. Hoppe-Seyler, 371 (1990), in press. 6. Sakuda, S., Isogai, A., Matsumoto, S., Suzuki, A. & Koseki, K., Tetrahedron Lett., 27

7. Sakuda, S., Isogai, A., Makita, T., Matsumoto, S., Koseki, K., Kodama, H. & Suzuki, A,, Agric. Biol. Chem., 51 (1987) 3251-9.

8. Koga, D., Isogai, A,, Sakuda, S., Matsumoto, S., Suzuki, A., Kimura, S. & Ide, A., Agric. Biol. Chem., 51 (1987) 471-6.

9. Legler, G., Pure Appl. Chem., 59 (1987) 145744.

(1986) 2475-8.


10. Widerstein, H., Diploma Thesis, University of Bonn, 1987. 11. Burkhardt, A. E., Russo, S. O., Rinehart, C . G. & Loudon, G. M., Biochemistry, 14

(1975) 5465-9.

Chemistry and Inhibition of Insect Cuticle Sclerotization

Martin G. Peter, Lothar Grun & Merle Miesner

Institut fur Organische Chemie und Biochemie der Universitat, Gerhard-Domagk-Str. 1, D-5300 Bonn 1, Germany

Insects construct their exoskeleton from macromolecular components which consist principally of chitin and proteins. Stabilization of the hydrated soft matrix of those biopolymers is called sclerotization. It results from oxidation of N- acetyldopamine (NADA) and N-P-alanyldopamine (NBAD) within the cuticle, and loss of water.’ It is often accompanied by darkening of the cuticle. The mechanisms of sclerotization have been the subject of numerous studies, both out of interest and for investigating a promising biochemical target for the development of selective insecticides. This communication summarizes the current views on the chemistry of sclerotization and the possibilities of inhibiting the process.

Reactive intermediates are quinones and quinone methides: Enzymatic oxidation of the diphenolic precursors NADA and/or NBAD within the cuticular matrix generates reactive electrophilic intermediates. These are o-quinones and their tautomeric p-quinone methides. o-Quinones are thought to crosslink proteins via nucleophilic groups. Indeed, solid state NMR of insect cuticle’ has shown addition of histidine to dopamine quinone3 but proved neither crosslinking in, nor the molecular size of, the aromatic component. p-Quinone methides were suggested after isolation of racemic N-acetylnoradrenaline from reactions containing NADA and insect c ~ t i c l e . ~ An enzyme activity has been described that isomerizes the o- quinone of NADA to its corresponding p-quinone met hide.'^^ Exposure of cuticle to NADA results in incorporation of the precursor into insoluble material4 from which ‘large amounts’ of N-acetylnoradrenaline are released upon acid hydrolysis.’ However, covalent binding of oxidation products via the Pcarbon atom was not confirmed by solid state NMR.3,8 Also, the enzymatic studies do not yield information on the quantities of phenolics or on the nature of the end products of diphenol oxidation in sclerotized insect cuticle.

Tanning substrates are oxidatively transformed into complex oligoiners: Only few quantitative data are available concerning the fate of the sclerotization precursors in cuticle. In Manduca sexta, the principal tanning agent is NBAD.9 The amounts of NBAD (including conjugates) and its incorporation into metabolites as calculated from literature data are listed in Table 1. Only a minor fraction (well below 1 %) of NBAD metabolites is extractable, in the form of chemically defined catechols, from sclerotized cuticle under relatively mild conditions.” Interestingly c . 1000-fold more P-alanine than catechols was found after exhaustive hydrolysis of elytra of the


TABLE 1 NBAD and Catecholic Metabolites in Manduca Sexta Fraction Investigated

Amount found (prnol individual-')

1. Haemolymph of pharate 2. Haemolymph of 1-da-old pulass9 3. Incorporation into cuticle (difference between 1 and 2) 4. Incorporation of [IL4C]NBAD, injected into pharate pupa" 5. Cuticle of 14-h-old pupa (cold 1.2 M HC1)" 6. Cuticle of 24-h-old pupa (1.2 M HCl, 100°C, 10 min)" 7. Cuticle of 24-h-old pupa (6 M HCl, reflux, 24 h)" 8. Cuticle of 24-h-old pupa, fraction insoluble in 6 M HCl"

2.2-6.3 0.6

1.65.7 1.4

0~01-0~04 0.06 0.77 0.61

beetle Tribolium." From Manduca pupal cuticle, a large portion of the metabolites (55 %) is released by harsh hydrolysis in the form of highly complex, unidentified components. Another fracton (44%) appears as acid-insoluble, melanin-like material.' Since ['4C]NADA, ['4C]tyrosine, and (['4C]tyrosine)-arylphorin are also incorporated into melanin-like material in Manduca pupal cuticle, this fraction must consist of a mixed-type polymer containing indolic as well as catecholic and polypeptide sub-structures." This is supported by results of additional in-vitro experiments: enzymatic oxidation of a mixture of NADA and tyrosine yields mixed- type melanin polymer^'^ and oxidation of NADA or NBAD in the presence of arylphorin leads to high-molecular-weight, cross-linked protein-polyphenol c~mplexes. '~ The structures of the cross-links still have to be established. Actually, it is possible that only a few covalent bonds are formed and the essential mechanism of diphenol oxidation is hydrophobic coating of the protein surface.' Non-covalent complexes of polymeric tanning agents and insect proteins have been characterized previously.'6

Inhibition of sclerotization: A limited range of compounds has been studied with respect to their effect on insect cuticle sclerotization. Some act at an early step of the metabolic pathway: DOPA decarboxylase inhibitors interfere with NADA and NBAD biosynthesis. Application of carbidopa to Lucilia larvae results in a lethal disturbance of water permeability of the cuticle, causing rapid dehydra t i~n . '~ Interfering chemically with B-alanine metabolism by such isosteric analogues of p- alanine as ethyl hydrazinoacetate causes development of black Manduca pupae that otherwise are sclerotized normally.'* This contrasts somewhat with the effects of impaired p-alanine biosynthesis or of its transport through the epidermis in Drosophila mutants, the cuticles of which show reduced stiffness and puncture- resistance as compared with the wild type." The anti-oxidant MON-0585 [(2,6-di- tert-butyl(cc,u-dimethylbenzyl)phenol] is lethal to mosquito larvae at low concentrations, probably due to inhibition of the oxidative activation of sclerotization precursors. The toxicity has been explained in terms of an interference with cuticular stabilization and malformation of the breathing tubes.20 In combination with diflubenzuron, MON-0585 causes incomplete crosslinking and interference with protein synthesis and active transport." Inhibition of chitin


deposition is not directly connected with catechol metabolism in the cuticle. However, diflubenzuron alone reduces flexural stiffness 16-fold and cuticle thickness 3.3-f0ld.~’ Another approach towards inhibition of sclerotization is directed at inhibiting cross-linking and polymerization: it was found recently that analogues of NADA with alkyl substituents in ring position 6 or in the B-position of the side chain are oxidized by tyrosinase and affect cross-linking of arylphorins in ~ i t r o . ~ ~ This result casts some doubt on the theory that quinones or quinone methides react essentially as acceptors of nucleophilic functional groups of proteins. When oxidation of NADA is performed by means of tyrosinase in the presence of 2- h ydroxy -N-acetyltyramine (2-OH-NATA), a deep-red coloured heterodimer forms by coupling of the resorcinol and the oquinone of NADA. In this case, not only cross-linking of arylphorin is inhibited but also in-vitro incorporation of NBAD into pharate pupal cuticle of M ~ n d u c u . ~ ~ This reaction serves as a valuable model for the oligomerization of sclerotization agents. It is the first example of the inhibition of protein cross-linking caused by trapping of intermediates in an oxidative phenolic coupling reaction.

Conclusions: The mechanisms of insect cuticle sclerotization have been reviewed many times and, concerning the metabolic fate of catechol precursors, some of the authors favour ‘a little bit of everything’. However, it is necessary to consider the relative contributions of the mechanistic variations more seriously in quantitative terms. There is no doubt that quinones and quinone methides occur as reactive intermediates, but all studies dealing with enzymatic diphenol oxidation in aqueous solutions have so far described only the first step of catechol activation. None of them has conclusively resolved the question of how these intermediates interact within the cuticular matrix to form the sclerotized exoskeleton of insects with its unique mechanical and chemical properties. Hopefully, correlations of the mode of action of suitably structured sclerotization inhibitors with the properties of the cuticle will yield useful answers. However, this approach is presently only in its infancy,

The work cited from our laboratory was supported by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie.

References

1. Andersen, S. O., Comprehensive Insect Physiology, Biochemistry and Pharmacology. Vol. 3, ed. G. P. Kerkut & L. I. Gilbert. Pergamon Press, Oxford, 1985, pp. 59-74.

2. Peter, M. G. & Forster, H., Angew. Chem. Int. E d . Engl., 23 (1984) 638-9. 3. Schaefer, J., Kramer, K. J., Garrow, J. R., Jacob, G. S., Stejskal, E. O., Hopkins, T. L.

& Speirs, R. D., Science (Washington), 235 (1987) 1 2 W . 4. Peter, M. G., Insect Biochem., 10 (1980) 221-7. 5. Saul, S. & Sugumaran, M., FEBS Lett., 237 (1988) 155-8. 6. Andersen, S. O., Insect Biochem., 19 (1989) 803-8. 7. Sugumaran, M., Arch. Insect Biochem. Physiol., 8 (1988) 73-88. 8. Williams, H. J., Scott, A. I., Woolfenden, W. R., Grant, D. M., Vinson, S. B., Elzen,

G. W. & Baehrecke, E. H., Comp. Biochem. Physiol., 89B (1988) 317-21. 9. Hopkins, T. L., Morgan, T., Aso, Y. & Kramer, K. J., Science (Washington), 217 (1982)

3646. 10. Griin, L. & Peter, M. G., 2. Natiirforsch., 39c (1984) 1066-74.


11. Morgan, T. D., Hopkins, T. L., Kramer, K. J., Roseland, C. R., Czapla, T. H., Tomer, K. B. & Crow, F. W., Insect Biochern., 17 (1987) 255-63.

12. Roseland, C. R., Kramer, K. J. & Hopkins, T. L., Insect Biochem., 17 (1987) 21-8. 13. Peter, M. G., Z. Physiol. Chem., 361 (1980) 313. 14. Grun, L. & Peter, M. G. In The Larval Serum Proteins of Insects, ed. K. Scheller.

Thieme Verlag, Stuttgart, 1983, pp. 102-15. 15. Vincent, J. F. V. & Ablett, S., J . Insect Physiol., 33 (1987) 973-9. 16. Hackman, R. H. & Goldberg, M., Insect Biochem., 7 (1977) 175-84. 17. Turnbull, I. F., Pyliotis, N. A. & Howells, A. J., J. Insect Physiol., 26 (1980)252-32. 18. Ujvary, I., Hiruma, K., Riddiford, L. M., Matolcsy, G., Roseland, C. R. & Kramer,

K. J., Insect Biochem., 17 (1987) 389-99. 19. Jacobs, M. E., J . Insect Physiol., 31 (1985) SO!-15. 20. Semensi, V. & Sugumaran, M., Pestic. Biochem. Physiol., 26 (1986) 22&30. 21. Zomer, E. & Lipke, H., Pestic. Biochem. Physiol., 16 (1981) 28-37. 22. Wolfgang, W. J. & Riddiford, L. M., J . Exp. Biol., 128 (1987) 19-33. 23. Peter, M. G., Grittke, U., Grun, L. & Schafer, D., In Endocrinological Frontiers in

Physiological Insect Ecology, ed. F. Sehnal, A. Zabza & D. L. Denlinger. Wroclaw Technical University Press, Wroclaw, 1988, pp. 519-30.

i.

Cellular Microtubules: Targets for the Fungicides Pencycuron and Zarilamide?

Pierre Leroux, Veronique Droughot & Michel Gredt

INRA, Station de Phytopharmacie, 78000 Versailles, France

The cellular microtubules are proteinaceous organelles found in the mitotic spindle which plays a predominent role during cell or nuclear division. As components of the cytoskeleton, these microtubules participate also in the biosynthesis of cell walls. Several groups of pesticides interfere with the formation and/or the functioning of cellular microtubules. As a consequence, they generally inhibit mitoses and produce such morphological alterations as swelling of root tips in plants or distortions of germ tubes in fungi.'*2 Among the agricultural fungicides, benzimidazoles and compounds which are metabolised to them (benomyl, carbendazim, thiabendazole, thiophanate-methyl) and phenylcarbamates (diethofencarb, MDPC) have been described as specific antimicrotubular agents.334 The antibiotic griseofulvin, used against human mycoses, also acts on fungal micro tubule^.^

Resistance to benzimidazoles in fungi is a widespread problem and is assumed to be caused by the presence of tubulin (the main protein of microtubules) with a reduced binding affinity to these fungicide^.^ Some benzimidazole-resistant strains exhibit an increased susceptibility towards phenylcarbamates and various other toxicants (including several herbicides and insecticides). This phenomenon is associated with hyphal distortions (especially twisting of germ tubes) similar to those produced by benzimidazoles on sensitive strain^.^ Whatever the exact mechanism, it is clear that all such chemicals which present a negative cross- resistance towards benzimidazoles can affect the functioning of cellular microtubules in fungi. The aim of this paper is to show that two recently discovered

TAB

LE 1

Ef

fect

s of V

ario

us T

oxic

ants

on

the

Mor

phol

ogy"

and

the

Elon

gatio

n of

Ger

m T

ubes

of

Diff

eren

t Phe

noty

pes

of B

otry

tis c

iner

ea a

nd

Pseu

doce

rcop

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lla h

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tric

hoid

es S

ensi

tive

(S) o

r R

esis

tant

(R

) to

Ben

zim

idaz

oles

(for

Det

aile

d M

etho

ds s

ee R

ef. 2

)

EC

,, (m

g lit

re-

') of

phe

noty

pes 08

To

xica

nt

B. c

iner

ea

P. h

erpo

trich

oide

s

BS

BR

I B

R2

PS

PRI

PR2

PR3

PR4

Car

bend

azim

0.

03 (

+)

>25

(-)

4 (+

I 0.

03 (

+)

25 (

+)

>50

(-)

27 (

+)

5 (-

) D

ieth

ofen

carb

>

25 (

-)

0.06 (

+)

>25

(-)

>

25

(-)

. 0.

03(+

) 0.

2(+

) >

25

(-)

>25

(-)

MD

PC'

6 (-1

0.

07(+

) 7

(-)

lo(-

) 0.3

(+)

0.4

(+)

1.0(

+)

12(-

) Pr

opyz

amid

e 12

(-1

10 (-

1 10

(-1

30(-

) 24

(-)

32 (-

1 25

(-)

22(-

) Za

rilam

ide

>50

(-)

1

5(+

) >S

O(-)

4

0(-

) 7.

5 (+

) 2

0(+

) lo

(+)

36(-

) G

riseo

fulv

in

0.15

(+

) 0.

06 (

+)

0.16

(+

) 4

(+)

I(+

) 5

(+)

5(+)

4

(+)

Penc

ycur

on

1.5

(+)

0.8

(+)

1.2

(+)

12

(+)

3(+

) 12

(+I

3(+

) lo

(+)

( + ) p

rese

nce

and

(- )

abs

ence

of

typi

cal d

isto

rted

ger

m tu

bes.

In

hibi

tion

of g

erm

tube

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ngat

ion

dete

rmin

ed a

fter 2

4 h

incu

batio

n at

20°

C fo

r B. c

iner

ea a

nd a

fter 4

8 h

incu

batio

n at

18°

C fo

r P.

herp

otric

hoid

es (

in b

oth

fung

i the

tem

pera

ture

was

15°

C fo

r pe

ncyc

uron

and

gris

eofu

lvin

). Th

ey a

re m

eans

of

two

to fo

ur is

olat

es.

MD

PC:

met

hyl N

-(3,5-dichlorophenyI)carbamate.

W

P

\D


fungicides, pencycuron and zarilamide, also probably exhibit antimicrotubular activities in fungi.

Pencycuron [ 1 -(4chlorobenzyl)-1 cyclopentyl-3-phenylurea] is a non- phytotoxic substituted urea which is highly effective against some diseases caused by Rhizoctonia soluni (especially on potato and rice) but it is not active against Rhizoctonia cereulis. When cultivated on solid media containing pencycuron, the sensitive strains of R. solani present particular hyphal branchings. Similar symptoms are produced by carbendazim and griseofulvin but not by carboxin, mepronil, iprodione or tolclofos-methyl. Pencycuron and griseofulvin cause similar distortions of germ tubes on both benzimidazole-resistant and benzimidazole- sensitive strains of Botrytis cinerea and Pseudocercosporella herpotrichoides. However, some resistant phenotypes exhibit an increased susceptibility to pencycuron or griseofulvin (Table 1). With both toxicants the previous effects are observed at lower concentrations when temperature decreases.

Zarilamide [(RS)-4chloro-N-(cyano(ethoxy)methyl)benzamide] is a new systemic fungicide mainly active against Oomycete pathogens. Its chemical structure is similar to that of the herbicide propyzamide which shows antimicrotubular effects in plant cells.’ However, this herbicide is not toxic to Oomycetes and it never induces twisting of germ tubes in other fungi. By contrast, zarilamide produces such types of symptom in some benzimidazole-resistant phenotypes of B. cinerea or P . herpotrichoides which simultaneously are highly sensitive to this fungicide. Such effects are similar to those observed with the phenylcarbamates MDPC and diethofencarb (Table 1 ).

In conclusion, although pencycuron and zarilamide have different chemical structures and antifungal activities, both fungicides can be considered to produce antimicrotubular effects in fungi. This mode of action, deduced from the results of simple biological tests, has to be confirmed by cytological studies. Biochemical experiments must be done to elucidate the exact mechanism of action of these two fungicides and to determine the causes of their selectivity. Finally, the risk of development of strains resistant to pencycuron or zarilamide must not be underestimated; this phenomenon has been reported, not only with benzimidazole fungicides but also with griseofulvin in the dermatophyte Arthrodermi simii5 and with herbicidal dinitroanilines in the weed Eleusine indica,6 all of which act on cellular microtubules.

References 1. Corbett, J . R., Wright, K. & Baillie, A. C. , The Biochemical Mode of Action of Pesticides.

2. Leroux, P. & Gredt, M., Neth. J . PI. Path., 95 (1989) 121-7. 3. Davidse, L. C., Ann. Rev. Phytopathol., 24 (1986) 43-65. 4. Suzuki, K., Kato, T., Takahashi, J. & Kamoshita, K., Nihon Noyaku Gakkaishi

5. Dekker, J. , In Mode of Action of Antijiungal Agents, ed. A. P. J . Trincy & J. F. Ryley.

6. Vaughn, K. C., Marks, M. D. & Weeks, D. P., Plant Physiol., 83 (1987) 956-64.

Academic Press, London, 1984, pp. 202-23.

( J . Pestic. Sci.), 9 (1984) 497-501.

Cambridge University Press, Cambridge, 1984, pp. 89-1 1 1 .


Benzoylphenyl Ureas: Detoxification, Synergism and Modes of Resistance in Tribolium castaneum and Spodoptera littoralis

Isaac Ishaaya

Department of Entomology, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel

The search for potent acylureas led to the development of chlorfluazuron, teflubenzuron and hexaflumuron, which are considerably more potent than diflubenzuron (DFB) against various agricultural pests.'-4 The high toxicity of these recent acylureas results from their high retention in the insect as a result of rapid transport from the gut into the larval tissues and/or of lower detoxificati~n.~?~ In assays carried out with Tribolium castaneum, hexaflumuron, teflubenzuron and chlorfluazuron were 4- to 23-fold more toxic than DFB and exhibited similar toxicity on both malathion-susceptible and -resistant strains. On the other hand, DFB was considerably less toxic to the resistant strain, which seems to be due to DFB's susceptibility to the relatively high oxidative and hydrolytic activities present in this strain (Table 1). Assays using radiolabelled DFB and chlorfluazuron applied to fourth-instar Tribolium larvae revealed a rapid elimination of DFB (Ti = 7 h) as compared with chlorfluazuron (Ti> 100 h). This was followed by an increase in labelled residues of DFB, as compared with chlorfluazuron, in the faeces5 Addition of hydrolase inhibitors such as phenyl saligenin cyclic phosphonate and S,S ,S- tributylphosphorothioate (DEF) to the diet, increased the retention time (Ti) of DFB in Tribolium larvae from 7 to 9.5 and 18 h, respectively. Furthermore, an addition of l00mg kg-' DEF to the diet increased the toxicity of DFB to T. castaneum and Spodoptera littoralis, which was probably due to inhibition of DFB hydrolase a~t ivi ty .~

Hydrolytic cleavage seems to be a major route for DFB detoxification in S .

TABLE 1 LC,, and LC,, Values (mg kg- ') and Potency Relative to Diflubenzuron (in Parentheses) of Four Chitin Synthesis Inhibitors obtained with Malathion-Susceptible (bb) and -Resistant

(CTC-12) Strains of Triboliurn castaneum'

Compound bb CTC-12

Lc50 LC95 LC50 LC95

Hexaflumuron 0.068ab (7.1) 0.108a (9.8) 007Oa (10.2) 0.116a (22.6) Teflubenzuron 0.092b (5.2) 0.148b (7.2) 0.104b (6.9) 0.188b (14.0) Chlorfluazuron 0.106b (4.5) 0.156b (6.8) 0.108b (6.7) 0.176b (14.9) Diflubenzuron 0.480C 1.060C 0.720~ 2 .63~

Data are mean of 15-20 replicates of 15 first-instar larvae in each case. Cumulative larval and pupal mortality was determined after Ishaaya et al.' * Data followed by different letters are significantly different from each other at P = 0.05 within the same group and column.


littoralis, and may serve to monitor insect resistance to DFB or other benzoylphenyl ureas. Hence an optimized assay for determining larval gut DFB hydrolase activity has been de~e loped .~ The optimal conditions for the enzyme activity were 0.9 mg protein of the post-mitochondria1 supernatant fraction incubated for 2 h at 37°C and ['"C] DFB (0.5 nmol; uniformly labelled on the aniline ring) in glycine-NaOH buffer (0.05 M; 0.4 ml; pH 9.0). The radiolabelled metabolites were separated and evaluated quantitatively using TLC and autoradiography. Chloroaniline, chlorophenylurea and polar materials were the major metabolites. The enzyme activity was totally inhibited in the presence of lop5 M DEF or profen~fos.~

Resistance studies revealed that a field strain of S . littoralis, which was over 100- fold more resistant to pyrethroids and organophosphorus compounds than a susceptible laboratory strain, showed a mild cross-resistance to teflubenzuron.' An addition of DEF (0.1 g litre-') to various concentrations of teflubenzuron considerably enhanced the toxicity against the field strain, indicating that a large part of its resistance is due to increased metabolic activity.*

Benzoylphenyl ureas affect the larval stages which are actively synthesizing chitin."." Hence, the adults of non-target species, e.g. parasites and predators, are seldom affected. Parasites of the housefly Musca domestica and of the gypsy moth Lymantria dispar are not appreciably affected by diflubenzuron."-'3 In some cases, parasite larvae inside treated hosts are sensitive to diflubenzuron but the adults are not affected.'"^'^ Predatory mites and adult predators are not appreciably affected when fed on treated l a r ~ a e . ' " ~ ' ~ ~ ' '

The high potency of benzoylphenyl urea compounds, especially the recent ones, on various lepidopterous pests such as Spodoptera and Heliothis spp. along with their favourable persistence and low toxicity to natural enemies and mammals, render these compounds valuable components in Integrated Pest Management programmes. Strategies aiming at preventing or slowing down the development of resistance are of utmost importance in prolonging the life span of these new selective control agents for the benefit of agriculture.

References

1. Ishaaya, I., Yablonski, S. & Ascher, K. R. S. In Proceedings of the Fourth International Working Conference on Stored-Product Protection, ed. E. Donahaye and S. Navaro. Caspit, Jerusalem, 1987, pp. 613-22.

2. Ishaaya, I. In Pesticides and Alternatives, ed. J . E. Casida. Elsevier Science Publishers BV, Amsterdam, 1990, pp. 365-76.

3. Becher, H.-M., Becker, P., Prokic-Immel, R . & Wirtz, W., Proc. 10th I n t . Congr. Plant Prot., Brighton, Vol. 1 , 1983, pp. 408-15.

4. Sbragia, R . J., Bisabri-Ershadi, B., Regterink, R . H., Clifford, D. P. & Dutton, R. , Proc. 10th Int. Congr. Plant Prot., Brighton, Vol. I , 1983, pp. 417-24.

5. Gazit, Y., Ishaaya, I. & Perry, A. S., Pestic. Biochem. Physiol., 34 (1989) 103-10. 6. El Saidy, M. F., Auda, M. & Degheele, D., Pestic. Biochem. Physiol., 35 (1989) 211-22. 7. Ishaaya, I. & Degheele, D., Pestic. Biochem. Physiol., 32 (1988) 180-7. 8. Ishaaya, I . & Klein, M., J . Econ. Entomol., 83 (1990) 59-62. 9. Ishaaya, I . & Casida, J . E., Pestic. Biochem. Physiol., 4 (1974) 48490.

10. Post, L. C., de Jong, B. J . & Vincent, W. R., Pestic. Biochem. Physiol., 4 (1974) 473-83. 1 1 . Ables, J . R., West, R . P. & Shepard, M., 1. Econ. Entomol., 68 (1975) 6 2 2 4 . 12. Shepard, M. & Kissam, J . B., J. Georgia Entomol. Soc., 16 (1981) 222-7.


13. Skatula, V. U., Aufeiger f u r Schadlingshunde Pfanzenschutze Umweltschutz, 48 (1975)

14. Broadbent, A. B. & Pree, D. J., Enuiron. Entomol., 13 (1984) 133-6. 15. Granett, J. & Weseloh, R. M., J . Econ. Entomol., 68 (1975) 577-80. 16. Anderson, D. W. & Elliott, R. H., Canad. Entomol., 114 (1982) 733-7. 17. Jones, D., Snyder, M. & Granett, J., Ent . Exp. Appl., 33 (1983) 2 9 M .

145-7.

Atypical Inhibition of Phytoene Desaturation by 2-(4-Chloro-2-nitrobenzoyl)-5,5- dimethylcyclohexane-l,3dione

Gerhard Sandman, Peter Boger

Lehrstuhl fur Physiologie und Biochemie der Pflanzen, Universitat Konstanz, PO Box 5560, D-7750 Konstanz, Germany

Izumi Kumita

Odawara Research Centre, Nippon Soda Co. Ltd, Takada, Adowara, 252002, Japan

All relevant bleaching herbicides known so far interfere with desaturation of phytoene, leading to decreased formation of carotenoids and subsequent photo- bleaching of chlorophyll (see Ref. 1 for review). In-vitro studies with norflurazon and other herbicides demonstrated that the enzyme phytoene desaturase is non- competitively inhibited by such herbicides.’ For several benzoyl cyclohexane-l,3- diones and related compounds, accumulation of phytoene has been demonstrated as the herbicidal mode of action.394 The present study of the mode of action was performed to investigate the interference of 2-(4chloro-2-nitrobenzoyl)-5,5- dimethylcyclohexane-1,3-dione5 in phytoene desaturation.

The effect of this compound on the formation of coloured carotenoids and on accumulation of phytoene was assayed with 3-day-old cress seedlings (Lepidiurn satiuum). Seeds were germinated on filter paper discs treated with herbicide solutions (Table 1). Decrease of carotenoid content was observed in light-grown as well as in dark-grown seedlings, and this was accompanied by accumulation of phytoene. Carotenoid bleaching was stronger in the light than in the dark and less phytoene was detected. This might be due to additional secondary photo-oxidative effects. Furthermore, chlorophyll, formed in the light, was also decreased in herbicide-treated seedlings. The percentage composition of coloured carotenoids in dark-grown seedlings was not greatly changed under the influence of the herbicide.

The effect of this cyclohexane- 1,3-dione was also assayed with soybean seedlings and cell-suspension cultures, as well as with eukaryotic and prokaryotic microalgae (Table 2A). When soybeans were germinated in the presence of herbicide, the same results were obtained as for cress seedlings (Table 1) when treated in the same way: carotenoids of primary leaves were decreased and phytoene was accumulated. However, when the cotyledons of 3-day-old seedlings were soaked for 3 h in


TABLE 1 Carotenoid and Chlorophyll Content (jig mg-' Dry Weight) of Cress Seedlings grown for 3 Days in the Presence of 2-(4-Chloro-2-nitrobenzoyl)-

5,5dimethylcyclohexane-l,3dionea

Herbicide concentration ( M )

o 10-7 10-6 10-5

A . Light-grownb Chlorophylls Phytoene Coloured carotenoids

B. Dark-grown Phytoene Coloured carotenoids

Carotenoid distribution (%) Neoxant hin Violaxanthin Antheraxanthin Lutein 8-Cryptoxanthin 8-Caro t ene

6.188 0.001 1.213

0.005 0.235

1 .o 25.7 1.7 56.0 1.2 14-3

0.45 1 0.021 0.21 1

0996 0.192

0.4 20.6 1.1

55.1 1.4 21.3

0.137 0.113 0.111

0.155 0.163

0.6 24.9 1.9 57.4 1.1 14.0

0.029 0.21 1 0.005

0.479 0.090

0.1 23.0 1.9 57.7 2.9 14.4

Details of determination procedures and growth conditions are given in Ref. 6. bLight intensity was 60 pE m-' sec-'.

herbicide solution, pigments of primary leaves emerging thereafter were very little affected in the following growth period of 6 days. Treatment of soybean cell suspension cultures with up to lop5 M herbicide over a period of one week resulted also in a very weak decrease of carotenoids. There is no reason why uptake in the cell suspension should be restricted nor was this observed for any other herbicide using Synechococcus PCC 7942 or Scenedesmus acutus cultures. Both unicellular species showed either no change in carotenoid content with the herbicide or, in the case of Scenedesmus, a high concentration M) was needed to effect some decrease. However, it should be emphasized that, even when a 40% decrease of coloured carotenoids was observed, no accumulation of phytoene was detectable.

The use of in-vitro systems, in the present case for phytoene-desaturase activity, is the only way of demonstrating a direct interaction of a herbicide inhibitor with a potential enzyme target. For phytoene-desaturase assays, corn chloroplasts or chromoplasts from petals of Cheiranthus cheiri with [14C]phytoene as substrate and Anacystis thylakoids with [ 14C]geranylgeranyl pyrophosphate as substrate were used.' After termination of the reaction, the conversion rate was calculated as end- point analysis from radioactivity found in the labelled (- and /?carotene divided by the sum of radioactivity in phytoene, <carotene plus /?-carotene (Table 2B). The results were not significantly different in all systems. Even concentrations of up to

M showed no interference with the phytoenedesaturase reaction. However norflurazon, a typical inhibitor of phytoene desaturase used in parallel experiments as a control, gave the expected inhibition.


TABLE 2 Effect of 2-(4-Chloro-2-nitrobenzoyl)-5,5dimethylcyclohexane-1,3dione on (A) Carotenoid

Content and (Bj In-vitro Phytoene Desaturation

Control Cyclohexanedione Norflurazon 10-5 M 10-5 M

( A ) Content of colouretl carotenoids ( p g mg-' dry weight) Soybean primary leaves 0.924 0.05 1

(seed treatment)

(cotyledon treatment) Soybean primary leaves 0.870 0.731" -

Soybean cell culture 0.815 0.808" -

Scenedesmus acutus cells 5.384 3.338" - Synechococcus PCC 7942 cells 4.996 4.691" -

(B) In-vitro activity of phytoene desaturase; conversion rate (YO) Synechococcus PCC 7942 thylakoids 53.9 47.8 5.5 Cheiranthus chromoplasts 60.4 61.1 4.9 Corn chloroplasts 41.3 40.7 11.3

" No accumulation of phytoene detected.

Like the 2,4-dichlor~benzoyl~ and the 2-chloro-4-methylsulfonylbenzoyl (SC- 0051)4 analogues the 4-chloro-2-nitrobenzoyl derivative used in the present investigation inhibited formation of coloured carotenoids and caused accumulation of phytoene, provided it was applied to the roots and taken up by them. Application via leaves or to single cells did not show this effect. As phytoene desaturase is not directly inhibited by this herbicide, it is assumed that a modification of these cyclohexanediones to an active form might be necessary. Obviously, this activation is dependent on the uptake and/or translocation system of higher plants.

References 1. Sandmann, G. & Boger, P. In Target Sites of Herbicide Action, ed. P. Boger & G.

2. Sandmann, G. & Kowalczyk, S., Biochem. Biophys. Res. Commun., 163 (1989) 916-21. 3. Soeda, T. & Uchida, T., Pestic. Biochem. Physiol., 29 (1987) 3542. 4. Mayonado, D. J., Hatzios, K. K., Orcutt, D. M. & Wilson, H. P., Pestic. Biochem.

5. Eur. Pat. Appl. EP 135,191 (1985).

Sandmann. CRC Press, Boca Raton, FL, 1989, pp. 25-44.

Physiol., 35 (1989) 13845.

6. Sandmann, G., Ward, C. E., Lo, W. C., Nagy, J. 0. & Boger, P., Plant Physiol., in press (1990).

A Comparison of Mammalian and Insect GABA Receptor Chloride Channels

Daniel B. Gant," Jeffrey R. Bloomquist,b Hafez M. Ayad" & Alison E. Chalmers"

"Rhbne-Poulenc AG Co.. Research Triangle Park, NC, 27709, USA bVirginia Polytechnic Institute, Entomology Department, Blacksburg, VA, 24061, USA

4-Aminobutyric acid (CiABA) acts as an inhibitory neurotransmitter in both the vertebrate and invertebrate CNS and at the invertebrate neuromuscular junction.''2


GABA receptors are an important site of action for insecticides, and detailed knowledge of the pharmacology of insect and vertebrate receptors may help in the design of potent and possibly insect-specific insecticides.

TBPS (4-tert-butyl-l,l-dihydro-2,6,7-trioxa-l-phosphabicyclo[2.2.2]octane- 1-thione and p-CN-tBUOB (4-tevt-butyl-1-(4-cyanophenyl)-2,6,7-trioxabicyclo [2.2.2]octane) have been used as radiologand probes to investigate GABA receptor chloride channel^.^ Specific C3%]TBPS binding sites were identified from membrane homogenates of both rat brain and housefly heads. Rat brain binding was approximately 90% specific, compared to 35% specific in the housefly preparation. Scatchard plots of equilibrium binding indicated one site in both tissues with KD = 30 nM, B,,, = 5.34 pmol mg-' protein and KD = 113 nM, B,,,=0.53 pmol mg-' protein in rat and housefly heads, respectively. Hill coefficients were approximately 1.0 for both membrane preparations. [35S]TBPS binding in the rat was inhibited by the insecticides endrin (IC,,= 1 3 n ~ ) and p- CN-tBUOB (ICso = 1-5 nM). However, C3%]TBPS binding in the fly preparation differed from that of the rat in several ways. Housefly C3%]TBPS binding was inhibited by endrin (ICso = 13 nM) but not by p-CN-tBUOB. Equilibrium and kinetic analysis indicated that both endrin and p-CN-t BUOB bound competitively in the rat, whereas in the fly, equilibrium analysis indicated that endrin bound non- competitively. Finally, unlike the rat, insect [35S]TBPS binding was not allosterically modulated by GABA or benzodiazepines.

Topical application bioassays were carried out on a cyclodiene-resistant strain of housefly, Musca domestica. The response of the adult female flies to dieldrin and to p-CN-t BUOB was measured and compared with the response of a susceptible strain. Dieldrin LD,, values were 0.3 pg g-' body weight and 867.3 pg g-' body weight, and p-CN-tBUOB LD,, values were 20.3pgg-' body weight and > 609.8 p g g- ' body weight for susceptible and resistant houseflies, respectively. Resistance ratios of 2891 for dieldrin and > 30 for p-CN-tBUOB suggest that the flies possessed a high resistance to dieldrin and moderate cross-resistance to

Physiological studies were performed on third-instar housefly larvae from both susceptible and cyclodiene-resistant strains. CNS and peripheral nerves were removed and transferred to a bath filled with saline. Spontaneous nerve activity was monitored by attaching a recording suction electrode to a peripheral nerve trunk and placing a silver reference electrode in the bath. Preparations were exposed to 10 mM GABA to abolish nerve activity and provide a baseline against which to measure the effects of dieldrin and p-CN-tBUOB. 1 p~ dieldrin elicited an increase in spike frequency in susceptible preparations, whereas resistant larvae showed little or no response to concentrations below 10 p ~ . The threshold concentration of p- CN-tBUOB to elicit a response was lOOnM in susceptible larvae, and 1 PM in resistant larvae. This indicates that a change in sensitivity of the nervous system is partially responsible for the observed resistance.

The binding assay results demonstrate that there are differences between the GABA receptor chloride channels of mammalian and insect nervous systems. TBPS, endrin and p-CN-tBUOB bind to the same site in the mammalian system, whereas p-CN-tBUOB appears to bind to a separate site in the fly GABA receptor

p-CN-tBUOB.


chloride channel. Further investigations of changes occurring in cyclodiene- resistant insects may give additional insight into insect GABA receptor pharmacology.

References 1. Olsen, R . W. & Venter, J. C. (eds). BenzodiazepinefGABA Receptors and Chloride

2 . Lummis, S . C. R., Comp. Biochem. Physiol., 95C (1990) 1-8. 3. Casida, J. E., Nicholson, R. A. & Palmer, C. J., Neurotox '88: Molecular Basis of Drug

and Pesticide Action, ed. G. G. Lunt. Elsevier Science Publishers, Amsterdam, 1988, pp. 12544.

Channels: Structurul and Functional Properties. Alan Liss, NY, 1986.

Mode of Action of Sterol Biosynthesis Inhibitors in Obligate Parasites

Rolf Pontzen; Birgit Poppeb & Dieter Berg"

"Bayer AG, Agrochemical Division, PF-Zentrum Monheim, 5090 Leverkusen, Germany bInstitute of Biology 111, RWTH Aachen, 5100 Aachen, Germany

So far, all studies on the mode of action of sterol biosynthesis inhibitors have been performed with yeasts or plant pathogens which can be easily cultured in uitro. Obligate parasites have not yet been included in such studies although they are the main target organisms for these fungicides. Therefore the mechanism of action of triadimenol and tebuconazole in Erysiphe graminis f. sp. hordei (barley powdery mildew) and Puccinia graminis f. sp. tritici (wheat stem rust) was investigated.

A new experimental approach was used for the analysis of powdery mildew sterols. Mildew lipids were extracted from epidermal strips of severely infected barley leaves and not from mycelium scraped off the leaf surface or from conidia. This unique technique yields mildew mycelium plus haustoria which was expected to be very sensitive to azole treatment.

Powdery mildew sterols were isolated from epidermal strips five days after inoculation. At that time the developing mildew mycelium had not yet started to produce conidia. Triadimenol and tebuconazole were applied three days after inoculation. The epidermal strips were shock frozen in liquid nitrogen, pulverized and freeze-dried (dry weight: 8&120 mg). Lipids were extracted with chloroform + methanol (2 + 1 by volume) and saponified. The unsaponifiable lipid fraction was further purified by means of silica cartridges and analysed by GC-MS.

Stem rust sterols were analysed using axenic cultures of the pathogen.' Dependent on growth conditions, different colony types developed. Sterols were extracted from spores and from rust colonies (4-5 weeks old) composed of vegetative or sporulating mycelium. Triadimenol(2 pg litre-') was added to the agar medium prior to inoculation with spores. The sterols were purified and analysed as described above. Some sterols were further purified by HPLC and identified by GC-MS and NMR.

Large quantities of mildew sterols could be isolated from severely infected

w

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359 7th International Congress of Pesticide Chemistry 359

Fig. 1. Gas-liquid chromatograms of the sterol fractions isolated from axenic culture of wheat stem rust: 1 : A5.22-stigmastadieno1; 2: 8’-ergostenol; 3: As-stigmastenol; 4: lanosterol; 5: A7-stigmastenol; 6:

A7.24(2s)-stigmastadienol; 7: 24-methyl dihydrolanosterol.

epidermal strips of barley: 15-22% of the total sterol fraction originated from the pathogen. A5.24(28)-Ergostadien~l was the predominant sterol of mildew mycelium, and not ergosterol. The same sterol was isolated from conidia.2 Application of triadimenol or tebuconazole induced an accumulation of 14a-methyl sterols (obtusifoliol and 24-methylene dihydrolanosterol) with a concomitant reduction in A5324(28)-ergostadienol. This indicated an inhibition of sterol 14a-methyl demethylase, the well-known target of azole fungicides in non-obligate plant pathogens and yeasts. The increased obtusifoliol content of treated epidermal strips, however, was due only partly to an inhibition of mildew sterol biosynthesis: azole application to leaves without mildew infection also induced a minor, but significant, accumulation of obtusifoliol (Table 1).

Untreated mycelium of stem rust contained A7~24(28)-stigmastadienol, A7- stigmastenol and minor amounts of A5-stigmastenol, A’-ergostenol and A5,22- stigmastadienol (Fig. 1). The sterol profiles differed according to the colony type of the axenic culture. The predominant sterol of vegetative mycelium was A7*24(28)- stigmastadienol (59.9%) which has also been identified in spores of flax rust and bean Sporulating mycelium, on the other hand, contained only 24.7% of that sterol. The amount of A7-stigmastenol in sporulating mycelium was relatively high (25.7 %)compared with the vegetative mycelium (7.0%). Very remarkable was the high concentration of lanosterol in rust mycelium, especially in sporulating colonies (18.4 %). This may be due to a slow sterol C-14 demethylati~n.~ Treatment of axenic rust cultures with triadimenol (2pg litre-’) resulted in a strong accumulation of 24-methylene dihydrolanosterol (50.2 % of total sterols).

The results presented reveal that the obligate parasites barley powdery mildew and wheat stem rust both have a slightly modified sterol biosynthetic pathway compared with other fungi in that they do not produce ergosterol. Powdery mildew synthesized A5324(28)-erg~~tadien~l and wheat stem rust mainly A’-sterols. Nevertheless, the mode of action of triazole fungicides is identical with that found in


non-obligate parasites: the cytochrome P-450-dependent sterol 14a-methyl demethylase is inhibited, as indicated by the accumulation of 14a-methyl sterols in the treated mycelium.

References 1. Kuck, K. H. & Reisener, H. J., Physiol. Plant Pathol., 27 (1985) 25948. 2. LoeMer, R . S. T., Butters, J . A . & Hollomon, D. W., Proc. I984 Brit. Crop Prot. Conf.

3. Lin, H.-K., Langenbach, R. J. & Knoche, H. W., Phytochem., 11 (1972) 2319-22. 4. Jackson, L. L. & Frear, D. S., Phytochern., 7 (1968) 6514. 5. Pontzen, R. & Scheinpflug, H., Neth. J . PI. Path., 95 (1989) Supplement 1, 151-60.

Pests and Diseases, 3 (1984) 911-16.

Mode of Action of Fenpropimorph, Fenpropidin and Tridemorph in Fusarium spp.

Daniele Debieu, Claude Gall, Jocelyne Bach, Alexandrine Lasseron, Michel Gredt, Christian Malosse & Pierre Leroux

INRA, Station de Phytopharmacie, 78026 Versailles Cedex, France

Fenpropimorph, fenpropidin and tridemorph, which are used in agriculture as fungicides, inhibit sterol biosynthesis. They have two potential targets; the A I 4 -

sterol reductase and the As-+A7-sterol isomerase. These two enzymes are inhibited differently, depending on the fungus and on the compound.‘ Since few studies have been done on filamentous fungi, these fungicides have been tested on several Fusarium spp., using laboratory trials to determine the effects of these toxicants on mycelial growth and sterol content. These experiments were carried out with fungi cultivated on agar medium and in shake culture, respectively. The sterol content was analysed by GC and the structure determined by UV and MS.

According to their response towards fenpropimorph, it was possible to define ‘sensitive’ and ‘tolerant’ Fusarium spp. The ‘sensitive’ ones included F. nivale, F . solani f. sp. pisi, F . solani var. minus, F . javanicum var. javanicum and F . decemcellulare. Their EC,, values (fungicide concentration causing 50 % growth inhibition) were <0.2 mg litre-’ (Table 1). The ‘tolerant’ species included F . solani var. creruleum, F. lateritium, F. roseum var. culmorum, F. roseum var. samhucinum, F . roseum var. gsaminearum, F . roseum var. arthrosporioides, F . semitectum, F. monoliforme var. subglutinans and F . oxysporum f. sp. melonis; their EC,,, values were >2mg litre-’ (Table 1). All the species appeared ‘tolerant’ towards tridemorph except F. nivale, which was highly sensitive (EC,, <0.05 mg litre-’). With fenpropidin, which was less active than fenpropimorph, the highest efficiency was with ‘sensitive’ Fusarium species, except F . nivale which showed a reduced sensitivity (data not shown).

Among the ‘sensitive’ species, F. solani f. sp. pisi was chosen for a comparison of the effect of the three fungicides on its sterol composition. Ergosterol was the major sterol in untreated mycelium, representing 95 % of 4desmethyl sterols, which

TAB

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themselves represented 90% of total sterols. Fecosterol (ergosta-8,24(28)-dien-3j?- 01) and lichesterol (ergosta-5,8,22-trien-3P-o1), both A'-sterols, were characterized as minor 4desmethyl sterols (2 %). No A8,14-sterols were detected. With fenpropimorph at concentrations ranging from 0.03 to 5 mg litre- ', the ergosterol relative amount decreased from 84 to 1 % of 4-desmethyl sterols and, simultaneously, A','4-sterols appeared and increased from 7 to 91 % of 4-desmethyl sterols. These A8*14-~tero1~ were identified as ergosta-8,14,24(28)-trien-3fl-ol, ergosta-8,14-dien-3fi-ol (ignosterol) and ergosta-5,8,14,22-tetraen-3j?-ol. At low concentrations of fenpropimorph (0.03 and 0.15 mg litre-'), a slight increase of lichesterol, a A8-sterol was observed. Such results suggest that the two enzymes are good targets for fenpropimorph in F. solani f. sp. pisi. The prevailing accumulation of A'~'4-sterols is due to the fact that AI4-sterol reductase acts before A8+A7-sterol isomerase in sterol biosynthesis. With fenpropidin, only A8"4-sterols were detected; this suggests that AI4-sterol reductase is the most sensitive target of fenpropidin. With tridemorph concentrations ranging from 0.6 to 12 mg litre-', the ergosterol relative amount decreased from 60 to 30% of 4-desmethyl sterols whereas the A'- sterols increased from 32 to 65 % of 4desmethyl sterols. These accumulated A'- sterols were identified as fecosterol, lichesterol and ergosta-8-en-3fi-01 in a lower amount. A8,'4-sterols were detected only with tridemorph concentrations above 2.4 mg litre- ' ; at 12 mg litre- ', they represented 2.5 % of 4-desmethyl sterols. These observations indicate that the most sensitive target of tridemorph in F . solani f. sp. pisi is A8+A7-sterol isomerase. All these results are consistent with those obtained with Saccharomyces cerevisiae on mycelial sterol composition and enzymic inhibition in cell-free enzyme systems.'-2

As with F . solani f. sp. pisi, ergosterol was the major 4-desmethyl sterol in all the other untreated Fusariuni species. Also, as shown in F. solani f. sp. pisi, fenpropimorph treatment led to a great amount of mainly A8*14-sterols in the mycelium of the other 'sensitive' Fusarium species ( F . solani minus, F . javanicum var. javanicum and F . decemcellulare), except in F . nivale which accumulated only A'- sterols. Among the 'tolerant' species, there was either an accumulation of mainly A'-sterols ( F . roseum var. arthrosporioides, F . monoliforme var. subglutinans and F . oxysporum f. sp. melonis) or of both A' and A8.14-sterols (F. solani var. caruleum, F. lateritium, F . roseurn var. culmorum, F . roseum var. sambucinum, F . roseum var. graminearum and F . semitectum). In all cases the ergosterol relative amount decreased (Table 1).

In conclusion, fenpropimorph may inhibit A14-sterol reductase and/or A8-A7- sterol isomerase, depending on the Fusarium species. However, A'4-sterol reductase inhibition seems to result in the maximum fungitoxicity, except for F . niuale. Differences in sensitivity of this enzyme may explain fenpropimorph selectivity among Fusarium species. This hypothesis is strengthened by the fact that fenpropidin, described as a potent A14-sterol reductase inhibitor,'-3 led to a similar selectivity among Fusarium species. The results obtained with tridemorph suggest that the A'-A7-sterol isomerase from the various Fusarium spp. exhibits a similar sensitivity towards this fungicide. However, for F. nivale, the fenpropimorph fungitoxicity seems determined by A8+A7-sterol isomerase sensitivity. On the phylogenic tree, F . nivale is the most distant species4 among all those studied. This may explain such differences in fungitoxicity with regard to enzyme sensitivity.


References 1. Baloch, R. I., Mercer, E. I., Wiggins, T. E. & Baldwin, B. C., Phytochemistry, 23 (1984)

2. Baloch, R. I. & Mercer, E. I., Phytochemistry, 26 (1987) 663-8. 3. Debieu, D., Akallal, R., Lasseron, A., Malosse, C. & Leroux, P., C . R . Acad. Agric. Fr. ,

4. Guadet, J., Julien, J., Laffay, J.-F. & Brygoo, Y., Mol. Biol. Evol., 6 (1989) 22742.

2219-26.

75(6) (1989) 179-81.

Mode of Action of the Phenylurea Fungicide Pencycuron in Rhizoctoniu soluni

Isao Ueyama," Yasuo Araki," Shin Kurogochi," Katsuyoshi Yoneyamab & Isamu Yamaguchi'

"Nihon Tokushu Noyaku Seizo K.K., Yuki Research Center, Yuki 9511-4, Ibaraki 307, Japan bMeiji University, Kawasaki, Kanagawa 214, Japan 'The Institute of Physical & Chemical Research, Hirosawa, Wako, Saitama 351-01, Japan

Pencycuron ['Monceren'@; l-(4chlorobenzyl)-lcyclopentyl-3-phenylurea] is a phenylurea fungicide which has specific activity for the control of plant diseases caused by Rhizoctonia solani, such as rice sheath blight and potato black scurf. The antifungal activities are extremely selective, even within the Anastomosis Groups (AG) of R. solani.' Pencycuron induces abnormal branching of the hyphae of the sensitive strains and its activity is static. The mode of action of pencycuron has been reported already in part,* but the evidence was inconclusive.

In this study the mode of action of pencycuron has been further investigated using ['4C]pencycuron and four strains of R. solani. These were C-423 (AG-1) and RC (AG-4) as pencycuron-sensitive, and Rh-131 (AG-4) and SH-1 (AG-5) as tolerant strains.

Uptake of ['4C]glucose was examined by the method of Nagashima et aL3 using a hyphal suspension of C-423. [ '4C]Gluc~~e was transformed mainly into trehalose, but this reaction was not inhibited by pencycuron at the concentration tested (0.5 p g ml-I). In addition, trehalose-6P-synthase activity was measured in a cell-free ~ y s t e m , ~ using ['4C]UDP-glucose and [14C]GDP-glucose as glycosyl donors. The results indicated that pencycuron had no direct effect on the biosynthesis of trehalose in the strains tested. Further experiments on trehalose metabolism in R. soluni were carried out in accordance with the method of Shigemoto et ~ l . , ~ and showed that pencycuron did not inhibit trehalase activity at 5 x M, in contrast to the findings that Validamycin A inhibited it at even lower concentrations (0.4-5 x M).

Next, the uptake of [' 4C]alanine and [14C]phenylalanine by the fungi and their incorporation into the fungal protein fraction were tested. Treatment with pencycuron, however, did not show any significant change in these items. The incorporation of ['4C]thymidine into DNA was then determined in strain C-423; pencycuron had no effect on DNA biosynthesis even at 2-0 pg ml- ', where growth of C-423 was inhibited completely.

The effect of pencycuron on the chitin biosynthesis system in R. solani was then

364 7th lnternationnl Congress of Pesticide Chemistry

TABLE 1 Growth Inhibition by Pencycuron and MBC in R. solani and Protein Binding Constant K

Strain AG" Conc. for 50% inhibition on PDA (pg r n 1 - I ) Protein binding constant K

Pencycuron MBC (mole-')b

C-423 1 0.05 0.50 1.0 x 104 Rc 4 0.025 0.75 5.3 x 104 Rh-131 4 c . 10 0.75 5.9 x 1 0 3 SH-1 5 c. 10 1.0 2.0 x 103

a Anastomosis Group. bObtained from 48 0009 supernatant from R. solani strains (22 mg ml-' protein) and pencycuron.

investigated by the method of Hori et aL6 Pencycuron inhibited ['4C]glucosamine incorporation into the chitin of C-423, but did not interfere with the reaction in Rc, another sensitive strain. Moreover, accumulation of ['4C]UDP-GlcNAc in C-423 was not observed in C-423 hyphae with pencycuron under conditions where significant accumulation was observed with polyoxin D. Thus the effect of pencycuron on chitin synthesis shown in the C-423 strain might not represent a primary mode of action.

The morphological change in hyphae of R. solani was studied by the method of Roberson et al.' Abnormal branching was seen in pencycuron-treated fungi and this was quite similar to that with MBC-treated fungi. This abnormal branching was not observed in the resistant strains (Rh-131 and SH-1) even at high concentration of pencycuron.

The affinity of pencycuron for the soluble fractions (48 O O Q supernatant) of the test strains was then examined and the binding constant ( K ) determined according to the method described by Davidse et a1.8 Pencycuron was bound more to the proteins existing in the 48 0009 supernatant from sensitive than to those from resistant strains (Table 1).

To summarize, from the results so far obtained, pencycuron hardly affected trehalose biosynthesis, trehalase activity, chitin, protein and DNA biosyntheses. Further biochemical and morphological studies using anti-R. solani chemicals such as Validamycin, Flutolanil and Polyoxin D, suggested that pencycuron had a different mode of action from those compounds. The morphological changes resembled those caused by MBC. Since the incorporation rate of ['4C]pencycuron into mycelium of sensitive and non-sensitive strains was almost identical, the presence of a specific binding site for pencycuron in the sensitive strains is suggested.

References 1. Yamada, Y., Saito, J. & Takase, I . , Nihon Noyaku Gakkaishi ( J . Pestic. Sci.), 13 (1988)

2. Kuck: K. H., Ueyama, I., Kurogochi, S., Yamada, Y. & Schneider-Christians, J.,

3. Nagashima, H., Ozaki, H., Nakamura, S. & Nishizawa, K., Bot. Mag. Tokyo, 82 (1969)

3 75-87.

Abstracts 5th International Congress of Plant Pathology, p. 22, Kyoto, 1988.

462-73.


4. Elbein, A. D., J . Bacteriol., 96 (1968) 1623-31. 5 . Shigemoto, R., Okuno, T. & Matsuura, K., Ann. Phytopath. SOC. Japan, 55 (1989) 23841. 6. Hori, M., Kakiki, K. & Misato, T., Agric. B i d . Chem., 38 (1974) 691-8. 7. Roberson, R. W., Fuller, M. S. & Grabski, C., Pestic. Biochem. Physiol., 34 (1989) 13W2. 8. Davidse, L. C. & Flach, W., J . Cell Biol., 72 (1977) 174-93.

Effects of Phosphonate on Phosphate Metabolism in Phyrophrhora cirrophrhora

Thierry Barchietto," Patrick Saindrenanb & Gilbert Bompeixb

"Biotransfer Ltd, 12 Avenue du General Leclerc, 75014 Paris, France bLaboratoire de Biochimie et Pathologie Vegetales, Universite P. et M. Curie, Tour 53, 2eme et., 4 Place Jussieu, 75252 Paris Cedex 05, France

The phosphonate ion (H,PO;) is the active metabolite of the phloem-translocated anti-Oomycete compound, fosetul-aluminium. The activity of this anion towards mycelial growth of several Phytophthora species was reversed by its structural analogue orthophosphate (Pi) in uitro as well as in uiuo. In a previous study, Pi was shown to be a partial competitive inhibitor of phosphonate uptake in P . cryptogea and P , citrophthora.' Thus, the antagonistic effect of Pi may not be due only to competition between the two ions at the uptake level. The present study reports the effects of phosphonare on several enzymic reactions which use Pi as a substrate of phosphorylation and as a regulator of their activities, or which are involved in metabolic pathways regulated by Pi in Phytophthora citrophthora.

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12), an enzyme of the glycolytic pathway, utilizes Pi and phosphonate as Physiological substrates. Though the enzyme shows an affinity for phosphonate ( K , = 0.60 mM) similar to that for phosphate ( K , = 0.38 mM), the maximum velocity for the former ion (V,,,=8 nKat mg-I protein) is six times lower than for the latter (V,,,= 46 nKat mg-' protein). The interaction between both anions for the catalytic site of this enzyme could lead to the reduction of the metabolic flux of precursors and explain the decrease in the ATP concentration, as well as the decrease in the lipid biosynthesis and in the respiratory activity observed after treatment of the organism.

Phosphonate treatment causes an increase, by a factor of two, in the intracellular Pi content of Phytophora citrophthora, and greatly enhances the activity of intracellular alkaline phosphatase (EC 3.1.3.1), a Pi starvation-induced enzyme.

Phosphonate enhances hexokinase activity (EC 2.7.1.1), the activity of both key enzymes of the pentose phosate pathway, glucose 6-phosphate- (EC 1 .I .1.49) and 6- phospho gluconate dehydrogenase (EC 1 .I. 1.44), and the activity of uridine-5'- diphospho-glucose pyrophosphorylase (E3 2.7.7.9) which is involved in the biosynthesis of P-glucans (Table 1). The pentose phosphate pathway is involved in secondary metabolism, whose expression, like that of primary metabolism, was regulated by the phosphate availability inside the cell. A phosphate limitation led to

366 7th Internutionul Congress of Pesticide Chemistry

TABLE 1 Effects of Phosphonate (0.5 mM) on the Activities of Hexokinase, Glucose 6-phosphate-, 6- Phospho gluconate dehydrogenase and Uridine-S’diphospho-glucose pyrophosphorylase

from P. citrophthora

Enzymes Specific activities (pKat . mg- protein) (f SD)”

Control (0 h) Control (24 h ) Phosphonate (24 h)

Hexokinase 0.046 ( f 0.005) 0.034 ( f O.OO4) 0.071 ( f 0.007) Glucose 6-P dehydrogenase 0.01 8 (k 0.010) 0.038 (k 0.008) 0.401 (k 0018) 6-P gluconate dehydrogenase 0.009 (k 0.004) 0.045 ( & 0.007) 0.479 (& 0-022) UDP-glucose pyrophosphorylase 1.090 (f 0.051) 0.980 (f 0.044) 8.700 (f0.060)

a Mean of 3 replicates.

the derepression of genes coding for the synthesis of the enzymes induced in these pathways.

The results indicate that not only are both anions potent substrates for phosphorylating enzymes, but also that phosphonate interacts with Pi as an anti- repressor of the activity of several enzymes. Thus, the interaction between both anions leads to a physiological state similar to a phosphate starvation.

after a primary site of action in the fungus, through an induction of the overproduction of metabolites with elicitor activity (activation) in culture filtrate^.^ It has been shown that some products of metabolic pathways involving enzymes like UDP-glucose pyrophosphorylase, such as P-glucans and glycoproteins, which have elicitor activities, were secreted in the culture filtrates of several fungi like Colletotrichum lindemuthianum and Phytophthora Another fungus, Pyricularia oryzae, produces in culture filtrate a secondary metabolite, tenuazonic acid, which induces a resistant-type response in rice tissue.’ Thus, it is suggested that the activation of the pathogen by phosphonate may be due to the deregulation of the activity of some enzymes involved in primary metabolism (e.g. glucan biosynthesis) and/or in secondary metabolism.

It is proposed that phosphonate enhances the host defence

References 1. Barchietto, T., Saindrenan, P. & Bompeix, G., Arch. Microbiol., 15 (1989) 54-8. 2. Saindrenan, P., Barchietto, T., Avelino, J. & Bompeix, G., Physiol. Mol. Plant Pathol., 32

3. Saindrenan, P., Barchietto, T. & Bompeix, G.. Plant Science, 58 (1988) 245-52. 4. Guest, D. I . , Physiol. Plant Pathol., 25 (1984) 125-34. 5 . Saindrenan, P., Barchietto, T. & Bompeix, G.. Plant Science, 67 (1990) 245-51. 6. Anderson-Prouty, A. J. & Albersheim, P., Plant Physiol., 56 (1975) 28691. 7. Keenan, P., Bryan, I . B. & Friend, J., Physiol. Plant Pathol., 26 (1985) 343-55. 8 . Farmer, E. E. & Helgeson, J . P., Plant Physiol., 85 (1987) 733-40. 9. Lebrun, M.-H., Orcival, J. & Duchartre, C., Rev. Cytol. Bid. Veget. Bot., 7 (1984) 249-59.

(1988) 425-35.

7th international congress of pesticide chemistry

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