Effect of some sterol-biosynthesis-inhibiting fungicides on the biosynthesis of polyisoprenoid compounds in barley seedings

EFFECT OF SOME STEROL-8lOSYNTHESlS-INHIBITING FUNGICIDES ON THE BIOSYNTHESIS OF POLYISOPRENOID COMPOUNDS IN BARLEY SEEDINGS Eric I. Mercer,

iqtidar

A. Khaiii;

and Zhi-Xin

Wang+

Department of Biochemistry, University College of Wales, Aberystwyth, Dyfed SY23 3DD, UK Present addresses: ‘Department of Agricultural Chemistry, N.W.F.P. Agricultural University, Peshawar, Pakistan; ‘Department of Agronomy, Agricultural University of Hebei, Baoding, Hebei, China Correspondingauthor: Eric I. Mercer, Ph D Received July 9, 1988 Revised April 17, 1989

ABSTRACT The effect of five sterol-bios~thesjs-inhibiting (SBI) fungicides, and triadimefon, triarimof, diclobutrazol, tridemorph, fenpropimorph on the germination, growth, and chloroplast pigment and sterol content of barley seedlings has been studied. Triadimefon, triarimol, and diclobutrazol at 250 MM depressed germination and growth, caused the accumulation of 14a-methyl sterols, but had no effect on the formation of chlorophylls or carotenoids. Tride~rph and fenpropimorph at 250 FM had no effect on germination or the formation of chlorophylls and carotenoids growth and caused the accumulation of but depressed 96,19-cyclopropyl sterols. INTRODUCTION One

of

the

fungicides the

most in

the

important past

two

advances

in

decades

has

sterol-biosynthesis-inhibiting

(SBI)

the

field

been

the

of

systemic

discovery

fungicides.

of

These

compounds are used widely to control the fungal diseases of cereal crops

and

appear

to

owe

their

fungitoxicity

to

their

ability

to inhibit the synthesis of the major fungal sterol, which, for the majority of fungi, is ergosterol (1).

STEROIDS

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1989 (393-4121

This

sterol

is

located

393

Mercer et al: EFFECT

394

predominantly where

it

in the

appears

interrelated function. of

OF SBI FUNCTIONS

ON BARLEY

plasma and endoplasmic

reticular membranes

be

maintenance

to

properties

essential of

for

the

fluidity,

permeability,

and

of

the

enzyme

The sterols that accumulate as a result of the action

these

fungicides

are

incorporated

into the fungal membranes

(2,3) in place of the normal membrane sterol and cause an increase in fluidity behavior these

(4,5) and abnormalities in permeability (6) and the

of

membrane-bound

membrane

enzymes

abnormalities

are

(5).

the

It

ultimate

believed

is

cause

of

that

fungal

stasis and death. The

SBI fungicides used in agriculture fall into two classes

on the basis of their site of action in fungal sterol biosynthesis. The

demethylation

inhibitors

triarimol,

and

diclobutrazol,

and

the

accumulation

cause

second

class,

fenpropimorph,

typified inhibit

by two

(DMIs), inhibit

of the

such

the

sterol

14a-methyl

the

triadimefon, 14-demethylase

sterols

morpholines,

enzymes,

as

(7-9).

The

tridemorph,

and

sterol

Albreductase

and the sterol A8+A7-isomerase (10-13).

Since 14-demethylation,

A14-reduction

and A8+A7-isomerization

are processes that occur in the biosynthesis of sterols in higher plants (14) as well as fungi, it is of interest to know the effect of these fungicides on the plants that they are used to protect. This

paper

reports

on

the

effect

of

triadimefon,

triarimol,

diclobutrazol, tridemorph, and fenpropimorph on the germination,

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Mercer et al: EFFECT

the of

OF SBI FUNCTIONS

growth barley

and

the

chlorophyll

sterol, The

seedlings.

395

ON BARLEY

chloroplast

and

carotenoid

pigments

were

content included

in the study because (i) DMI fungicides have been shown to cause the leaves of treated plants to become more intensely green (15,16) and

(ii)

it

is

of

some

cytochrome

14a-demethylase

possible

that

P-450

(17)),

DMI fungicides,

mono-oxygenases

might

also

inhibit

being

(e.g., the

inhibitors the

sterol

mono-oxygenase

catalyzed conversion of carotenes into xanthophylls. EXPERIMENTAL Instrumentation HPLC and liquid scintillation counting were carried out on PU4001 Intertechnique instruments, Pye-Unicam and SL-33 GC was carried out on a Pye-Unicam 204 modified respectively. for use with capillary columns by the addition of a SGE OCI-3 on-column injector and equipped with a flame ionization detector whose output drove a Pye-Unicam PU4810 computing integrator. GCMS was carried out on a Carlo Erba/Kratos MS-25 instrument equipped with a DS-55 data system. Fungicides Triadimefon (Bayer AG), triarimol (Eli Lilly & Co), diclobutrazol (ICI PLC), tridemorph (BASF AG), and fenpropimorph (BASF AG) were gifts from the respective companies. They were administered to the plants at a concentration of 250 pM. Their solutions were prepared by dissolving the appropriate weight of fungicide in 5 mL of methanol, thoroughly mixing in 1 mL of Tween 80 and diluting with distilled water to 1L. Control plants were treated with distilled water/methanol/Tween 80 (99.4:0.5:0.1 v/v/v). Growth of Seedlings Barley seeds, a gift from the Welsh Plant Breeding Station, Aberystwyth, were soaked for 10 h in control or fungicide solution and then planted about 2-cm apart and at a depth of about 1 cm in John Innes No. 2 compost. They were allowed to germinate and grow in a cabinet which maintained the following conditions: 16 h at 6000 Lux and 22 C followed by 8 h of darkness at 18 C. The compost was moistened each day with control or fungicide solutions; care was taken to avoid wetting the shoots with the latter. Morphological Measurements The percentage germination was recorded on the 4th day after

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396

Mercer et al: EFFECT

OF SBI FUNCTIONS

ON BARLEY

sowing. The following growth parameters (seedling height; coleoptile length; primary leaf length, area and wet wt; primary root length; shoot wet wt) were measured on the 10th day after sowing on random samples of seedlings. Each sample contained at least 10 seedlings and the average value of a given parameter was taken as a replicate. Four such replications were used for statistical analysis. Analysis of Chloroplast Pigments About 20-30 orimarv leaves from each batch of seedlinos were randomly selected and cut into small pieces (0.5 cm).' These were thoroughly mixed and sub-samples (1 g wet wt) were withdrawn for chloroplast pigment analysis and dry wt determination. The drying was performed overnight at 105 C. The extraction of the sub-samples taken for pigment analysis was carried out under low illumination. They were homogenized in ice-cold, redistilled acetone and filtered through a sintered funnel. The solid residues were re-extracted with acetone and re-filtered until they were colorless. The acetone extracts from each sub-sample were combined and taken to dryness by rotary evaporation at 40 C. The green residue from each was redissolved in 20 ml of acetone and 20-uL aliquots were analyzed by HPLC. An ODS-Hypersil (5 um) reverse-phase column (20 cm x 4.5 mm i.d.) was used along with two solvent systems, A [CH2C12/CH30H/H20 (21:63:15.8 v/v/v)] and B [CH2C12/CH30H (15:85 v/v)] which flowed through it at 1.5 mL/min (18). The chromatogram was developed for 10 min with solvent A, during which time the polar xanthophylls, neoxanthin, violaxanthin, and lutein were eluted in that order, and then for 6 min with solvent B when chlorophyll a, chlorophyll a, and the non-polar carotene, B-carotene, were eluted in that order. The column effluent was monitored at 400 nm and the peak areas determined. Each peak area was converted into the corresponding quantity of pigment using a calibration curve relating peak area to pigment quantity which had been prepared previously with a pure authentic sample of the relevant pigment; the calibration curve for each of the pigments was linear over the range that was required for this work. Analysis of Leaf Sterols The shoots of lo-day-old seedlings from each treatment were cut just above the compost level. About loo-150 excised shoots from the same treatment and of uniform length were gathered into a bundle held loosely with an elastic band and their cut ends trimmed level. The bundle was then placed, cut ends first, into a lOO-mL beaker containing 10 ml of incubation solution. This consisted of the control or 250 pM fungicide solution corres onding to their manner of growth containing 10 uCi of sodium Cl-P4Clacetate (Amersham International PLC) per 10 mL. The bundle was allowed to stand in the incubation solution for 24 h under constant illumination at room temperature. A gentle stream of air was

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Merceretal:EFFECTOFSBIFUNCTlONSON

BARLEY

397

passed over it to increase transpiration and thereby induce a greater rate of uptake of the incubation solution; as the volume of the latter decreased it was made up to the original level in the beaker by the addition of distilled water. washed free of the seedlings were incubation After [1-14Clacetate and homogenized in acetone. The homogenate was filtered through a sintered funnel and the residue twice re-extracted with acetone and re-filtered. The acetone extracts were combined, diluted with water, and extracted four times with The combined ether extracts dry, peroxide-free diethylether. were washed with water, dried over anhydrous Na2S04, and taken The green lipid residue was to dryness in a rotary evaporator. then saponified and the unsaponifiable lipid extracted with ether The yellow unsaponifiable lipid was then in the usual way. chromatographed on a column of acid-washed, Brockmann grade 3 alumina to remove the carotenoids from the sterols. The column was developed successively with (i) light petroleum, bp 40-60 C, which eluted B-carotene, (ii) diethylether/light petroleum (15:85 v/v), which eluted all the sterols and left the xanthophylls to the column. The sterol-containing eluate was adsorbed evaporated to dryness under nitrogen, vacuum desiccated, and subjected to TLC on Whatman LK6 silica gel concentration zone This plates using benzene/ether (9:l v/v) for development. procedure separated the sterols into three structural classes: the 4,4-dimethyl sterols (Rf 0.4), the Q-methyl sterols (Rf 0.35), and the 4-demethyl sterols (Rf 0.2) which relate closely normal sequence of demethylation in to the higher plant biosynthesis, which is: 4,4,14-trimethyl sterols + 4a,l4-dimethyl sterols + C-methyl sterols + 4-demethyl sterols (14). Thus the second two components of the demethylation sequence fall into the &-methyl sterol TLC class. The three TLC zones were located by radioautography, scrapped off the plate, eluted with ether, and l/lOOth aliquots assayed for radioactivity using NE 216 as the liquid scintillator; measurements were automatically corrected for quench and expressed as disintegrations/min (dpm). The following ratios were then calculated: (i) dpm in 4,4-dimethyl sterols/ dpm in 4-demethyl sterols and (ii) dpm in 4a-methyl sterols/ dpm in 4-demethyl sterols. The sterol that remained in each of the TLC fractions was then analyzed by GLC and GCMS. GC was carried out on a SGE BP-5 capillary column (12 m x 0.33 mm i.d.); carrier gas was helium flowing at 1.5 mL/min and the make-up gas was nitrogen. The column temperature was raised ballistically from 30 C to 275 C and held there for 25 min. Cholesterol was used as the internal standard and retention times expressed relative to it. GC-MS was carried out using the same GC conditions and an ionizing energy of 70 eV. The sterols were identified by the coincidence of their relative retention times and mass spectral fragmentation patterns with

STEROIDS 53/3-S

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Mercer

398

et al: EFFECT OF SBI FUNCTIONS

1iterature values and those determination of the percentage was based on GC peak areas.

of authentic composition of

ON BARLEY

samples. The sterol mixtures

RESULTS Effect of Fungicides on Germination and Growth The data in Table 1 show that treatment of barley with the three DMI fungicides at a concentration of 250 uM reduced the germination rate by about In contrast,

15% and the various growth parameters by 20-40%. the

data

in Table

2

show

that

similar

treatment

with the two morpholine fungicides has a less marked effect.

The

figures for the germination rate, coleoptile length, and primary root

length,

though

without

exception

lower

than those of

the

control plants, are not significantly different at a probability level of 5%; however, all the other growth parameters are 20-30% lower. Effect of Fungicides on Chlorophyll and Carotenoid Content The

data

(a, b, of

the

in Table

3

show that when the content of chlorophyll

and total) is expressed on a dry weight basis the effect three

DMI

fungicides

is

mixed:

triadimefon

causes

a

decrease, whereas triarimol and diclobutrazol cause no significant change at a probability level of 5%.

However when the chlorophyll

content is expressed per unit area of leaf all three DMI fungicides cause a 15-201 increase. that the two morpholine

In contrast, the data in Table 4 show fungicides do

not cause any change

in

the chlorophyll content that is significant at the 5% probability

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Mercer

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TABLE 1.

ON BARLEY

EFFECT OF DMI FUNGICIDES OF BARLEY

Parameterasb

399

Control

Triadicsf

86 21.2 2.5 11.0 18.0 165 93 6.5

72 14.0

% Germination Seedling height (cm) Coleoptile length (cm) Primary leaf length (cm) Primary root length (cm) Shoot wet wt (mg)

AND GROWTH

ON GERMINATION

ft.: 13:5 125 78 4.8

Triarid3f

Diclobepf

75 17.0

78 12.8

fi:“5 14.8 127 74 4.5

i-i 15:2 138 80 4.2

a % Germination determined on the 4th day after sowing. b Growth parameters measured on the 10th day after sowing. C Triadimefon; d Triarimol; e Diclobutrazol (2R,3R-isomer). f Seeds soaked in a compost moistened daily z&water containing a 250 uM concentration of the fungicide.

TABLE 2.

EFFECT OF MORPHOLINE AND GROWTH OF BARLEY

Parameterasb

FUNGICIDES

Control

% Germination Seedling height (cm) Coleoptile length (cm) Primary leaf length (cm) Primary root len th (cm) Shoot wet wt (mg4

88 18.9 ;*; 15:5 182 98 5.9

ON

Tridemorphc 82X 12.7 1.3x 7.5 14.2X 133 82 4.6

THE

GERMINATION

Fenpropimorphc 84X 14.5 1.4x 7.2 13.8X 155 78 4.3

% Germination determined on the 4th day after sowing. Growth parameters measured on the 10th day after sowing. Seeds soaked in and compost moistened daily with water containing a 250 uM concentration of the fungicide. In a given row values followed by the superscript letter x are not significantly different from the control value at probability level of 5% (i.e., P = 0.05).

STEROIDS 53/3-5

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1989

400

TABLE 3.

Mercer

et al: EFFECT OF SBI FUNCTIONS

ON BARLEY

EFFECT OF DMI FUNGICIDES ON CHLOROPHYLL AND CAROTENOID CONTENT OF THE LEAVES OF lo-DAY-OLD BARLEY SEEDLINGS

Parametera

Pigment

Control

Triadib

Triaric

Diclod

Chl ae Chl bf Tot Chlg

12.84x

Wt(mg)/g dry wt

1;':::: .

11.41 4.04x 15.45

12.20x 4.41x 16.61X

13.10x 4.47x 17.87x

Wt(ug)/cm2 leaf area

Chl ae Chl bf Tot Chlg

22.95 8.33 31.28

26.99 9.58 36.57

25.67 9.36 35.03

27.67 10.03 37.70

Wt(mg)/g dry wt

Wt(ug)/cm2 leaf area

8-Carh Neoxani Violaxj

B-Carh Neoxani violaxj Lutein Tot Car4

0.941x ;*;;",: .

1.673x 0.504x 0.545 1.618X 4.340x

0:278

0.863x 0.251x 0.293

0.889x 0.272x 0.314x

0.784 2.134

0.830 2.237

",j;:: .

1.951 0.626 0.659 1.857 5.093

1.876X 0.514x 0.662 1.739x 4.791x

2.034 0.584 0.673 1.926 5.217

;*;i,":

Seeds soaked in and compost moistened daily with water containing a 250 uM concentration of the fungicide; Triadimefon. c Triarimol. d Diclobutrazol. e Chlorophyll 2. Chlorophyll b. g Total chlor phyll. h B-Carotene. Neoxanthin. 7 Violaxanthin. ! Total carotenoid. In a given row values followed by the superscript letter x are not significantly different from the control value at a probability level of 5% (i.e., P = 0.05).

STEROIDS

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Mercer et al:EFFECT

OF SBI FUNCTIONS

401

ON BARLEY

level regardless of whether

it is expressed on a dry weight or

a leaf area basis. The data in Table 3 also show that when the content of the various species

of

is

carotenoids

expressed

on

a

dry

weight

basis

triadimefon and triarimol generally cause a small reduction whereas diclobutrazol probability carotenoid DMI

species

cause

carotenoids

morpholine it

is

that

when

expressed increases

is significant at the 5%

the per

content unit

that

are

of

leaf in

the

area

the

various

all

range

three

15-20%.

in Table 4 show that when the content of the various

species of

when

However

level.

fungicides

The data

two

produces no change

is

is expressed

fungicides

expressed

on a dry weight

generally

per

unit

cause

area

of

a

basis the

decrease

leaf

they

whereas cause

no

significant change at the 5% probability level. Effect of Fungicides on Sterol Content Data in Tables 5 and 6 show that the DMI and morpholine fungicides markedly

increase

the

radioactivity,

derived

biosynthetically

from Cl-I4C]acetate, in both the 4,4-dimethyl and the b-methyl sterols relative to that in the 4-demethyl sterols. Data

in Table

sitosterol,

5 show that whereas the 4-demethyl sterols,

stigmasterol,

and

campesterol,

constitute about 85%

of the total sterol in the shoots of control barley plants they are

decreased

triadimefon

or

to

lo-17%

triarimol,

in

those

this

that

decrease

of a considerable increase in the &-methyl

STEROIDS

53/3-S

March-May

1989

had

been

being

at

treated the

with

expense

sterols, cycloeucalenol

Mercer et al: EFFECT

402

TABLE 4.

OF SBI FUNCTIONS

ON BARLEY

EFFECT OF MORPHOLINE FUNGICIDES ON THE CHLOROPHYLL AND CAROTENOID CONTENT OF THE LEAVES OF lo-DAY-OLD BARLEY SEEDLINGS

Parametera

Pigment

Control

Tridemorb

Fenpropimorc

Wt(mg)/g dry wt

Chl ae Chl bf Tot Chlg

16.60X 4.58x 21.18x

14.46x 4.39x 18.85x

15.02x 4.20x 19.22x

Chl ae Chl bf Tot Ch19

23.82x 6.40x 30.22x

25.61x 8.11x 33.72x

17.10x 7.60x 34.70x

t+Carh Neoxani violaxj Lutein Tot Cark

1.379 0.339 0.403x 1.347 3.468

1.162 0.284 0.328 1.083 2.857

1.193 0.297 0.362X 1.164 3.016

t+Carh Neoxani Violaxj Lutein Tot Cad

1.976X

2.215X

2.213X

;*";",;: . 1.940x 4.983X

;*,":;:: * 2.143x 5.499x

;*,";;:

Wt(pg)/cm2

leaf area

Wt(mg)/g dry wt

Wt(j.ig)/cmz

leaf area

’ 5.. :::::

Seeds soaked in and compost moistened daily with water containing a 250 pM concentration of the fungicide; Tridemorph. c Fenpropimorph. e Chlorophyll a. Chlorophyll b. g Total chlorophyll. h B-CarTtene. Neoxanthin. 7 Violaxanthin. k Total carotenoid. In a given row values followed by the superscript letter x are not significantly different from the control value at a probability level of 5% (i.e., P = 0.05).

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Merceretal:EFFECTOFSBIFUNCTlONSON

TABLE 5.

403

BARLEY

EFFECT OF DMI FUNGICIDES ON THE INCORPORATION OF 14C FROM [l-14ClACETATE INTO THE 4,4-DIMETHYL, 4a-METHYL AND 4-DEMETHYL STEROL FRACTIONS OF lo-DAY-OLD BARLEY SHOOTS AND ON THEIR PERCENTAGE STEROL COMPOSITION

Parametera 4,4-Di/4-De ratioe 4cr-Me/4-De ratiof

Cycloartenol 24-Methylenecycloartanol Cycloeucalenol Obtusifoliol Campesterol Stigmasterol Sitosterol Other sterolsh Total 4,CDimethyl sterols Total h-Methyl sterols Total 4-Demethyl sterols

Control

Triadib

Triaric

Diclod

0.509 0.509

2.86 2.34

2.20 2.78

1.13 2.72

2.40 5.52 8.77 38.05 14.05 10.09 15.03 3.09 10.92 48.82 39.17

2.83 13.60 8.96 30.54 11.26 10.90 17.98 3.93 16.43 39.50 40.14

0.62 4.47 3.44 7.26 20.50 21.20 41.25 1.26 5.09 10.70 82.95

2.84 5.71 2.70 1.58 17.84 24.01 42.80 2.52 8.55 4.28 84.65

a Seeds soaked in and compost moistened daily with water containing a 250 NM concentration of the fungicide; cut ends of excised loday-old shoots immersed in water containing 10 MCi of [l-I4C]acetate and a 250 bM concentration of the fungicide; b Tridimefon. c Triarimol. cl Diclobutrazol. e This ratio is derived from exposure of the excised shoots to [1-14C]acetate and is "dpm in the 4,4-dimethyl sterol TLC fraction"/"dpm in the 4-demethyl sterol TLC fraction." f As for "e" but the ratio is "dpm in the C-methyl sterol TLC fraction"/"dpm in the 4-demethyl sterol TLC fraction." g The ratios "e" & "f" in the control shoots were in the range 0.4-1.5 but have been adjusted to 0.5; the corresponding ratio in the fungicide-treated shoots have been adjusted in proportion. h The minor sterols probably include some pentacyclic triterpenes.

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404

TABLE 6.

Mercer et al: EFFECT

OF SBI FUNCTIONS

ON BARLEY

EFFECT OF MORPHOLINE FUNGICIDES ON THE INCORPORATION OF I4C FROM [l-I4CIACETATE INTO THE 4,4-DIMETHYL, 4a-METHYL AND 4-DEMETHYL STEROL FRACTIONS OF lo-DAY-OLD BARLEY SHOOTS AND ON THEIR PERCENTAGE STEROL COMPOSITION

Parametera

Control

4,4-Di/4-De ratioe 4a-Me/4-De ratiof

0.509 0.509

Cycloartenol 24-Methylenecycloartanol Cycloeucalenol 24-Methylenepollinastanol 24-Methylpollinastanol Cyclofontumienol Obtusifoliol 5a-Ergost-8,22-dien-36-01 5a-Ergost-B-en-38-01 Campesterol Stigmasterol Sitosterol Other sterolsh Total 9 ,19-cycle sterols Total A1 -sterols Total A5-sterols

1.52 4.85 2.36 0 : 1.65 : la.19 21.97 47.56 I.89 a.73 1.65 87.72

Tridemorb

Fenpropimorc

1.70 2.23

2.30 2.87

2.90 7.20 50.40 trace la.70 2.30 0 3.40 2.05 2.60 0 4.70 5.75 84.30 5.45 7.30

2.55 8.80 40.02 trace 19.92 2.00 0 3.25 1.90 5.20 0 6.30 6.06 78.99 5.15 11.50

a Seeds soaked in and compost moistened daily with water containing a 250 uM concentration of the fungicide; cut ends of excised lo-day-old shoots immersed in water containing 10 uCi of [1-I4Clacetate and a 250 uM concentration of the fungicide. b Tridemorph. c Fenpropimorph. e This ratio is derived from exposure of the excised shoots to [1-I4Clacetate and is "dpm in the 4,bdimethyl sterol TLC fraction"/"dpm in the 4-demethyl sterol TLC fraction." f As for "e" but the ratio is "dpm in the C-methyl sterol TLC fraction"/"dpm in the 4-demethyl sterol TLC fraction." g The ratios "e' & "f" in the control shoots were in the range 0.4-1.5 but have been adjusted to 0.5; the corresponding ratios in the fungicide-treated shoots have been adjusted in proportion. h The minor sterols probably include some pentacyclic triterpenes.

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Mercer et al: EFFECT

and

OF SBI FUNCTIONS

and

obtusifoliol, 24

sterol,

a

405

ON BARLEY

smaller

increase The

methylenecycloartanol. did

diclobutrazol,

not

significantly

in

the

4,4-dimethyl

other

DMI

fungicide,

decrease

the

proportion

of 4-demethyl sterols and caused only a relatively small increase in that of mixture

the &-methyl

of

4-demethyl &-methyl

diclobutrazol sterols

to

The

sterols. at

250

70.4%

of

uM, the

2!,3& +

2&3_S

however,

decreased

the

increased

the

total

and

isomeric

sterols to 19.3% (data not shown in the table).

Data shown in Table 6 show that both the morpholine

fungicides

markedly decrease the proportion of 4-demethyl sterols and cause a

vast

increase

principally

as

that

of and

cycloeucalenol,

the A8-sterols. such

in

The

the a

9B,19-cyclopropyl

smaller

9s,19-cyclopropyl

24-methylpollinastanol,

that

increase

sterols are

not

sterols,

in

that

of

include several, normally

present

in significant amounts in barley shoots. DISCUSSION Both

classes

of

fungicides,

when

administered

concentration of 250 PM, retarded growth. with

the

DMI

fungicides

which

reduce

to

barley

at

a

This was most apparent

the germination

rate and

all the parameters of shoot and root growth that were measured. The DMI fungicide-treated plants had a more compact growth habit than The

the

morpholine

coleoptile

STEROIDS

controls,

53/3-5

or

being

fungicides root

length

March-May

1989

shorter, had

no

though

stiffer, effect other

on

and

darker

green.

germination

growth

parameters

or

on

were

Mercer et al: EFFECT

406

decreased. a more

Morpholine

compact

OF SBI FUNCTIONS

fungicide-treated

growth habit,

nor were

plants

did

they darker

ON BARLEY

not

have

green.

The

growth-retarding effect of the DMI fungicides is almost certainly due

to their ability

oxidation

of

to

kaurene

in

inhibit the cytochrome P-450 catalyzed the

gibberellin

biosynthetic

pathway.

The consequent decrease in the production of gibberellins, whose function it is to stimulate cell elongation, would have the effect of

reducing

gibberellin

growth. content

Triadimefon of

barley

has

been

seedlings

shown

to

(19) and

lower

the

ancymidol,

a

structural analogue of triarimol, has been shown to inhibit kaurene oxidation

in

peas

(20).

The

metabolic

cause

of

the

growth

retarding effect of the morpholine fungicides is not yet clear. Neither

class

of

fungicide

appears

formation.

The

increased greenness of the DMI fungicide-treated suggestive

of

an

or

specific

inhibitory

is

chlorophyll

any

or

which

on

have

stimulatory

barley,

effect

to

increased

carotenoid

synthesis

of

chlorophyll, is almost certainly a secondary consequence of growth reduction because the chlorophyll content is virtually the same as

that

of

control

leaves when

expressed

per

unit

dry

but is 15-20% higher when expressed per unit leaf area.

weight However

the fact the carotenoid content of DMI fungicide-treated barley, when

expressed

content, also that

it

is

per

unit

leaf

15-20% greater

the

same

area,

is,

like

the

chlorophyll

than that of the control suggests

quantity

of

chloroplast material,

STEROIDS

53/3-5

rather

March-May

1989

Mercer et al: EFFECT

OF SBI FUNCTIONS

than

just the chlorophyll, that

leaf

area.

and

morpholine

whether the

The

chlorophyll

latter

is being packed into a smaller

content

fungicide-treated

expressed

per

being

407

ON BARLEY

unit

the

barley was

dry

consistent

of

weight

with

the

or

leaves of virtuafly

per

fact

unit

that

control the

same

leaf area,

the

leaves of

morpholine fungicide treated barley were no more green than those Similarly the carotenoid content of morpholine

of control plants. fungicide-treated is

the

same as

when

barley,

expressed

per

the control, which

that of

unit

leaf

area,

is consistent with

an unchanged packing of chloroplast material in the leaves. Neither class of fungicide has any effect on the ratio of s-carotene

to

xanthophylls. might

total

xanthophyll

or

to

any

of

the

individual

This rules out the possibility that DMI fungicides

inhibit

mono-oxygenase-catalyzed

conversion

of

carotenes

into xanthophylls. The to

observation

5-fold

increase

that the

classes

radioactivity,

of

fungicide

cause a

biosynthetically

in the 4,4-dimethyl- and &-methyl

from [l-l’k]acetate, relative to

that

inhibit

sterol

the

both

4

derived sterols

in the 4-demethyl sterols indicates that they biosynthetic

pathway

in

barley

at

a

stage

prior to the removal of the C-4 and C-14 methyl groups. The

marked

cycloeucalenol barley

STEROIDS

shows

5313-S

accumulation

and

of

obtusifoliol,

that

March-May

these

7989

in

the the

fungicides

lrla-methyl DMI

sterols,

fungicide-treated

inhibit

the

sterol

Mercer et al: EFFECT

408

14a-demethylase and

(21)

suspension

cultures

the

DMI

three

having

the

demethylase. potent

who of

studied

bramble

fungicides

least

ON BARLEY

Fig. l), thus confirming the work of Schmitt

(see

Benveniste

OF SBI FUNCTIONS

the fungal

of

on

has

fenarimol

fructicosus)

diclobutrazol

effect

diclobutrazol

inhibitor of

effect

(Rubus

tested,

inhibitory

Since

the

the

been

sterol

cells.

stands

barley shown

out

sterol

on Of as 14a-

to be a more

lrla-demethylase than

the

other two (17), there is a prima facie case for concluding that it shows a greater selectivity in its toxicity than they do. The

vast accumulation

decrease

an

98,19-cyclopropyl

sterols and the

in the obtusifoliol content in barley treated with the

morpholine the

of

fungicides

inhibition

of

in

biosynthesis (see Fig. that accumlated (eg. 24-methyl

it seems

green

of

their

primary

plant,

but

effect

is

isomerase

(COI),

fungal,

sterol

not

Some of the 96,19-cyclopropyl sterols

1).

cyclofontumienol, 24-methylenepollinastanol,

pollinastanol)

not members

that

cycloeucalenol-obtusifoliol

involved

enzyme

indicates

are

the normal

96,19-cyclopropyl

phytosterol

reasonable to assume

sterols that are

biosynthetic

pathway and

that they result from metabolism

of the rapidly accumulating cycloeucalenol by enzymes whose normal substrates pathway The

are

and

which

increase

indicates

sterols

the

in

do the

in not

the have

AS-sterol

morpholine

post-cycloeucalenol absolute content

fungicides

substrate of

also

STEROIDS

the

part

53/3-5

the

specificity.

treated

inhibit

of

the

barley sterol

March-May

1989

Mercer et al: EFFECT OF SBI FUNCTIONS ON BARLEY

+

24-Me

Figure

STEROIDS 53/3-5

409

Key: a =cycloartenol b = 24-methylenecycloartanol c = cycloeucalenol d = obtusifoliol e = 4ti-methyl-5=ergost-8,24(28)dien-3p-ol f q cyclofontumienol g = 24-methylene pollinastanol h = 24 -methyl pollinastanol

& Et -A5-sterols

1. Primary sites of inhibition of sterol biosynthesis in barley by DMI and morpholine fungicides. (Sterols to the left of the dashed line are on the normal biosynthetic pathway, those to the right are not.)

March-May 1989

Merceretal:EFFECTOFSBIFUNCTIONSON

410

A8+A7-isomerase, A8-SterOlS

did

as

they

not

98,19-cyclopropyl

do

in

accumulate

fungi. to

sterols is most

the

The same

reason extent

BARLEY

that

the

as

the

likely to be due to the fact

that the COI-catalyzed step precedes that catalyzed by the A~+ATisomerase (see Fig.

1) and was so strongly inhibited that only

small amounts of normal sterol precursors were able to leak past it to reach the subsequent blockage. the

work

of

Benveniste's

group

who

These observations support examined

the

effect

of

tridemorph on sterol biosynthesis in cultured bramble cells (22) and maize

seedlings

(23).

The

basis of the selective toxicity

of the morpholine fungicides towards fungi rather than the plants they are used to protect appears to be due to the fact that the A8 - and A8*14-sterols that accumulate in fungi alter the behavior of the fungal membranes far more than the 9B,19-cyclopropyl sterols do in green plants

(5,24).

APPENDIX The following trivial names are used in this paper: Triadimefon = l-(4-chlorophenoxy)-3,3-dimethyl-(1!$1,2,4triazol-1-yl)butanone Triarimol = a-(2,4-dichlorophenyl)-a-phenyl-5-pyrimidine methanol Diclobutrazol = (2&,3R_)-l-(2,4-dichlorophenyl)-4,4-dimethyl-2(ltJ-1,2,4-triazol-1-yl)pentan-3-01 Tridemorph = Reaction mixture of CII-Cl4 4-alkyl-2,6-dimethylmorpholine homologues containing 60-70% of 4-tridecyl isomers Fenpropimorph = cis-4-[3-(4-tert-butylphenyl)-2-methylpropyll??$dimethylmorpholine Ancymidol = a-cyclopropyl-a-(4-methoxyphenyl)-5-pyrimidine methanol Fenarimol = a-(2-chlorophenyl)-a-(4-chlorophenyl)-5pyrimidine methanol

STEROIDS 53/3-5

March-Mav1989

Mercer

et al: EFFECT OF SBI FUNCTIONS

Brassicasterol Campesterol Stigmasterol Sitosterol Cycloartenol

ON BARLEY

411

= = = = =

(24R)-Z4-~thylcholest-5,22-dien-3%-ol (24~)-24-~thylcholest-5-~n-3%-ol (24~-24-ethylcholest-5,22-dien-3%-01 (24R)-24-ethylchofest-5-en-36-01 4,4~14a-trimethyl-9%,19-cyclo-5a-cholest-24-en38-01 24-Methylenecycloartanol = 4,4,14a-trimethyl-9%,19-cycle-5aergost-24-en-3%-01 Cycloeucalenol = ~,l~-di~thyl-9%,19-cyclo-~-ergost-24(28)-en38-01 = 4a,l4a-dimethyl-5a-ergost-8,24(28)-dien-3%-ol Obtusifoliol 24-Methylenepollinastanol = 14a-methyl-9%,19-cycle-5a-ergost24(28)-en-38-01 24-Methylpollinastanol = 14a-methyl-9%,19-cyclo-5a-ergastan-3%-ol Cyclofontumienol = &,I&-dimethyl-9%'19-cycle-5a-stigmast-f24(28)-en-38-01 REFERENCES JD (1980). Lipid Biochemistry of Fungi and Other Organisms, Plenum Press, New York, pp.233-242. Weete JD, Sancholle M, Touze-Soulet J-M, Bradley J, and Dargent 2. R (1985). Effect of triazoles on funqi. III. Composition of .a plasma ~mbrane-enriched fraction bf Taphrina deformans. BIOCHIM EIOPHYS ACTA 8123633-642 Steel CC and Mercer EI (1987). Glucose permeability of liposome 3. vesicles prepared with sterol extracts from fenpropimorph:grown fungi. BIOCHEM SOC TRANS 16:350-351. Baloch RI (1984) The effects of fungicides on sterol 4. biosynthesis. Ph.D. Thesis, University of Wales. The mode of action of morpholines. In: Mercer EI (1988). 5, Biosynthesis Inhibitors: Sterol Pharmacological and Aqrochemical Aspects (Berg D and Plempel M, eds) Ellis Horwood, Ltd., Chichester (UK), pp. 120-150. Thomas PG, Haslam JM, and Baldwin BC (1983). The effects 6. of the funaicide diclobutrazol on membrane function in Saccharomyces" cerevisiae and Ustilago maydis. BIOCHEM SOC TRANS 11:713. Ragsdale NN (1975). Specific effects of triarimol on sterol 7. biosynthesis in Ustilago maydis. BIOCHIM BIOPHYS ACTA 380:81-96. Kato T, Tanaka S, Ueda M, and Kawase Y (1975). 8. Inhibition of sterol biosynthesis in Monilinia fructigena. AGR BIOL CHEM 39: 169-174. Buchenauer H (1977). Mode of action of triadimefon in Ustilago 9. avenae. PESTIC BIOCHEM PHYSIOL 7:309-320. 10. Kato T, Shoami M, and Kawase Y (1980). Comparison of tridemorph with buthiobate in antifungal mode of action. 1.

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J PESTIC SC1 5:69-79. 11. Kerkenaar A, Uchiyama M, and Versluis GG (1981). Specific effects of tridemorph on sterol biosynthesis in Ustilago maydis. PESTIC BIOCHEM PHYSIOL 16:97-104. 12. Baloch RI, Mercer EI, Wiggins TE and Baldwin BC (1984). Inhibition of biosynthesis in Saccharomyces ergosterol cerevisiae and Ustilago maydis by tridemorph, fenpropimorph and fenpropidin. PHYTOCHEMISTRY 26:2219-2226. 13. Baloch RI and Mercer EI (1987). Inhibition of sterol A8+A7-iSOIIWaSe and sterol 14a-demethylase by fenpropimorph, tridemorph and fenpropidin in cell-free enzyme systems from Saccharomyces cerevisiae. PHYTOCHEMISTRY 26:663-668. 14. Goodwin TW (1979). ANNU REV Biosynthesis of terpenoids. PLANT PHYSIOL 30:369-404. 15. Hardison JR (1971). Chemotherapeutic eradication of Ustilagv striiformis and Urocystis aqropyri in ~Pao pratensis Merion by root uptake of a-(2,4-dichlorophenyl -a-pynmidine-methanol (EL 273). CROP SC1 11:345-347. 16. Buchenauer H Triadimefon: mode and Grossman F (1977). of action in plants and fungi. NETH J PLANT PATH 83 (Suppl 1):93. 17. Gadher P, Mercer EI, Baldwin BC, and Wiggins TE (1983). A comparison of the potency of some fungicides as inhibitors of sterol 14-demethylation. PESTIC BIOCHEM PHYSIOL 19:1-10. 18. Mashonganyika CN (1981). Pigment variation in rye-grass strains M.Sc. Thesis, University of (Lolium perenne; Graminae). Wales. Effect of triadimefon 19. Buchenauer H and Rohner E (1981). and triadimenol on growth of various plant species as well as on gibberellin content and sterol metabolism in shoots of barley seedlings. PESTIC BIOCHEM PHYSIOL 15:58-70. 20. Coolbaugh RC and Hamilton R (1976). Inhibition of entkaurene oxidation and growth by a-cyclopropyl-a-(e-methoxyphenyl)-5pyrimidine methyl alcohol. PLANT PHYSIOL 57:245-248. 21. Schmitt P and Benveniste P (1979). Effect of fenarimol on sterol biosynthesis in suspension cultures of bramble cells. PHYTOCHEMISTRY 18:1659-1665. 22. Schmitt P, Benveniste P, and Leroux P (1981). Accumulation of 9B,19-cyclopropyl sterols in suspension cultures of bramble cells cultured with tridemorph. PHYTOCHEMISTRY 20:2153-2159. Manipulation by 23. Bladocha M and Benveniste P (1983). tridemorph, a systemic fungicide, of the sterol composition of maize leaves and roots. PLANT PHYSIOL 71:756-762. Sterol structure and membrane function. 24. Block KE (1983). CRC CRIT REV BIOCHEM 14:47-92.

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1989