Effects of sterol biosynthesis-inhibiting fungicides and plant growth regulators on the sterol composition of barley plants

PESTICIDE

BIOCHEMISTRY

Effects

AND

PHYSIOLOGY

27,

%‘)-3t)fl

( 1987)

of Sterol Biosynthesis-Inhibiting Fungicides and Plant Growth Regulators on the Sterol Composition of Barley Plants RAYMOND

S. BURDEN,TERENCECLARK,ANDPETER

Received A number

of sterol

July

21.

19X6:

biosynthesis-inhibiting

accepted (SBI)

October

fungicides

J. HOLLOWAY

17. 1986 and

plant

growth

regulator

analog5

were applied as root drenches to barley seedlings and their effect on the total sterol composition of the roots and shoots was measured by gas-liquid chromatography. Prochlorar ~a\ found to be inactive in this system. probably hecause of poor uptake. while the other compounds could be divided into morpholines isomerase the enzyme isomers fungicidal

two groups tridemorph

according to their and fenpropimorph

whereas triadimenol. responsible for the and enantiomers and anti-gibberellin

nuarimol. removal

mode of action inhibited the

paclobutrarol. of the C-14

methyl

of some compounds on sterol propertie\. Shoot growth

a\ as\e\sed by sterol profiling. enryme cycloeucalenol-obtusifoliol and triapenthenol group. Effects

The

(RSW 041 I) inhibited of individual diastereo-

profile5 were compared was reduced by all the

with their compounds

known tested,

paclobutrazol. nuarimol. and triapenthenol being the most effective. As well as inducing accumulation of abnormal sterols. SBI fungicide treatment changed the ratio of campesterol to stigmasterol and sitosterol. It is hypothesired that thi\ may reflect changes in membrane architecture and may offer tide-treated

an

explanation for the incre;lsed plants. ( 1w: :\~;idclnl~ PlC\\. lm

fro\t

INTRODUCTION In recent years the Crop Protection Industry has developed a large number of fungicides which operate by inhibiting the biosynthesis of fungal terminal sterols. usually ergosterol (I, 2). Although these sterol biosynthesis-inhibiting (SBI) fungicides are structurally diverse. most of them share a common mode of action. that of blocking the cytochrome P-450-dependent C-14 demethylation process (Z-4). The resulting depletion of ergosterol and accumulation of C-14 methyl sterols cause a disruption of membrane activities and inhibition of fungal growth (5). A smaller but increasingly important group of SBIs are the morpholines (6). These do not affect fungal sterol biosynthesis at the C-14 demethylation stage but inhibit the h14-reductase and L,*+h’ isomerase processes (7). Again abnormal mycelial membranes result. Some of the C-14 demethylating SBI fungicides have also been found to induce morphological effects in higher plants t8- 13) and this property has been exploited

hardinea

sometime\

observed

with

SBI

fungi-

in the development of structurally related plant growth retardants t 14- 16). Here the principal mode of action appears to be on gibberellin biosynthesis and the treated plants are compact with short internodes. In the gibberellic acid (GA) biosynthetic pathway the cytochrome P-450-dependent oxidation of rnt-kaurene to rnf-kaurenoic acid is specifically inhibited (I I, 17). Although SBI fungicides and plant growth regulators are applied to plants. their effects on the biosynthesis of the host sterols is relatively unexplored. Plant sterols are thought to function as regulators of membrane permeability and possibly as hormones or hormone precursors (18, 19). Nontarget interference with their biosynthesis might therefore be expected to be deleterious. However, this is by no means certain and already there is evidence for beneficial physiological effects such as increased cold tolerance in plants following SBI treatment (14. 20). This may be related to an alteration of the structure and performance of the membrane sterols. In this paper we report quantitative and qualitative effects of a range of SBI fungi-

290

BURDEN,

CLARK,

AND

(250 ml per pot) as a root drench after which they were watered with nutrient solution. Three weeks after emergence the seedlings were harvested, shoot lengths measured, morphological effects noted, and roots and crowns separated from the rest of the shoots. The tissue was then frozen prior to extraction and analyis. Greenhouse plants of the barley cultivar Triumph were grown similarly. For fieldgrown plants of this cultivar. the seeds received prior treatment with individual enantiomers of triadimenol, formulated as for Baytan (25% active ingredient) and applied at the normal field rate of I .S mg ai per gram of seed. The plants were harvested 7 weeks after sowing. Ctrt~ttricds. The compounds used were triadimenol. nuarimol. prochloraz. tridemorph. fenpropimorph, paclobutrazol. and the experimental plant growth regulator RSW 041 1 or “triapenthenol” (Fig. I). All were gifts from the manufacturers and were

tides and plant growth regulators (PGRs) (structures in Fig. 1) on the total sterol composition of barley seedlings. In view of the important relationship between SBI stereochemistry and biological activity (6. 17, 21-241, the effects of available diastereoisomers and enantiomers were assessed separately. The results are discussed in relation to SBI stereochemistry and enzyme inhibition, plant morphology, and membrane integrity. MATERIALS

AND

HOLLOWAY

METHODS

Plunt muter.ial. Barley (Hordeurn \lul,qare cv. Egmont) seeds were germinated and grown in moist sand/vermiculite in 6-in. pots in a greenhouse at 18°C. The light regime was a 16 hr day/8 hr night with sup-

plementary lighting provided by 400-W mercury vapor lamps. Seedlings were treated at emergence with chemicals. normally at 20 mg liter-’ but sometimes at lower dosages, in 1% ethanolic solution

C,3H,-N

AH3 o

‘(,H

A

P”

CIk(&CH,-CH-kH-C(CH,3

ri

NJ

N-N

3 4

(CH,),CvCH,-CH-CH2-h

2

~

5

Cl

STEROL

BIOSYNTHESIS-INHIBITING

obtained as their technical grades. Diastereoisomers of triadimenol, paclobutrazol, and tridemorph were obtained from the technical samples by TLC (25, 26). The preparation of enantiomers of triadimenol is described elsewhere (27). Stem1 nnalysis. Analysis for total sterols was carried out using a combination of previously published procedures (28-30). Frozen tissue was lipophilized, ground, and weighed. Samples were then extracted with acetone in a Soxhlet apparatus for 34 hr and each extract was divided into halves. One-half was analyzed qualitatively for total sterols while the other was prepared for quantitative analysis of campesterol, stigmasterol, and sitosterol by the addition of an internal standard of cholesterol at the concentration of 1.0 mg/g dry wt of tissue (30). In each case the acetone extracts were evaporated to dryness and 10 ml of 95% ethanol containing 0.05 ml of concentrated sulfuric acid was added. The mixture was refluxed for 2 hr, made alkaline by the addition of 10 ml of IO% KOH in 95% ethanol. and refluxed for a further 2 hr. This procedure was necessary for the hydrolysis of sterol glycosides and esters (28) and also had the practical advantage of destroying most of the coextracted chlorophyll present. The solution was then cooled, diluted with HzO. and extracted three times with n-hexane. The hexane extracts were then dried and evaporated. Samples for qualitative analysis of total sterols were now acetylated by treatment for 24 hr with 4 drops each of pyridine and acetic anhydride. The reagents were then removed in a stream of nitrogen and the residue was dissolved in ethyl acetate. GLC analysis was performed on a Varian Mode1 3700 gas chromatograph fitted with a 25 m x 0.25 mm SE-52 bonded capillary column. a splitter, a flame ionization detector, and an electronic integrator. Nitrogen was used as carrier gas and a program of 12O”C-+295”C at YCimin produced an optimum separation of sterol acetates. Extracts for quantitative analysis were

291

FUNGICIDES

applied to Merck silica gel FZs4 thin-layer plates and developed three times with CH,CI,. The bands of 4.4-dimethyl,4methyl, and 4-demethyl sterols which separated in this system were located by cutting off the edge of the plate, spraying it with vanillin-H,PO, reagent, and heating. The 4-demethyl sterol zone was removed, eluted, and acetylated as described above. GLC analysis was performed on a 10 m x 0.32 mm bonded OV-I capillary column and the amounts of campesterol, stigmasterol. and sitosterol were calculated from a comparison of the peak area with that of the cholesterol standard. A standard mixture of sterols was used to calculate correction factors for variation in the detector response to the different sterols. The use of cholesterol as an internal standard was justified (30) since it is present in barley at very low levels (28). Mass spectra were obtained on a Kratos MS-80 RFA instrument using the 10 m x 0.32 mm OV-1 bonded capillary column. The instrument was set at 70 eV in the EI mode with helium used as carrier gas. The relative retention times (25 m x 0.25 mm bonded SE-52 column. 12O”C+295”C at S”C/min, t,, cholesteryl acetate = 1.OO) and M+ - 60 ions as determined by GLCMS (10 m x 0.3 mm bonded OV-l column) for the sterol acetate derivatives were as follows: campesterol 1.08, 382 (26%): 14amethyl-A*-ergostenol I .09, 396 (13); stigmaster01 1.1 I, 394 (1.8); obtusifoliol 1.13, 408 (12); dihydroobtusifoliol 1.135, 410 (20); 24-methylpollinastanol 1.14, 396 (2 I); sitosterol 1.16, 396 (25); cycloeucalenol 1.19, 408 (I 3): 24-methylenecycloartenol 1.27. 422 (5.8). RESULTS

AND

DISCUSSION

The effects of the SBI-type xenobiotics on the normal 4-demethyl sterols of barley shoots and roots are given in Tables 1 and 2, respectively. Although full replicate analyses are not reported here, statistical

292

BURDEN,

Control tuntreated) IRS.XR-Triadimcm>l lRS.‘RS-Triadimenol ‘R.S,3SR-Paclobutrazol ~RS.3R.S-Paclobutrnrol Nuarim~>l Triapenthrnol Tridemurph Fenpropimorph PI-ochloraz

lY.6 19.3 3 7 Il.6 12.5 17.h IO.7 I’ 4 Fix 31.7

CLARK.

AND

HOLLOWAY

0.44 t1.3 0.3’ 0. I7 0.35 0.17 O.?Y O.Oh

(I.33 0.27 0.30 O.OY 0.2’) 0. I!, 0 21 (1.0 O.(F 0.34

(1.07 0.4Y

(’ Mean 01‘ five \erdling\ SED 140 (I/ t I .95. h mg of \terol ;I\ meanred by GLC:g dry wt ot’tt~\nc.

variations of the order of i_S% were generally found. The percentage composition of the control tissue was found to vary somewhat from previously published values (28, 31). However. this may be at least partially explained in terms of the use of different cultivars. tissues of different ages, and different growth conditions. Also in the present study, crown tissue was included with the roots. All compounds with the exception of the imidazole prochloraz affected the composition of the normal barley sterols but to considerably different extents. Larger effects TABLE The Ejfkl

of1nhihiror.v

OII the -6fktrlrriryl -.

were observed on the shoot sterols than on those of the root tissue; this is consistent with the systemic nature of most of the xenobiotics tested and the likelihood of their eventual accumulation in the shoot tips. Prochloraz was included in the study as its antifungal spectrum differs considerably from that of other SBI fungicides (32). However. it has little systemic activity in plants (32) and hence when applied. as here, as a root drench it was probably not translocated to the sites of sterol biosynthesis. Hence it should not be inferred that prochloraz intrinsically lacks the capacity 2

Srerrd C‘~ttrp~~.sit;otl Roar Applic rl/irur

t!f’~trriczy

((‘1’.

~,q:,pl,~tlt)

Total Campesterol Compound Control

(untreated)

lRS,3SR-Triadimenol lRS.?R.S-Triadimenol ~RS.3SR-Paclobutrazol 2RS.3RS-Paclobutrazoi Nuarimol Triapenthenol Tridemorph Fenpropimorph Prochloraz n mg of sterol

as measured

Stigmasterol

Sitosterol

Ratio campesteroli

three

sterols as ? of control

citosterol \tigmasterol

(m&Y

(m&V

(I.59

0.36

0.96

0.41 0.46 0.35

0.3 0.19 0.22

0.73 0.92 0.70

0.4s 0.43 0.3x 0.3x

0.53 0.X 0.77

0.38 0. IX 0.21

0.87 0.5x 0.58

0.46 0.37 0.34

0.23 0.36

O.Ih 0.76

0.47 0.72

0.62

0.37

I .09

0.37 0.37 0.42

by GLCig

dry

wt of tissue.

(mg&

~~)~)f.\ ,fi~[/c,~,~;~lg

+

STEROL

BIOSYNTHESIS-INHIBITING

to affect the enzymes of plant sterol biosynthesis. The morpholine fungicides tridemorph and fenpropimorph had the most dramatic effect, reducing in each case the normal plant sterols to 12% of the control. This was accompanied by a large increase in “abnormal” sterols as revealed by the total sterol profile for fenpropimorph (Fig. 3, Table 3). The inhibition of plant sterol biosynthesis by morpholine fungicides was first noted by Benveniste and his coworkers and the discovery was made that in bramble cell cultures the primary site of action was the enzyme cycloeucalenol-obtusifoliol isomerase (39). This enzyme operates at a relatively early stage in the biosynthesis of plant sterols ( 18) and its inhibition leads to an accumulation of “abnormal” cyclopropyl sterols. This finding has been confirmed for carrot, tobacco. and soybean suspension cultures (34) and also for the shoots and roots of maize seedlings (29). Fenpropimorph is specifically the isomer having the methyl groups cis with respect to the morpholine ring and also possesses an additional asymmetric

FUNGICIDES

293

carbon atom (6). Although in our experiments the racemic form was used, it has been demonstrated that it is the S enantiomer which has the greater fungitoxicity (6) while in bramble cell cultures the S enantiomer was also found to be more effective than the R enantiomer in causing the accumulation of cyclopropyl sterols (33). Unlike fenpropimorph, tridemorph is marketed as a ci.r-trans mixture of isomers. These can be separated by TLC (26) and tests have revealed that both isomers are equally fungitoxic (6). The effects of the separated isomers at 10 and 7 mg liter-’ on the sterols of barley shoots are shown in Table 4. The data demonstrate that while both isomers reduce the normal sterol levels, the greater activity appears to reside in the cis form. The pyrimidine methanol nuarimol and the triazole alcohols triadimenol. paclobutrazol. and triapenthenol (RSW 0411) are known or suspected to inhibit C-14 demethylation in fungal sterol biosynthesis (3). The results demonstrated here (Tables 1 and 2) show that their application to barley seedlings leads to a diminution in the levels of “normal” plant sterols. I’revious work with the pyrimidine fungicides fenarimol and triarimol in plant suspension cultures has also demonstrated an impairment of sterol biosynthesis (29. 34) although this did not appear to occur with triarimol-treated whole plants of P/~tr.sc~olr~.s \,rrlgaris ( I I ). In work similar to that described here Buchenaur and Rohner have applied triadimenol and its keto analog triadimenfon as a soil treatment to barley and analyzed the 4-demethyl sterols by GLC (9). Although the three main sterols were incompletely separated by the packed column technique used and quantitative results were not reported, it can be inferred from the chromatograms reproduced that the effects of the inhibitors on the barley sterols were similar to those observed in the present work. Triadimenol possesses two chiral centers and consequently can exist in four enantio-

STEROL

Control (untreated) RS 2 mg liter-’ 10 mg liter- ’ SR 2. mg liter-’ 10 mg liter-’ RR 2 mg liter-’ 10 mg liter-’ ss ? mg liter-’ 10 mg liter’ Cl mg of sterol

measured

BIOSYNTHESIS-INHIBITING

Control RS SR RR s.7

(untreated)

2%

0.26

0.28

0.24 0.3

0.31 0.39

0.60 0.41

83 75

0.24 0.29

0.13 0.31

0.50 0.46

70 77

0.26 0.23

0.33 0.36

0.65 0.55

90 x2

0.26 0.24

0.33 0.44

0.65 0.57

90 91

by GLCig

dry

wt of tissue.

activity (17) while the RR enantiomer is reported to possess fungicidal activity against cereal mildews and rusts (23). The discovery that the RR enantiomers of paclobutrazol and its dichloro analog are the major fungitoxicants (22) is surprising in view of the fact that the SR enantiomer of triadimenol is most fungitoxic. As both series of compounds are believed to inhibit fungal sterol biosynthesis at the same site of action it has been concluded that as fungicides they may have different modes of binding at the active centers (21). In the present experiments, a large difference was noted in the influence of the two enantiomeric pairs of paclobutrazol on

Enantiomer

FUNGICIDES

Campesterol (mgigl”

normal plant sterols (Tables 1 and 2). By far the more effective was the 2RS,3SR diastereoisomer which indeed was the most active of all the C-14 demethylation inhibitors tested. The difference between paclobutrazol diastereoisomers was confirmed at lower dose rates with plants harvested at the slightly later time of 4 weeks after application. The 3RS,3SR form administered at 10 and 2 mg liter-’ as a root drench decreased normal shoot sterols by 75 and 46’S, respectively, while with the 2RS,3RS form the corresponding figures were 15 and 5%. The relative contribution of the two enantiomers of the 2RS.3SR pair is not yet known. However. in view of these results

Stigma5terol

0.28 0.33 0.25 0.23 0.31

No/c>. Three plants were combined for each analysis. cm with no significant differences between enantiomer ‘I mg of sterol measured by GLCig dry wt of tissue.

(mgig)” 0.24 0.32 0.21 0.20 0.3 Plants analyzed treatments.

Sitosterol (mgigla

Total three sterolh ah Q of control

I.16 0.90 0.84 0.x0 I.12 had heights

100 92 77 73 100 varying

between

45 and SX

296

BURDEN,

CLARK,

and with the knowledge that it is the SS enantiomer which is primarily the anti-gibberellin component it does seem probable that with paclobutrazol anti-GA and anti-plant sterol effects can be distinguished at the enantiomer level. Thus these enantiomers may prove to be most useful probes for studying the mode of action of plant growth regulators. The likely sites at which the SBI fungicides blocked the synthesis of normal barley sterols were located from a consideration of the total shoot sterol profiles (Fig. 2). Identification of abnormal sterols was by relative retention time. order of elution, and GLC-MS compared with literature data (29). For all the compounds tested there was an excellent correlation between the diminution of the levels of the normal sterols and the accumulation of “abnormal” ones, exemplified by the total sterol profiles obtained with fenpropimorph and nuarimol (Fig. 2, Table 3). As expected from the work of Benveniste and his colleagues (29) fenpropimorph appeared to inhibit the enzyme cycloeucalenol-obtusifoliol isomerase leading to an accumulation of cycloeucalenol and also of a cyclopropyl 4-demethyl sterol, 24-methylpollinastanol (Table 3). A similar profile (not shown here) was observed with the other morpholine tridemorph, whereas the other SBI fungicides, with the exception of prochloraz, gave profiles of the type obtained with nuarimol. Here the major “abnormal” sterols were identified as obtusifoliol, dihydroobtusifoliol and 14~-methyl-~8-ergostenol (Table 3). This is excellent evidence for the inhibition of a C- 14 demethylase enzyme at a position in plant sterol biosynthesis (Fig. 3) equivalent to that involved in the biosynthesis of ergosterol in fungi. It should be emphasized, however, that the dosages employed here were higher than those which would normally be supplied to field-grown plants and hence the fungal C-14 demethylase is likely to be far more sensitive to these xenobiotics than that of the host plant. Nuarimol analogs. the py-

AND

HOLLOWAY

rimidine methanols fenarimol and triarimol, have previously been shown to inhibit C-14 demethylation in plant cell cultures (29. 34). Some triazole herbicides with an unknown mode of action have also been reported (35) and it is possible that these could operate by inhibition of the plant C-14 sterol demethylase. The mean shoot heights recorded for the barley seedlings are given in Table 1. All treatments significantly reduced plant height compared to control and within the treatments there appeared to be three distinct groups. Nuarimol, UK-140, and both diastereoisomers of paclobutrazol showed the greatest growth reduction; prochloraz showed the least: and tridemorph. fenpropimorph. and the two diastereoisomers of the triadimenol were intermediate. Although the 2RS.3SR diastereoisomer of paclobutrazol has been stated to have low plant growth regulatory activity (23). in the present experiment it surprisingly reduced plant height to approximately the same extent as the 2RS.3RS form. However. the seedling morphology was quite different. for the 2RS,3RS produced stocky green and healthy plants with greatly increased tillering while the 2RS,3SR were much weaker plants with little tillering and pronounced scorching at the leaf tips. In view of the previous discussion it is possible that the 2RS,3RS-induced symptoms are largely the result of interference with the gibberellic acid pathway while the stunting caused by the 2RS.3SR may be related to the depletion of normal sterols and the accumulation of C-14 methyl sterols such as obtusifoliol (29) which may perturb membrane structure leading to “toxic” effects. The morpholine-treated plants. although shorter. remained green and apparently healthy despite the accumulation of “abnormal” 9.19-cyclopropyl sterols. In this case it is thought (29, 36) that although the C-14 methyl group is present. its adverse effects on membranes are moderated by the “bent” sterol structure resulting from the presence of the cyclopropyl ring.

STEROL

BIOSYNTHESIS-INHIBITING

FUNGICIDES

297

HO

An additional factor which may be important in membrane performance following xenobiotic treatment is a change in the ratio of the three “normal” sterols: campesterol, stigmasterol, and sitosterol. There is now good evidence that physical and chemical changes in cell membranes, particularly plasma membranes, are involved in such phenomena as the acclimation of plants to low temperatures and their ability to withstand freezing stress (37). Frost injury may be caused by the intracellular formation of ice crystals (38) and hence the ability of plasma membranes to

remain fluid at low temperatures may facilitate the efflux of water and reduce the chances of injury. In the most commonly accepted model for biological membranes, complex lipids are thought to form a bilayer in which integral proteins are embedded (39). The maintenance of the fluidity of the lipid bilayer is essential for life processes but a phase change from a liquid crystalline to a gel state can, however, take place at temperatures within the range of plant growth (40). The function of sterols, particularly cholesterol, in the regulation of the fluidity of the bilayer has received much at-

298

BURDEN,

CLARK,

tention (36, 39, 41). With membranes in the liquid crystalline state cholesterol has a condensing effect on the acyl chain region of the phospholipid components, producing a more ordered structure and reducing fluidity. However, at low temperatures cholesterol increases membrane fluidity by remaining intimately associated with the phospholipids and preventing them from adopting the all-tr.ans configuration characteristics of the gel phase. It has been convincingly argued by Bloch that cholesterol possesses the optimum structural features for membrane regulation and that in animals it is the “end product of directed evolutionary pressures” (36). However. for reasons which are not fully understood. cholesterol is a minor sterol in plants which have instead the C-24 alkylated analogs. Studies with plant root membranes have indicated that the control of permeability decreases in the order cholesterol, campesterol, stigmasterol, and sitosterol (31, 42-44). From this it may be hypothesized that any change in sterol balance toward a “more planar” cholesterol-type structure might enhance the control of permeability, particularly at low temperature, and hence contribute to cold acclimation and frost hardiness. Support for this emerges from the work of Sikorska and Farkas on frost hardening in winter rape (38). In frost-hardened leaves the relative contribution of sitosterol to the total sterols was reduced while the proportion of both campesterol and cholesterol increased substantially. In the present work the major objective has been an assessment of the effect of xenobiotics on the composition of total barley sterols. However, the changes observed may also apply to the functional sterols of membranes. In the shoots (Table I) but not the roots (Table 2) there appeared to be a reduction in the C-24 ethyl sterols sitosterol and stigmasterol as compared to the C-24 methyl sterol campesterol. This apparent inhibition of the second alkylation step has been noted previously with tridemorph and fenarimol (an analog of nuar-

AND

HOLLOWAY

imol) in plant tissue culture studies (34). While the biochemical reason for this remains obscure (both alkylations are effected by methyltransferases involving Sadenosylmethionine) a practical consequence might be an alteration in the sterol balance of membranes toward a more “cholesterol-like” structure with concomitant changes in fluidity at low temperature. The cold resistance of cabbage and barley plants treated with triadimefon as a root drench has previously been noted and an explanation offered in terms of an alteration of hormone balance (20). An alternative explanation may be a change in membrane architecture brought about by an alteration of sterol levels and composition. However. on present data this must be regarded as highly speculative and more information must be obtained on the sterol and phospholipid composition of purified membrane fractions, particularly the plasmalemma. in relation to plant physiological performance. ACKNOWLEDGMENTS We thank Mr. R. F. Hughes and Mr. J. B. Woodley for growing plant material. Mr. M. J. Lewis for GLCMS determinations and. Mrs. G. M. Arnold for statislical analysis. Dr. J. R. Lenton. Dr. D. N. Butcher, and Mr. D. T. Cooke contributed much valuable diszussion. Long Ashton Research Station is financed through the Agricultural and Food Research Council. REFERENCES I. F. J. Schwinn. Ergosterol biosynthesis inhibitors. An overview of their history ad contribution to medicine and agriculture. Pesric. Sci. 15. 40 (1984). 2. P. Langcake. P. J. Kuhn. and M. Wade. The mode of action of systemic fungicides, Proa. Pesfic Bioc97erl7. 7inicol. 3, I (1983). 3. H. D. Sisler and N. N. Ragsdale, Biochemical and cellular aspects of the antifungal action of ergosterol biosynthetic inhibitors, in “Mode of Action of Antifungal Agents.” p. 257. Cambridge Univ. Press. Cambridge. 1984. 4. H. D. Sisler and N. N. Ragsdale, Fungitoxicity and growth regulation involving aspects of lipid biosynthesis. Nrrl~. J. Plnnf Parhd. 83fSuppl. I). 81 (1977). 5. B. C. Baldwin. Fungal inhibitors of ergosterol

STEROL

BIOSYNTHESIS-INHIBITING

biosynthesis. Bioclre,tr. Sot. Trrrns. 11, 659 t 1983). 6. E.-H. Pommer. Chemical structure-fungicidal activity relationships in substituted morpholines. Pestic. Sci. 15, 285 (1984). 7. R. I. Baloch. E. I. Mercer, T. E. Wiggins. and B. C. Baldwin, Inhibition of ergosterol biosynthesis in S~cc~~crrorn~ces err-evisiar and Usii/allo muydis by tridemorph, fenpropimorph and fenpropidin. Phyroclzewzistryv 23, 2219 (1984). 8. H. Buchenauer. Mode of action and selectivity of fungicides which interfere with ergosterol biosynthesis it! “Proceedings. 1977 British Crop Protection Conference-Pests and Diseases.” p. 699. 1977. 9. H. Buchenauer and E. Rohner. Effect of triadimefon and triadimenol on growth of various plant species as well as on gibberellin content and sterol metabolism in shoots of barley seedlings, Pesti<,. Biochem. Physiol. 15, 58 ( 1981 l. IO. H. Buchenauer, B. Kutzner. and T. Koths, Effect of various triazole fungicides on growth of cereal seedlings and tomato plants as well as on gibberellin contents and lipid metabolism in barley seedlings. Z. ~flKt~nrl/r. @7Sclllrr;. 91, 506 t 1984). Il. J. B. Shive and H. D. Sisler. Effects of ancymidol (a growth retardant) and triarimol (a fungicide) on the growth, sterols and gibberellins of P/wseol~s r,ulgtrr-is CL.), Plant P/fy.sio/. 57, 640 t 1976). 12. R. T. Kane and R. W. Smiley. Plant growth-regulating effects of systemic fungicides applied to Kentucky bluegrass, Apron. J. 75, 469 (1983). 13. L. Buyrse. A. Callebaut. and D. De Craene. Inhibition of growth and DNA synthesis in pea epicotyls by fungicidal inhibitors of ergosterol biosynthesis and its reversal by GA,, Med. Fat. Ltrrldhou\lw~. Rijksrtnit~. Gent 49, 1019 (1984). 14. J. Dalziel and D. K. Lawrence. Biochemical and biological effects of kaurene oxidase inhibitors such as paclobutrazol, Brit. Plunf G~-ott,fh Re,y/rl. Group Mono. 11, 43 t 1984). 15. K. Izumi, I. Yamaguchi, A. Wada. H. Oshio, and N. Takahashi. Effects of a new plant growth retardant (E)-I-(4.chlorophenyl)-4.4-dimethyl-?t 1.2.4-triazol-I-yll-I-penten-3-01 (S-3307) on the growth and gibberellin content of rice plants. Plar~t Cell Physiol. 25, 61 I (1984). 16. K. Lurssen and W. Reiser, Chemistry and physiological properties of the new plant growth regulator RSW 041 I. in “Proceedings 1985 British Crop Protection Conference-Weeds.” p. 121. 1985. 17. P. Hedden and J. E. Graebe, Inhibition ellin biosynthesis by paclobutrazol homogenates of Cucwbita marirnrr

of gibberin cell-free endosperm

FUNGICIDES

18. 19. 70.

21.

21.

23.

24.

35.

76.

27.

28.

19. 30. 31.

32.

33.

299

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