Effects of triadimefon on the fine structure of germinating conidia of Botrytis allii

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

Effects of Triadimefon

21, 74-83 (1984)

on the Fine Structure of Botrytis allii D. V.

of Germinating

Conidia

RICHMOND

Long Ashton Research Station, University of Bristol, Bristol BS18 9AF, United Kingdom Received

July 6, 1983; accepted July 27, 1983

Conidia of Botrytis allii germinated in the presence of triadimefon to produce stubby and swollen germ tubes. Electron micrographs showed that organelles remained intact after treatment but in some cells there was an increase in vacuolation and in the numbers of lipid vesicles. The most striking changes were in the process of septum formation. Septa almost invariably remained incomplete in the presence of triadimefon but complete in its absence. In some hyphae septa were not produced but the wall became unevenly thickened. Vesicles occurred close to septal and hyphal walls and within swollen parts of walls. Tiiadimefon probably interferes with the mechanism of septal and hyphal wall construction.

endoplasmic reticulum than untreated hyphae (17). This paper describes the changes in fine structure that occur when conidia of Botrytis allii Munn germinate in the presence of triadimefon.

INTRODUCTION

Triadimefon [ 1-(4-chlorophenoxy)-3,3-dimethyl-l(lH-1,2,4-triazol-1-yl)-2-butanone] is a member of a group of systemic fungicides including triforine (l), fenarimol (2), and imazalil (3) which inhibits the biosynthesis of ergosterol by preventing sterol C-14 demethylation (4, 5). The activity of triadimefon appears to result from its conversion into isomers of triadimenol [ 1-(Cchlorophenoxy)-3,3-dimethyl-I-(1,2,4-triazol-lyl)butan-2-011 by fungi and plants (6-9). The fungicides which inhibit ergosterol biosynthesis suppress hyphal growth more effectively than spore germination. Germ tubes of treated spores are swollen and deformed (IO- 13), the separation of daughter cells from sporidia of Ustilugo is inhibited, and treated sporidia become branched and distorted (4, 14, 15). A mutant of Ustilago maydis deficient in sterol C-14 demethylation had sporidia closely resembling the wild type treated with fenarimol, grew slower than the wild type, but was unaffected by this fungicide thus confirming the site of action (16). Triadimefon is reported to inhibit formation of sclerotia of Sclerotinia sclerotiorum, and hyphae grown in the presence of the fungicide to contain more mitochondria and

MATERIALS

Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in anv form reserved.

METHODS

Fungus. B. allii (C.M.I. 42 078) was grown on malt extract agar (5% Oxoid). Conidia (2 x 106/ml) were suspended in distilled water or in a nutrient medium containing 0.2% malt extract broth (Oxoid) and incubated for 15 hr at 25°C on an orbital shaker at 50 rpm. Triadimefon (final concentration, 4, 10, and 20 p.g/ml) was added to the cultures in methanol solution. The final concentration of methanol did not exceed 1% in treated and untreated cultures. Electron microscopy. Conidia were fixed by adding glutaraldehyde (final concentration 2% w/v) in pH 7.0 sodium cacodylate buffer (final concentration 25 mM> directly to the culture flasks at 25°C. The flasks were then transferred to 0-4°C for 1 hr, centrifuged, washed with buffer at O-PC, and refixed for 1.5 hr in glutaraldehyde (2% w/v). The fixed cells were again washed with the same buffer and postfixed with osmic acid (2% w/v) for 1.5 hr at 0-4°C. The fixed conidia were washed with sodium 74

0048-3575/84 $3.00

AND

EFFECT

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TRIADIMEFON

cacodylate buffer, dehydrated in a graded series of ethanol and propylene oxide, and embedded in Spurr’s low-viscosity resin. Sections were cut with an LKB ultratome, stained with uranyl acetate and alkaline lead citrate, and examined with an AEI 801 transmission electron microscope. Light microscopy. Samples of the conidia prepared for electron microscopy in Spurr’s resin were placed on a microscope slide and covered with a coverslip, and the resin was polymerised at 70°C. RESULTS

Conidia did not germinate when incubated in distilled water and resembled resting conidia fixed directly from growing cultures (Fig. 1). They produced long germ tubes (Figs. 2, 3) in the presence of malt extract broth. A septum with Woronin bodies was formed at the base of the germ tube (Fig. 3). Nuclei were clearly visible with the light microscope in all conidia (Figs. 1, 3), but the storage bodies clearly present in resting conidia (Fig. 1) were not detected after germination (Figs. 3, 4). Germ tubes became progressively shorter as the concentration of triadimefon increased. At 20 Fg/ml the germ tubes were stubby and swollen (Fig. 4). The fine structure of resting conidia was indistinguishable from that of conidia incubated with distilled water (Figs. 5, 6). Storage bodies were characteristic features, occupying a considerable proportion of the conidium (Figs. 5, 6), nuclei and mitochondria were visible (Figs. 5-7) as well as the abscission scar left where the conidium became detached from the conidiophore (Fig. 6); Woronin bodies occurred beneath the delimiting septum (Fig. 6); the large number of ribosomes present obscured other organelles. When conidia germinated, the organelles became more distinct (Figs. 10, II), strands of endoplasmic reticulum developed, and the storage bodies became less conspicuous and more dispersed. Nuclei, mitochondria, endo-

ON

Botryris

u//ii

7-l

plasmic reticulum, and other structures were seen more clearly in freeze-etched replicas (Figs. 8, 9). After treatment with triadimefon, organelles remained intact but in some cells vacuolation increased (Figs. 20. 21, 25) and in others there was an increase in the number of lipid vesicles seen in thin skctions (Figs. 12, 15) and freeze-etched replicas (Figs. 13, 14). The most striking changes, however, occurred in the process of septum formation. A septum with a single central pore (Fig. 16) is formed in untreated conidia at the base of the germ tube soon after it has emerged. In the presence of triadimefon, septa almost invariably remained incomplete (Figs. 19-22). Complete septa were of uniform thicknes* throughout and had Woronin bodies (Fig 17) close to the septal pore (the section\ shown in Figs. 17 and 18 did not pass through this central pore). The early stages of septum formation frequently followed the sequence reported fol other fungi (18, 19) whether triadimefon was present or not; translucent material. triangular in cross section (Figs. 20,3 1. 321. was deposited on the inner surface of the hyphal wall, and multivesicular bodies (Figs. 20, 31) and vesicles, (Figs. 24. 31) occurred close to incomplete septa. Areas of low ribosome density (Fig. 24) were present where wall formation should take place. In what was probably an early stage of septum formation in untreated material, areas of low electron density (arrows) occurred close to the wall where a septum might be expected to form (Fig. 10). In some conidia treated with triadimefon the septa had grown symmetrically (Figs. 19, 20, 24, 29, 31), in others one side of the septum had developed further than the other (Fig. 25, 29), in others only one side had developed (Fig. 21). and finally, in others no septum had formed but wall thickenings had been produced (Figs. 92, 23, 29, 30). A whorled membranous body was found on both sides of a septum in Fig. 26. The concentric structure of this organelle was demonstrated in a freeze-etched

76

FIGS. l-7. Figs. 1-4, light micrographs; Figs. 5-7, thin sections. (I) Resting conidia showing storage bodies (SB) and nuclei (N) with nucleoli (NU). x 850. (2) Germinating conidium with typicai germ tube. X4@. (3) Germinating condium showing septum (S) with Woronin bodies ( WB) and nucleus (N). X 1700. (4) Conidium germinating in triadimefon (20 pgiml) showing short, stump\ germ tube. X 1300. (5) Conidium incubated in distilled water showing large storage bodies (SB). nucleus (NJ and mitochondria (IV). x8500. (6) Conidium incubated in distilled water, a large storage body is present (SB), ribosomes (R) are clearly visible, mitochondria (IV) are present, and Wortmir: bodies ( WB) lie close ro the abscission scar (AS). x23,OOU. (71 Enlargemenr of resting conidimv showing nucleus (IV). mitochondria (M). ribosomes (R). and endoplasmic reticulum (ER). x63,00.

replica from untreated material and in a thin section of a treated (Fig. 27). A similar membranous occurred close to a septum (Fig.

(Fig. 28) conidium structure 181, and

there may be continuity between this structure and the plasmalemma. Vesicles occurred parallel to the septal and hyphal walls (Figs. 24, 311, within hyphal wails

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TRIADIMEFON

ON Botrytis

a/Iii

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FIGS. 8-11. Figs. 8, 9. freeze-etched replicas; Figs. 10. II, thin sections. (8) Part of untreafed germinating conidium showing nucleus (N) with nuclear pores (NP) and mitochondria (M) in surface view and in section. ~34,000. (9) Untreated germinaring conidium showing storage body (SB), endoplasmic reticulum (ER), and cross-fractured lipid body (L) in surface view. ~25,000. (IO) Part 0.1 untreated germinating conidium probably showing an early stage of septum formation with translucent ureas (arrows) close to rhe constricted neck; nucleus (IV), mitochondria (M), ribosomes (R). and a Woronin bodv (WB) are present. x 17,000. (11) Part of a conidium treated with triadimefon (20 pgt m0: the cytoplasm is well preserved, mitochondria (IV) and ribosomes (R) are present. A spindle pole body occurs on the nucleus (N). x37.000.

(Fig. 12), and in large numbers in swollen parts of the wall (Figs. 22, 23, 29, 30, 32). Triadimefon even at 20 pg/ml usually had little effect on cytoplasmic and nuclear

structures. The nucleus (with a spindle pole body in Fig. 11) was usually well prese rved and the cytoplasm showed no sign of’ disorganisation; moreover, incomplete sep-

78

D. V.

RICHMOND

FIGS. 12-18. Figs. 12, 15,17,18, thin sections; Figs. 13, 14,16, freeze-etched replicas. (12) Hyphal tip of conidium germinating in triadimefon (20 &ml), showing numerous lipid bodies (L), mitochondria (M), and vesicles within the hyphal wall (arrows). ~17,000. (13) Conidium treated with triadimefon (20 pglml) showing fractured lipid bodies (L) and mitochondria (M). x 17,000. (14) Part of conidium treated with triadimefon (20 kglml) showing large number of vesicles (VE), vacuoles (V), and endoplasmic reticulum (ER). ~27,000. (15) Portion of germ tube from conidium treated with triadimefon (20 pglml), vesicles (arrows) occur within the thickened wall, lipid bodies (L) are present, and membrane profiles (MP) occur within the vacuole (v). ~30,000. (16) Surface view of septum from untreated conidium showing septal pore (SP). Small particles (P) occur on the plasmalemma face. X25,000. (17) Complete septum (S) f rum untreated material showing four Woronin bodies (WB) and mitochondria (M). ~21,000. (18) Septum (S) from material treated with triadimefon (20 u,glrnf) showing membranous structure (arrow) close to plasmalemma, Woronin bodies (WB) are present. x 24,000.

EFFECT

OF

TRIADIMEFON

ON

Borrvtis

ailii

FIGS. 19-25. All thin sections. Figs. 19-22, stages in development of incomplete septa from conidia treated with triadimefon (20 FglmL). (19) Septum (S) almost complete, Woronin Bodies (WB) and a storage body (SB) are present. x 7,500. (20) Septum (S) reduced to two opposite wedges, a Woroniu body ( WB) and multivesicular body (MVB) lie close to the septum initials. the large \~acuole (V) in t/u, ityphal tip contains membrane profiles (arrows). x 10,000. (21) Septum reduced to a single septum initial (arrow). x 10.000. (22) No septum produced but wall thickening is present (arrow). x II ,000. (23) Part of hypha from conidium treated with triadimefon (10 pglml) shonGg thickened nail w,ith ijesicles (arrows) within wall, nucleus (N) with nuclear membrane (double arrows). ond well preserved. unnltered cytoplasm with mitochondria (M) and endoplasmic reticulum (ER). ~55,000. (24) Enlargevesicles (arrows) close to septul and hyphal walls. An area of loua ribosome ment of (19) showing density (LRD) occurs within the enlarged septal pore, Woronin bodies ( WB) und mitochondria (M) are also present. ~24,000. (25) Hypha from conidium treated nsith triadimefon (IO kglml) show~inq incomplete septum (S). Woronin bodies (WB), mitochondria (M), and vucuoles Cv) with membratrt~ profiles (MP); light areas (arrows) occur close to the hyphal w,all. x 13.500.

D. V. RICHMOND

80

ta frequently well-preserved 23, 24).

occurred in hyphae with nuclei and cytoplasm (Figs. DISCUSSION

Some conidia treated with triadimefon showed increased vacuolation, accumulations of lipid vesicles, and mitochondria with swollen cristae. Related compounds which also act by inhibiting the biosynthesis of ergosterol(18) have shown similar effects on fungal tine structure. De Nollin and Borgers (19) suggested that miconazole acted primarily on the cell wall and plasmalemma of Candida albicans and Preusser (20) considered that econazole increased the permeability of the cell membrane of Trichophyfon rubrum. Freeze-fracture studies have shown damage to the plasmalemma of C. albicans (21) and Saccharomyces cerevisiue (22). Naftifine produced accumulations of lipid bodies in T. mentagrophytes (23) and C. parapsilosis (24). These effects are consistent with the mode of action of compounds which act by inhibiting the biosynthesis of ergosterol, resulting in an interference with membrane synthesis, and the accumulation first of sterol intermediates and later free fatty acids (18). The cytological response to toxicants depends, however, on the dose, time of exposure, and individual variations within the population. Some of the reported changes such as membrane disruption, swollen mitochondria, and increased vacuolation occurred only in the presence of fungicidal concentrations of toxicant (24) and are typical of the nonspecific changes that occur when cells die (25). In the present study concentrations of triadimefon were used which reduced growth but did not kill the spores. The most striking effects of triadimefon were on the structure of septal and hyphal walls. As the changes occurred in cells with well preserved and unaltered organelles typical of healthy living material, they probably represent specific effects of the

fungicides. Incomplete septa have also been illustrated in U. avenae treated with triadimefon (26) and nuarimol (27), in T. mentagrophytes treated with naftitine (23), and in Puccinia recondita and Uromyces viciae-fabae treated with triadimefon (28). Growth of fungal septa is usually very rapid (29), taking from 3.5 to 4 min in Neurospora crassa (30) and 3 to 7 min in Ascodesmis sphaerospora (3 I); consequently it is difftcult to study the fine structure of their development. Incomplete septa were not seen in untreated B. fabae (32, 33) although stages in the formation of septa of B. cinerea have been observed (34). In the present study most septa were complete in the absence of triadimefon and incomplete in its presence. These effects suggest that triadimefon interferes with the molecular mechanism of morphogenesis responsible for the architectural structure of septa and walls. Our understanding of the biochemical processes involved in the construction of hyphal and septal walls is incomplete (35). Chitin microfibrils may be synthesized by microvesicles or chitosomes either self-assembled, or formed from endoplasmic reticulum or multivesicular bodies, delivering a packet of chitin synthetase to the cell surface (36). Alternatively, a chitin synthetase zymogen may be uniformly distributed along the plasmalemma and be activated by a protease located in cytoplasmic vesicles (37). Young septa consist mainly of chitin, probably synthesized in situ rather than formed elsewhere and transported to the site (29). Hyphai walls are more complex, consisting of four layers; an outer glucan layer, followed by a glycoprotein reticulum, then a mainly protein layer, and finally an inner chitin layer. The glycoprotein reticulum proliferates in the hyphal wall adjacent to septa (29). Sterols regulate membrane permeability by altering the internal viscosity and molecular motion of other lipids and influence the activity of membrane-bound enzymes (38). Triadimefon may disrupt septum for-

EFFECT

OF

TRIADIMEFON

ON

Botrytis

&ii

FIGS. 26-32. Fig. 28, freeze-etched replica; remainder, thin sections. (26) septum from conidium treated with triadimefon (10 kglml) showing membranes structure (MS) passing through septal port. x 64.000. (27) Whorled membranous structure (MS) from material growing in triudimefon (20 ~giml). < 64,000. (28) Freeze-etched replica of membranous structure (MS) from untreated mcltericil. x 30.000. (29) Hypha from conidium treated with triadimefon (20 pglml) with incomplete septum (.S) and large wall swellings (arrows). Nuclei (I%? one with a nucleolus (NU), Woronin bodies (WB), (mui mitochondria (M) are present. x 17,000. (30) Part of snsollen hyphal MYI// containing many I.Es~c./c.\ (urrows) from conidium treated with triadimefon (20 Fglmi). x 30,000. (31) Enlargement (!f (201 .sho%c*ing incomplete septum (S), a Woronin body (WE), a mu/ti~~e.sicrrlar body (MVE), and ~~sic,/ea (rrrrow) close to the incomplete septum x 27,000. (32) Incomplete septum (S) .from conidium treutctl( tl,ith triadimefon (20 pgimf) showing vesicles (urrows) in septul x’tdl. X 36,000.

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D. V. RICHMOND

mation and produce abnormal hyphal wall thickenings by inhibiting sterol biosynthesis and thus altering the permeability and fusion properties of the microvesicles involved in wall synthesis. This may account for the large number of vesicles within incomplete septa and wall thickenings. Alternatively, a deficiency of ergosterol may limit the rapid membrane synthesis necessary during septum formation, the resulting imbalance between wall and membrane synthesis leading to wall swellings. Other compounds such as sorbose (39) and griseofulvin (40) produce thickenings and distortions in hyphal walls and alter wall structure. Cycloheximide (41) changes wall synthesis from extension at the apex to subapical thickening. Cytochalasin A produces abnormal wall deposits in N. CY~SS~ and may disorganise the cytoplasmic vesicles involved in wall synthesis (42). Caffeine produces irregular walls and wall protrusions as well as vesicles within plant cell walls. The effects of caffeine on root tips of Vicia faba, are remarkably similar to those produced by triadimefon in B. alfii (43). Caffeine may act in higher plants in a similar way to triadimefon in fungi by interfering with the cytoplasmic vesicles involved in wall formation.

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EFFECT

22.

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OF TRIADIMEFON

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ON Botr?‘tis a//ii

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