HORTSCIENCE 26(2):173-175. 1991.

Fine Structure of Apple LeavesTreated with the Sterol-inhibitingFungicide BitertanolS.V. Overton1, L.D. Moore1, and O.K. Miller2

Virginia Polytechnic Institute and State University, Blacksburg,VA 24061

Additional index words. Malus domestics, chloroplast, thylakoid

Abstract. Ultrastructural observations were made of leaves of apple (Malus domesticsBorkh. cv. Red Delicious) 12, 24, and 72 hours following a single foliar application ofthe sterol-inhibiting fungicide bitertanol. Thylakoids of chloroplasts from treated leaveswere swollen and irregular and chloroplasts had lost their integrity within 12 hoursafter treatment. Occasionally, mitochondria looked washed out, although no otherchanges in membrane or organelle structures were observed. Within 24 to 72 hours,moreover, thylakoids of chloroplasts from treated leaves returned to a state similar tothat of the controls. However, the numbers of starch granules in the chloroplasts oftreated leaves appeared to increase throughout the 72 hours and remained somewhathigher than levels in controls. Thus, bitertanol does not appear to have a lasting effecton apple leaves. Chemical name used: ß-([1,1´-biplenyl]-4-yloxy)- αα-(1,1-dimethyle-thyl)-1H-1,2,4-trizole-1-ethanol (bitertanol).

The inhibition of sterol biosynthesis byagricultural chemicals has been ‘studied ex-tensively in animal tissues (Schroepfer, 1981)and in microorganisms (Leroux, 1982;Oelschlager et al., 1980). Little research hasbeen-conducted with higher plants, althoughmany of the sterol-inhibiting compounds usedin agriculture reduce the growth of plants(Buchenauer and Grossman, 1977; Buch-enauer and Rohner, 1981; Shive and Sisler,1976). Moreover, triarimol, a sterol-inhib-iting fungicide (SIF), and ancymidol, a plantgrowth retardant, are reported to interfere withergosterol biosynthesis in fungi (Ragsdale,1975). These compounds reportedly retardedthe growth of Phaseolus vulgaris L. cv.Contender seedlings but had no noticeableeffect on the qualitative and quantitative dis-tribution of the main sterols present (Shiveand Sisler, 1976). The SIFs nuarimol, tri-adimenol, triadimefon, and imazalil inhib-ited the incorporation of 14C acetate into lipidsof barley plants. The effects of these SIFson the sterol fractions were analogous to thosereported to occur in fungal systems (Buch-enauer and Rohner, 1981), although much

—Received for publication 10 July 1989. This studywas supported, in part, by the Mobay ChemicalCorp. The cost of publishing this paper was de-frayed in part by the payment of page charges.Under postal regulations, this paper therefore mustbe hereby marked advertisement solely to indicatethis fact.1Dept. of Plant Pathology, Physiology and WeedScience.

2Dept. of Biology.

H O R TSCIENCE, VO L. 26(2), FEBRUARY 199

higher-concentrations were required to achievecomparable results.

Even though SIFs are often appliedthroughout a growing season on numerous

Figs. 1 and 2. Untreated ‘Red Delicious’ leaves.(1) Transmission electron micrograph of an un-treated leaf showing a uniform cuticular layerwith underlying epidermal cells; cuticle (C), cellwall (CW), epidermis (E), × 5600. (2) Paren-chyma cells showing internal arrangement oforganelles; chloroplast (Ch), mitochondrion (M),nucleus (N), rough endoplasmic reticulum (rER),× 5600.

1

field crops, ornamentals, and fruit crops, nostudies have been conducted concerning theeffects of these compounds on the ultrastruc-ture of leaves of higher plants. The purposeof this study has been to examine the effectsof the SIF bitertanol (Bayer) on the finestructure of leaves of the cultivar Red Deli-cious.

Four 10- to 12-year-old ‘Red Delicious’apple trees were used in this study. The trees,which were selected for uniformity of sizeand fruit set, had not previously been treatedwith SIF. Twelve groups of shoots per aspectlocated on the north, east, west, and southaspects of each tree were used. An equalnumber of shoots was treated with bitertanoland maintained as controls. The bitertanolwas applied with a hand sprayer to abaxialand adaxial leaf surfaces at a rate equivalentto 1.134 g·ha-1

. The six youngest fully ex-panded leaves of four sprayed and unsprayedbranches located at each aspect of each treewere used. Thus, there were 24 leaf samplesat each aspect on each of four trees.

Four 1- to 3-mm rectangular strips inter-mediate between a primary vein and marginof leaves, untreated and treated with the fun-gicide, were removed with a razor blade 12,24, and 72 hr after fungicide application.These strips were then processed for electronmicroscopy by being prefixed in 4% glutar-aldehyde in 0.1 M cacodylate buffer for 5 hr

Figs. 3 and 4. Untreated ‘Red Delicious’ leaves.(3) Transmission electron micrograph of pali.sade parenchyma cells showing peripheral elon-gated chloroplasts; chloroplast (Ch), thylakoid(T), x 5200. (4) Palisade parenchyma cellsshowing numerous chloroplasts containing mi-nute electron dense lipid bodies. Note sphericalto elongated mitochondria; mitochondrion (M),chloroplast (Ch), thylakoids (T), × 8450.

173

Fig. 5. ‘Red Delicious’ leaves 12 hr after appli-cation of bitertanol. Palisade parenchyma cellexhibiting membrane disruption and swelling ofthe thylakoids. Note the increase in the numberof starch granules; chloroplast (Ch), cell wall(CW), mitochondrion (M), starch (S), thyla-koids (T), × 6600.

Figs. 6-8. ‘Red Delicious’ leaves 12 hr after ap- Figs. 9 and 10. ‘Red Delicious’ leaves 24 andplication of bitertanol. (6) Higher magnification 72 hr after application of bitertanol. (9) Trans-than in Fig. 5 of chloroplasts showing mem- mission electron micrograph of a palisade pa-brane disruption and thylakoid swelling (ar- renchyma cell showing internal organellerows); chloroplast (Ch), cell wall (CW), nucleus arrangement 24 hr after leaf was treated. Chlo-(N), thylakoids (T), × 13,300. (7) Mitochon- roplasts and mitochondria are normal, althoughdria are washed-out; chloroplast (Ch), mito- the nucleus is less dense as compared to thosechondrion (M) × 30,800. (8) Nucleus is less of the controls. Note the increased numbers ofdensely stained; nucleus (N), × 9800. starch granules within the chloroplast; chloro-

plast (Ch), mitochondrion (M), nucleus (N),starch (S), × 9800. (10) Palisade parenchymacells 72 hr after treatment showing internal ar-rangement of organelles. Chloroplasts and mi-tochondria are normal, although the nucleusremains washed-out; mitochondrion (M), nu-cleus (N), starch (S), thylakoids (T), × 9100.

at 0C, washed in three changes of 0.1 M

sodium cacodylate buffer for 1 hr, and post-fixed in sodium cacodylate buffered osmiumtetroxide for 16 hr at 0C. The strips werethen washed in three changes of 0.1 M so-dium cacodylate buffer for 45 rein, dehy-drated in a graded ethanol series (30 mineach) followed by an additional change ofabsolute alcohol for 30 rein, and then byacetone to remove the water that might bepresent (two changes of 10 min each). Next,the strips were stepwise infiltrated overnightand embedded in Spurr’s low viscosity resin(Spurr, 1969). Blocks were cured at 70C for24 hr. Sections were cut on a Sorvall MT2Bultramicrotome with a DuPont diamond knife,collected on 200-mesh copper grids, anddouble-stained with 2% uranyl acetate for 30min and Reynold’s lead citrate (Reynold,1963) for 8 min.

Untreated leaves possessed a uniform cu-ticular layer that covered the outer surfaceof the epidermis (Fig. 1). Chloroplasts werefound primarily in the palisade and spongymesophyll cells of the leaf (Figs. 2-4). Pal-isade parenchyma consisted of long colum-nar cells (Figs. 3 and 4). Located around theperiphery of these cells were numerousspherical to elongate chloroplasts that mea-sured ≈ 2 × 6 µm. The internal structure ofthese chloroplasts was characterized by asystem of membranes, the basic subunit ofwhich was a thylakoid (Figs. 3 and 4). Tightlypacked stacks of thylakoids, parallel to eachother, were present-throughout the stroma ofthe chloroplasts. In addition, lipid and starchgranules were located within the chloro-plasts.

Twelve hours after treatment, the thyla-koids of chloroplasts were swollen and ir-regular (Figs. 5 and 6), which gave thechloroplasts a somewhat distorted appear-ance. Occasionally, mitochondria werewashed out (Fig. 7), while nuclei were less

174

densely stained (Figs. 6 and 8).After 24 (Fig. 9) and 72 hr (Fig. 10) the

thylakoids were tightly packed and no longerswollen. Mitochondria were normal al-though nuclei remained somewhat washedout (Figs. 9 and 10). There appeared to be,moreover, an increase in the size and numberof starch granules 12, 24, and 72 hr follow-ing treatment with the fungicide (Figs. 5, 9,and 10). This observation was based on theexamination of ≈ 50 samples selected at ran-dom. However, a morphometrical analysiswould be required to statistically substantiatethis observation.

treatment of ‘Red Delicious’ leaves withbitertanol may lead to a temporary alterationin photosynthetic efficiency immediately afterapplication as evidenced by the presence ofswollen and irregular thylakoid membranesand loss in chloroplast integrity 12 hr fol-lowing treatment. Ashton et al. (1963) ob-served the disorganization of chloroplasts andcellular membranes induced by the triazineherbicide atrazine, an electron transport in-hibitor. The structure of atrazine is similarto that of bitertanol in that both have N-con-taining rings. That bitertanol inhibited pho-tosynthesis was not established in this studyand awaits further investigation. However,the chloroplasts appeared to have recoveredfrom the effect of the fungicide and returnedto a normal state 24 and 72 hr after treat-ment. It was previously observed by Overtonet al. (1988) that the stigmasterol level ofbitertanol-treated ‘Red Delicious’ leaves haddecreased significantly 24 hr after treatment.Moreover, treatments with bitertanol af-fected the free sterol content of apple leavesonly within the initial 24 hr after treatment

H

(Overton et al., 1988). This correlated wellwith the observation that the thylakoids andthe chloroplasts regained their integrity inthe same time period.

It should be noted that in treated leaves,the nuclei were somewhat less densely stainedthroughout the treatment period. A more ex-tensive time course investigation would berequired to determine if such an effect is per-manent and if it is detrimental. Overall, itappears that bitertanol did not have a long-lasting effect on the plant sterol concentra-tion or on the structural integrity of appleleaves. However, one must be aware thatSIFs such as bitertanol might have an effecton plant metabolism if used continually forseveral years.

Literature Cited

Ashton, F.M., E.M. Gifford, and T. Bisalputra.1963. Structural changes in Phaseolus vulgarisinduced by atrazine. 11. Effect on fine structureof chloroplasts. Bot. Gaz. 124:336-343.

Buchenauer, H. and F. Grossman. 1977. Triadi-mefon: mode of action in plants and fungi. Neth.J. Plant Pathol. 83:93-103.

Buchenauer, H. and E. Rohner. 1981. Effect oftriadimefon and triadimenol on growth of var-ious plant species as well as on gibberellin con-tent and sterol metabolism in shoots of barleyseedlings. Pesticide Biochem. Physiol. 15:58–70.

ORTSCIENCE ,. VO L. 26(2), FEBRUARY 1991

Leroux, P. 1982. Phenomes de resistance de Bo-trytis cinerea aux fungicides. La Defense desVegetaux 36:3-18.

Oelschlager, A.C., H.D. Pierce, A.M. Pierce, R.H.Angus, E. Quantin-Martenot, A.M. Unrau, andR. Srinwasan. 1980. The use of mutants andazasterols in studies of yeast sterol biosynthesis,p. 395-403. In: P. Benveniste, C. Costes, R.Deuce, and P. Mazliak (eds.). Biogenesis aridfunction of plant lipids. Elsevier/North-Holland

Biomedical Press, Amsterdam.

H O R TSCIENCE , VO L. 26(2), FEBRUARY 1991

Overton, S.V., L.D. Moore, D.M. Oreutt, K.S.Yoder, and S.A. Meredith. 1988. Influence ofsterol-inhibiting fungicides on the free sterol andfree fatty acid composition of Red Deliciousand Jonathan apple cultivars. J. Hort. Sci.63:183-191.

Ragsdale, N.N. 1975. Specific effects of triarimolon sterol biosynthesis in Ustilago maydis.Biochim. Biophys. Acts. 380:81-96.

Reynolds, E.S. 1963. The use of lead citrate athigh pH as an electron opaque stain in electron

microscopy. J. Cell Biol. 17:208-212.Schroepfer, G.J. 1981. Sterol biosynthesis. Annu.

Rev. Biochem. 50:585-621.Shive, J.B. and H.D. Sisler. 1976. Effect of an-

cymidol (a growth retardant) and triarimol (afungicide) on the growth, sterols, and gibber-ellin of Phaseolus vulgaris. Plant Physiol.57:640-644.

Spurr, A.R. 1969. A low viscosity epoxy resinembedding medium for electron microscopy. J.Ultrastruct. Res. 26:31.

175