Analele ştiinţifice ale Universităţii Al. I. Cuza Iaşi Tomul LIV, fasc. 1, s. II a. Biologie vegetală, 2008 CONTRIBUTIONS TO THE HISTO-ANATOMICAL STUDY OF THE CALENDULA OFFICINALIS L. LEAVES TREATED WITH THIOPHANATE METHYL (TOPSIN M) LUMINIŢA HUŢANU-BASHTAWI, C. TOMA Abstract. The study analyzes the histo-anatomical modifications of the Calendula officinalis leaf, caused by the treatment with thiophanate methyl, applied 3 times, in two different concentrations, of 0.1% and, respectively, 0.4%. The cross-sections made at the three levels of the leaf, as well as the surface ones, evidenced some quantitative differences between the two variants of treatment and the reference, while the differences of qualitative type are minimum, referring to the different distribution of the pallisadic tissue on the two sides of the foliar limb; consequently, the leaf structure is different: bifacial unequally equifacial in the reference and bifacial heterofacial, respectively, in the treated samples. The quantitative type modifications are related to the prominence extent of the median nervure, thickness of the meristematic area, size and number of the conducting fascicles and the xylem vessels (which are intensely stimulating parameters in the two treatments), the presence of secretory hairs, width and thickness of the foliar limb which, at a concentration of 0.4%, are slightly inhibited, in spite of the fact that the median nervure is much more prominent, even comparatively with the 0.1% concentration treatment. Keywords: Calendula, Topsin M, cytokinin hormone-type action, foliar limb, histo-anatomical modifications. Introduction The influence of fungicides on plants productivity is usually attributed to the primary fungicide/fungistatic effect of such substances, although, quite frequently, they may show secondary physiological effects, which may be either toxic or, on the contrary, beneficial to the plants subjected to such treatments [3]. The thiophanate methyl, a systemic fungicide belonging to the benzimidazole class, is largely utilized, as due to its large spectrum of action, as a curative and protecting substance for the cultivation of alimentary, industrial as well as medicinal plants [9]. Involvement of benzimidazoles and of some of their derivatives in the regulation of certain physiological processes developed at plant level has been extensively studied, being usually defined as a cytokynin hormone-type action [6, 10]. In the case of both thiophanate methyl and carbendazime, the main metabolite and the fungicide s active substance, respectively, cytokinin-like effects have been demonstrated in some culture plants, being manifested by the inhibition of leaves senescence, lower degradation of chlorophyll, proteins and AND; more than that, synthesis of the photosynthetic pigments is stimulated, so that the treated leaves maintain their green colour over a longer period of time [1, 4, 5, 6]. The (20 g ml -1 ) carbendazime solution applied on the leaves of wheat Al. I. Cuza University, Faculty of Biology, 20A Carol I Bd., 700506, Iaşi, Romania 22
prevents the loss of electrolytes and amino acids, as well as disorganization of the cellular organites, the main mechanism of the antisenescence activity exercised by carbendazime being its protecting effect upon the membranary system. At higher concentrations (100 g ml -1 ), the fungicide losses its cytokinin-like activity; more than that, it even stimulates this loss of electrolytes and amino acids at the level of the membranary system of the treated leaves [7]. It is expected that the action exercised by carbendazime on the treated plants will be possibly extrapolated to the thiophanate methyl, in spite of the fact that, according to some authors [6], the cytokinin hormone-type action might be partially caused by carbendazime, in certain cases fungicides being more active, in this context, then pure carbendazim; on the other hand, the results of the experiments performed with various commercial formulae of the benzimidazolic fungicides are quite controversial as to their secondary effects [8]. Considering all these observations, the present paper analyzes the histo-anatomical modifications induced by thiophanate methyl and/or its main metabolite (MBC) upon the Calendula officinalis leaf, along their correlation with the cytokinin hormone-type effect of the fungicide, anatomically evidenced on the Cynara scolymus leaves [2], comparatively with the non-treated reference sample. Material and methods The experimental material, cultivated in the Anastasie Fătu Botanical Gardens of Iasi, was obtained from seeds of the Petrana kind, provided by the Research Station for Medicinal and Aromatic Plants of Fundulea. Besides the treated plants (TM70 0.1%, a concentration, applied in agriculture and, respectively, a TM70 0.4%, a concentration value recommended for fungicides similar to thiophanate methyl), a sample batch, formed of nontreated plants, was prepared for comparative purposes. The administration of fungicide, as a moisty powder, was made three times (at intervals of 7 and 10 days), in the moment of branching or of the first anthodium formation, the plants possessing 30-35 nomophyles. The vegetal material, harvested 10 days after the last treatment, was fixed and conserved in 70% ethanol, then processed according to the methods commonly applied in studies of vegetal anatomy. Measurements were performed on a photonic microscope, by means of a micrometer (ocular and objective), while the light micrographs were performed on a Novex (Holland) microscope, using a Cannon A95 camera. In this paper we used the following abbreviations: Ca. of. M - Calendula officinalis, control (untreated plants); Ca. of. TM 0,1% - Calendula officinalis, treated with Topsin M 0,1%; Ca. of. TM 0,4% - Calendula officinalis, treated with Topsin M 0,4%. Results and discussions Cross-sections - the basal leaf, the middle third In the reference the median nervure is visibly prominent on the inner side of the limb, evidencing only one conducting fascicle (Fig. 1, 2). The mesophyl shows 2 layers of low pallisadic cells, with sinuous lateral walls on the upper side, and lacunary tissue (6-7 23
layers of cells) on the inferior side, the structure of the limb being bifacial heterofacial (dorsi-ventrally) (Fig. 9, 10). In the TM 0.1% treated sample the median nervure strongly prominent on the inferior side of the limb, evidencing a semi-circular contour and including 3 large conducting fascicles which, in the case of Asteraceae, is an exception (Fig. 1, 2). The mesophyl includes 2 layers of law pallisadic cells, on the upper side, and 6 layers of lacunary tissue, so that the structure of the limb is bifacial heterofacial (dorsi-ventrally) (Fig. 9, 10). In the TM 0.4% treated sample the median nervure strongly prominent on the inferior side of the limb, with a semi-elliptical contour, forming 3 ribs: a large one in the middle and two, smaller, lateral ones (Fig. 1, 2). The secretory hairs are more numerous on the surface unit, similarly with the tectory ones, which form large bunches on the edge of the limb (Fig. 14 a). The mesophyl evidences 2 layers of low pallisadic cells on the upper side, and 5 layers of lacunary tissue, with isodiametric cells. The cells of the abaxial layer do not form a typical palisade, being rather square-shaped, their ratios being of 1/1.5, while the mesophyl is visibly thinner at this sample (Fig. 9, 10; Tab. IV). Cross-sections - the leaf from the mid strain, the middle third In the reference the median nervure is pronouncedly prominent on the inferior side of the limb and moderately prominent, respectively, on the upper side (Fig. 4). The mesophyl is of the pallisdic type under both epidermes, yet the cells from the adaxial side are taller; between the two pallisades, the cells of the assimilatory parenchyma are isodiametric, the structure of the leaf being therefore bifacial unequally equifacial (Fig. 11, 12). The median conducting fascicle is collaterally open, having a collenchyma girdle at each of the two poles (Fig. 5). All the other conducting fascicles are small, the latter ones possessing only phloem elements. In the TM 0.1% treated sample the median nervure, very thick and highly prominent on the inferior side of the limb, includes 3 conducting fascicles (of which, the median one is thicker), all of the open collateral type, each with a collenchyma girdle at both poles (Fig. 4, 5). The mesophyl from the adaxial side is of the pallisadic type, yet with lower cells, similar to those of the reference, while the one from the inferior side is mostly of lacunary type, which explains the different bifacial heterofacial structure of the limb, comparatively with the reference (Fig. 11, 12). The frequency of the secretory hairs is approximately equal to that recorded in the standard sample. In the TM 0.4% treated sample the median nervure is highly prominent on both sides of the limb, the hypodermic layer being of collenchymatic type (Fig. 4). The fundamental parenchyma of the median nervure evidences a single conducting fascicle of open collateral type (Fig. 4, 5); the lateral nervures of the first order show, too, relatively large conducting fascicles. The mesophyl is more lax, with the pallisadic tissue at its adaxial side (with visibly lower cells, as actually in the case of the untreated sample) and lacunary tissue on its inferior side, the limb s structure being bifacial heterofacial, as in the 0.1% treatment (Fig. 11, 12). 24
Cross-sections - the upper leaf, the middle third In the reference the mesophyl is almost wholly of the pallisadic type, yet with visibly higher cells in the hypodermic cells; the cells in the middle of the mesophyl are not always perpendicularly elongated on the epidermis, yet oblique; the structure of the limb is, at this level, too, bifacial equifacial (Fig. 13). In the TM 0.1% treated sample the median nervure is prominent on both sides of the limb, evidencing isodiametric epidermal cells, with an extremely thick, almost wholly cutinized wall (Fig. 6). The fundamental parenchyma of the median nervure includes a single conducting fascicle, of open collateral type, its wooden vessels occurring in parallel radial rows (8-10) as well as a thin girdle of mechanical fibers, in the course of formation in periphloemic position (Fig. 7); the generating area between the xylem and the phloem has 4 cell layers, comparatively with the reference which evidences only 2 layers (Fig. 8). The mesophyl is homogeneous, formed of isodiametric cells with large aeriferous spaces among them; it is only the cells of the adaxial hypodermal layer that appear slightly higher, reminding of the pallisadic form, with sinuous lateral walls; here and there, up to two layers of pallisadic cells may be observed (Fig. 13). The secretory hairs are thicker than in the reference. In the TM 0.4% treated sample the secretory hairs are more frequent on the median nervure, yet fewer then in the untreated and 0.1% treated samples. The mesophyl is almost homogeneous, of lacunary type, only the cells of the adaxial hypodermal layer being slightly taller (Fig. 13). In front of the median nervure, the epidermis has isodiametric cells, with their internal and external wall thicker than the others, the external one being covered by a thin cuticle. Long tectory hairs are visible at the edges of the limb (Fig. 14 a); where, too, the mesophyl appears typically lacunary within the whole thickness of the limb. The fascicle of the median nervure evidences numerous (10-12) parallel rows of woody vessels (Fig. 7); the generating area between the xylem and the phloem is thicker (5-6 cell layer) (Fig. 8), while the phloemic pole has a girdle of sclerenchymatic fibers with moderately thickened and lignified walls. Epidermis in front side view (the upper leaf) In the reference the upper epidermis is formed of polygonal cells with straight lateral walls. Here and there, stomatae of anomocytic type and secretory hairs may be observed. In the TM 0.1% treated sample, more stomatae occur on the unit of the surface. Besides the secretory hairs, very long tectory hairs, with a very long filiform terminal cell, have been also noticed. In the TM 0.4% treated sample, the epidermal cells are more numerous on the unit of surface, therefore they are smaller, yet the hairs have the same frequency as in the previous sample (Fig. 15 a). In the reference the inferior epidermis evidences some epidermal cells with slightly waved lateral walls. The stomatae and the secretory hairs have the same frequency on both sides of the limb, the gland showing secretory cells arranged on 3-4 levels, the ones situated at superior levels having a convex wall. In the TM 0.1% treated sample, the inferior side shows some cells with slightly waved walls. The frequency of hairs is the same on both sides, yet more numerous than in the untreated sample (3-4 in a microscopic field). In the TM 0.4% treated sample, all epidermal cells show slightly waved lateral walls. 25
The secretory hairs are more rare (1-2 in a microscopic field), yet the stomatae show the same frequency as in the TM 0.1% treatment (Fig.15 b). Conclusions The histo-anatomical modifications induced by the treatment with Topsin M depend on both the administered dose and the leaves development stage in the moment of fungicide s application. The response reactions of the tissues are more frequently of quantitative order, although mention should be also made of certain structural aspects that might influence the physiology and biochemistry of the plant and, consequently, the active principles synthesized by Calendula officinalis. The first and most evident effect of thiophanate methyl involves an increase of the foliar surface, a process to be decompensated by a weaker development of the pallisadic tissue, along with a more lax texture of the lacunary one (especially in the 0.4% treatments). At higher concentrations, the fungicide visibly reduces the thickness of the foliar limb, by reducing the sizes of the pallisadic cells (Tab. IV, V, VI), the ratio of which become 1.5/1 on the upper side (mainly in the terminal leaves which, in the moment of spraying, appear in an incipient stage of development), and 1/1.5, respectively, on the inferior side (almost quadratic cells); consequently, the structure of the foliar limb gets modified, from bifacial equifacial in the reference, to bifacial heterofacial in the leaves collected from the middle of the stem, and to an almost wholly lacunary (isofacial) structure in those from the top of the Calendula officinalis stem (Fig. 9-14). However, the median nervure is much thicker, with a modified (either triangular or semi-circular) contour in cross-section, the growth being intensely stimulated by the fungicide action, through an increased number of conducting fascicles (Tab. I, II, III). In Asteraceae, the presence of more numerous conducting fascicles in the median nervure constitutes an exception, to be possibly explained by inclusion of the first order nervures, as a result of the stimulating action exercised by the thiophanate methyl (the cytokinin hormone-type action of this fungicide being acknowledged). The woody conducting tissue shows a visible reaction to the treatment, by increasing the number of its vessels and, equally, of their diameter (TM 0.1%) (Tab. I, II, III); between the xylem and the phloem, the generating zone is thicker in the treated samples, including several cell layers (TM 0.4%) (Fig. 3, 5, 8). The epidermal cells are more numerous and smaller, with more waved lateral walls than in the reference (Fig. 15), while the stomatae are more numerous on the unit surface, the stomatic index recording higher values in the treated materials (Tab. 7); the frequency of secretory hairs increases in inverse ratio to the concentration of Topsin M and with the development stage of leaves in the moment of fungicide s application so that, in the terminal leaves, it records higher values in the TM 0.1% treatment (stimulating dose) and lower values, respectively, in the TM 0.4% one (inhibiting dose). 26
REFERENCES 1. EL MASHAD A.A.A., 2002 - Effect of thiophanate methyl on the growth and some metabolic activities of soybean plant. Egyptian J. Physiol. Sci., 24, 1: 83-102 2. HUŢANU-BASTAWI LUMINIŢA, TOMA C., 2006 - Considerations on the histo-anatomical study of the leaves of Cynara scolymus L. treated with thiophanate methyl (Topsin M). 4th Conference on Medicinal and Aromatic Plants of South-East European Countries - Iaşi, România, 28th 31st of May 2006 :126-132 3. PETRÓCZI, M.I., MATUZ, J., KÓTAI C., 2002 - Study of pesticide side-effects in winter wheat trials. Acta Biologica Szegediensis, 46, 3-4: 207-208 4. PRIESTELY, R.H., BAYLES, A. ROSEMARY, 1982 - Effect of fungicide treatment on yield of winter wheat and spring barley cultivars. Plant Pathology, 31, 1: 31 37 5. STASKAWICZ B., KAUR-SAWHNEY R., SLAYBAUGH R., ADAMS W., GALSTON A.W., 1978 - The cytokinin-like action of methyl-2-benzimidazolecarbamate on oat leaves and protoplasts. Pesticide Biochemistry Physiology, 8, 1:106-110 6. THOMAS T.H., 1974 - Investigations into the cytokinin-like properties of benzimidazole-derived fungicides. Ann. Appl. Biol., 76: 237-241 7. TRIPATHI R.K., TANDON, K., SCHLÖSSER E., HESS W.M., 1981 - Effect of fungicides on the physiology of plants. Part IV: Protection of cellular organelles of senescent wheat leaves by carbendazim. Pesticide Science, 13, 4: 395 400 8. VAN IERSEL, M.W., BUGBEE, B., 1996 - Phytotoxic effects of benzimidazole fungicides on bedding plants. J. Am. Soc. Hort. Sci., 121, 6: 1095-1102 9. YEOUNG-SEUK, B., BYEONG-YONG P., TAE-JIN A., BYEONG-YEON Y., SUNG-WOO L., NAK- SUL S., 2006 - Selection of potential fungicides for control of Ginseng seedling damping-off and research on fungicide application for disease control in farms. Treat. Crop Sci., 7: 679-698 10. YOSHIDA Y., 1970 - Effect of benzimidazole on the senescence of wheat chloroplasts and their boat shape transformation. Plant Cell Physiology, 11, 3: 435-444 Table I. Numerical values - basal leaves (median nervure, middle level) Thickness Length/width (μm Oc.10 x Ob.4) No. of fascicles No. woody vessels Diameter of vessels (μm Oc.10 x Ob.40) Ca. of. M 1200/1075 1500/1400 1 78-89 15-22,5 Ca. of. TM 0.1 % 1850/2350 1925/2475 3 81-94 22,5-32,5 Ca. of. TM 0.4 % 2075/3325 2250/3500 3 111-115 22,5-30 Table II. Numerical values - middle leaves (median nervure, middle level) Diameter Thickness No. No. of of vessels Length/width (μm woody fascicles (μm Oc.10 Oc.10 x Ob.4) vessels x Ob.40) Ca. of. M 1375/750 1500/1000 1 80-88 22,5-27,5 Ca. of. TM 0.1 % 1700/2250 1800/2600 3-5 95-109 25-32,5 Ca. of. TM 0.4 % 2000/1900 2200/2075 2-3 132-139 22,5-27,5 27
Table III. Numerical values - superior leaves (median nervure, middle level) Diameter Thickness No. No. of of vessels Length/width (μm woody fascicles (μm Oc.10 Oc.10 x Ob.4) vessels x Ob.40) Ca. of. M 950/975-1000/1000 1 50-56 20-22,5 Ca. of. TM 0.1 % 1075/1050 1250/975 1 61-72 25 Ca. of. TM 0.4 % 1375/1200 1600/1450 2 68-88 25-30 Table IV. Numerical values - basal leaves (limb, middle level) (Oc.10 x Ob.20) Thickness of the No. of layers in Thickness of the limb (μm) the limb pallisade (μm) Ca. of. M 375-450 7-8 135-160 Ca. of. TM 0.1 % 350-435 6-7 125-150 Ca. of. TM 0.4 % 325-375 7-8 115-130 Table V. Numerical values - middle leaves (limb, middle level) (Oc.10 x Ob.20) Thickness of the No. of layers in Thickness of the limb (μm) the limb pallisade (μm) Ca. of. M 350-400 6-7 150-175 Ca. of. TM 0.1 % 375-410 7-8 140-170 Ca. of. TM 0.4 % 300-350 6-7 110-120 Table VI. Numerical values - superior leaves (limb, middle level) (Oc.10 x Ob.20) Thickness of the No. of layers in Thickness of the limb (μm) the limb pallisade (μm) Ca. of. M 335-375 6 125-145 Ca. of. TM 0.1 % 325-375 7-8 125-135 Ca. of. TM 0.4 % 275-300 7 100-120 Table VII. Numerical values epidermis of the upper leaves, middle level (Oc.10 x Ob.20) Upper epidermis Inferior epidermis Samples No. of No. of Stomatic No. of No. of Stomatic cells stomatae index cells stomatae index Ca. of. M 63 9 11,11 76 13 12,74 Ca. of. TM 0.1 % 71 12 12,63 78 15 13,88 Ca. of. TM 0.4 % 75 15 14,28 88 17 13,93 28
Fig. 1. Cross-sections median nervure of the limb, basal leaf, middle third Fig. 2. Cross-sections median conducting fascicle, basal leaf, middle level Fig. 3. Cross-sections median conducting fascicle, basal leaf, middle level Fig. 4. Cross-sections median nervure of the limb, middle leaf, middle level 29
Fig. 5. Cross-sections median conducting fascicle, middle leaf, middle level Fig. 6. Cross-sections median nervure of the limb, superior leaf, middle level Fig. 7. Cross-sections median conducting fascicle, superior leaf, middle level Fig. 8. Cross-sections median conducting fascicle, superior leaf, middle level 30
Fig. 9. Cross-sections limb, basal leaf, middle level Fig. 10. Cross-sections mesophyl, basal leaf, middle level Fig. 11. Cross-sections limb, middle leaf, middle level Fig. 12. Cross-sections mesophyl, middle leaf, middle level 31
Fig. 13. Cross-sections mesophyl, superior leaf, middle level a b Fig. 14. Calendula officinalis: tectory hairs (a), uni- and biseriated secretory hairs (b) a b Fig. 15. Epidermis in front side view - upper (a) and inferior epidermis (b), superior leaf, middle level 32