Influence of S-Ethyl Dipropylthiocarbamate on Growth, Chlorophyll and Carotenoid Production and Chloroplast Ultrastructure of Germinating Polytrichum commune Spores

Germinating Polytrichum commune Hedw. spores showed great insensitivity to S-ethyl dipropylthiocarbamate (EPTC). At the lowest EPTC concentration (2 ppmw EPTC) the young protonema was usually more developed than the control material, with higher chloroplast pigment content on a basis of mg/dish. At 100 ppmw EPTC, the young protonema was less developed and production of chlorophylls and carotenoids was decreased by about 30%. However, dry weight was at the level of the control material. At 200 ppmw EPTC spores remained either ungerminated, stopped germinating at an early stage or developed into sporelings with few tightly-packed short cells containing chloroplasts. At this stage, growth processes in general were inhibited more than the chloroplast pigment production. Also, the following changes in ultrastructure of sporelings were noted: cytoplasm was very dense, nucleus large, amount of vacuolization low and chloroplasts were amyloplast-like. At 300 ppmw the spores were usually ungerminated. Species response to herbicides is of major ecological interest. Alternatively, a nontarget species at one location may become an undesirable plant (i.e. weed) at another location. Mosses demonstrate this ambivalence. In many portions of the world mosses are nontarget species whose preservation is sought. However, in the Scandinavian countries, mosses often become weeds in lawns and turf. A paucity of information exists on the response of mosses to herbicides. EPTC is known to reduce cuticle thickness of leaves by reducing the content of epicuticular wax components (Gentner, 1966; Wilkinson & Hardcastle, 1969, 1970; Still et al., 1970; Wilkinson, 1974). Kolattukudy and Brown (1974) suggested that this inhibitory influence of the thiocarbamate herbicides was on acyl elongation of epicuticular wax constituent precursors. Also, EPTC has been shown to interfere with the metabolism of chloroplast membrane components by inhibiting the fatty acid synthesis in isolated spinach (Spinacia oleracea L.) chloroplasts (Wilkinson & Smith, 1975a,b). Polytrichum commune spores contain chloroplasts with a well developed grana lamellae system (Karunen, 1971, 1972). The spores germinate rapidly and after three days develop into sporelings with numerous rapidly replicating chloroplasts. Synthesis ' Institute of Biology, Department of Botany, University of Turku, SF-20500 Turku 50, Finland. 2 Department of Agronomy, Georgia Station, Experiment, Georgia. 30212, USA. This content downloaded from 157.55.39.104 on Mon, 20 Jun 2016 06:23:30 UTC All use subject to http://about.jstor.org/terms 1976] KARUNEN, ET AL.: PHYSIOLOGY OF GERMINATING POLYTRICHUM SPORES 333 of the chloroplast membrane components is efficient based on chlorophyll production (Karunen, 1973). Therefore, it was of interest to measure the effect of EPTC on Polytrichum commune spore germination and sporeling growth and morphology, giving special attention to changes in the chlorophyll and carotenoid content and chloroplast ultrastructure. MATERIAL AND METHODS Procedures utilized were those reported by Karunen (1972) with minor modifications. The agar previously utilized absorbed the herbicide and no plant response to the herbicide treatment was observed; therefore, 20 ml sea sand (Merck 7712) per 9 cm diameter petri dish was substituted for the agar. Technical grade S-ethyl dipropylthiocarbamate (EPTC) was dissolved in 99.5% ethanol. Then, sufficient EPTC was added to 10 ml MS-solution (Waris, 1953) to give the desired concentration of herbicide/dish while retaining the ethanol concentration below 0.1%. The sand was covered by a cellophane membrane, and 2.5 mg spores were added in 1 ml MS-solution containing 0.01% Triton X-100. Triton X-100 at 0.01% had no effect on germination. Higher concentrations of Triton X-100 (0.1%) inhibited spore germination (0% germination) while at lower concentrations of this surfactant (0.001%) the water surface tension was not reduced sufficiently to allow the spores to submerge into the water. Spore germination and growth were about equivalent in petri dishes where spores were sown directly onto the membrane compared with spores sown in 1 ml 0.01% Triton X-100; the dry weight of the culture in one week was 12.3 + 0.94 mg/dish in the former and 11.6 + 0.84 mg/dish in the latter. After seven days in a growth-chamber at 250C and continuous light at 10,000 lux, the dry weight as culture was recorded. Seven to ten replicates were utilized for the dry weight data and eight replicates were utilized for chloroplast pigment determinations. Data were subjected to an analysis of variance on a randomized block design. Chlorophylls and carotenoids were extracted by grinding the material in a Potter-Elvehjelm homogenizer with 85% aqueous acetone. The optical density of the extracts at 663, 644 and 452.5 nm was measured in a Beckman spectrophotometer (Model G 2400). The chlorophyll and carotenoid contents were measured according to the nomogram of Robbelen (1957). For electron microscopy, the material was fixed in glutaraldehyde-osmium by methods described earlier (Valanne, 1971), dehydrated in an alcohol series and embedded in Epon 812. The thin sections were stained with Reynold's lead citrate and examined with an AEI EM6B electron microscope. RESULTS AND DISCUSSION S-ethyl dipropylthiocarbamate (EPTC) concentrations of 200-300 parts per million by weight (ppmw) inhibited the dry weight production of germinating Polytrichum commune spores (Fig. 1). This concentration level is near the water saturation level for EPTC and approximates 1.6 X 10-3 M EPTC. Since EPTC is normally incorporated into the top 7.5 cm soil layer in agricultural practice, 1 kg/ha approximates a 1 ppmw concentration (Herbicide, 1974). Thus, in agricultural use, normal EPTC concentrations are less than 6 ppmw and the latter rate, in maize (Zea mays L.), must include a safener (N,N-diallyl-2,2-dichloroacetamide) applied concomitantly (Herbicide, 1974). Therefore, germinating P. commune spores were about 50 times less sensitive to EPTC than the most resistant crop for which this herbicide is currently utilized. When the EPTC concentration was reduced to 100-150 ppmw, dry weight of the culture was equivalent to that of the untreated material. At 2 ppmw EPTC the dry weight production was slightly increased over that of the untreated material. Table 1 shows the influence of EPTC on chlorophyll and carotenoid production. There was an enhancement of chlorophyll a + b at 2 and 20 ppmw EPTC on a basis of mg/dish. A similar result occurred in carotenoids at 2 ppmw EPTC. Thus, the stimulation in the production of chloroplast pigments occurs concomitantly with the increase in the dry weight production. A concomitant increase in dry weight and This content downloaded from 157.55.39.104 on Mon, 20 Jun 2016 06:23:30 UTC All use subject to http://about.jstor.org/terms 334 THE BRYOLOGIST [Volume 79