Phyrochemis~ry.Vol. 35, No. 1, pp. 39 42, 1994 Printed in Gnat Britain.
003 1 -9422/w s6.00 + 0.00 0 1993 PergamonPressLtd
EFFECT OF OZONE ON THE BIOCHEMISTRY AND APHID INFESTATION OF SCOTS PINE PIRJO KAINULAINEN, JARMOK. HOLOPAINEN, HELI HYTTINEN and JARI OKSANEN Ecological Laboratory, Department of Environmental Sciences, University of Kuopio, P.O. Box 1627, SF-7021 1 Kuopio, Finland (Receioed in revisedfirm 23 June 1993)
Key Word Index--Pinus syluestris; Pinaceae; ozone; aphids; amino acids; carbohydrates; compounds; Schizolachnus pineti.
secondary
Abstract-One-year-old Scats pine (Pinus syloestris L.) seedlings were exposed to 0.1 and 0.15 ppm 0, for four weeks, and to 0.3 ppm 0, for six days in three separate experiments. Plants did not exhibit any visible injuries or differences in growth parameters. Ozone did not affect the numbers of nymphs produced by grey pine aphid [Schizolachnus pineti (F.)]. The levels of total essential amino acids for aphids were not significantly affected by 03. The levels of total amino acids were significantly reduced on exposure to 0.3 ppm 03. Reduced levels of starch in shoots and roots were found in OS-exposed seedlings. No changes in other sugars (glucose, fructose, sucrose) were detected. Ozone had no effect on secondary compounds: monoterpencs, resin acids, total phenolics and catechins. The results indicate that rising levels of atmospheric Oa may disturb primary carbohydrate and amino acid metabolism of Scats pine, while secondary metabolites remain unaffected.
RESULTS AND DISCUSSION
INTRODUCTION Ozone
is an important constituent of photochemical air pollution and it might be the most important single pollutant in remote areas. The surface concentration of 0, at mid and high latitudes has more than doubled in the past 100 years and the global tropospheric concentration of 0, is estimated to increase at a rate faster than during the past 100 years [l]. Ozone is considered to have serious effects on vegetation in central Europe and North America [2,3]. The growth and development of trees depend to a large extent on their photosynthetic capabilities, as well as on environmental factors. It is commonly believed that injury, caused by air pollutants, initially takes place at the biochemical level, subsequently progressing to the ultrastructural level and then to the cellular level, and finally visible symptoms appear [4]. Exposure of various trees to 0, has resulted in changes in, e.g. photosynthesis [S], carbon allocation 163 and amino acid metabolism [4,fl Among conifers, pine species are usually more sensitive to 0, than spruce [8,9]. Scats pine is economically the most important tree species in Finland, but its sensitivity to O9 is poorly investigated, although it is considered to be susceptible [8]. We studied the effects of 0, concentrations that represent slightly or moderately elevated levels compared with present naturally occurring peak concentrations [lo] on amino acid, carbohydrate and secondary metabolism of Scats pine, and on the reproduction of the aphid S. pineti. This aphid starts reproducing at the time of the annual peak OS concentrations in May.
Growth responses of seedlings
Ozone did not affect growth of the seedlings and dry weights of shoots or roots, and did not cause visible damage. The 0, dose given in the experiments was lower than reported to cause visible symptoms [l 1,123. Within populations of species, individual plant response to OJ may vary, often ranging from no observable symptoms to complete tissue necrosis 183. It is known that some pine individuals are more sensitive to O3 than others [13].
Aphid growth and amino acids
Final numbers of the produced nymphs (log (n+.l) -transformed data) slightly increased at 0.1 and 0.15 ppm Oj, and decreased at 0.3 ppm O,, but no statistically significant difference to the control was detected. The levels of total amino acids in OS-treated seedlings were significantly reduced at 0.3 ppm 0, and not affected at concentrations of 0.1 and 0.15 ppm Oj. The levels of total essential amino acids for aphids were not significantly affected by 0,. Amino acid spectra of seedlings appeared to be quite similar as in the needles of Scats pine trees [14,15], dominated by glutamic acid in experiments 1 and 2, and glutamine in experiment 3. Glutamic acid levels were significantly (P~0.05 by t-test) reduced in O,-exposed seedlings at 0.1 and 0.3 ppm 0,. Glutamine was not 39
40
P.
KAINULAINEN er al.
detected at 0.1 ppm OJ, and it was nearly significantly (P=O.O8) reduced at 0.3 ppm 0,. Levels of arginine and 1-methylhistidine were significantly (PcO.05) increased in O,-fumigated seedlings at 0.15 ppm, while proline and free ammonia were significantly (P ~0.05) reduced and isoleucine (PcO.05) increased in seedlings exposed to 0.3 ppm 0,. High concentrations of 0, (0.3 ppm) are known to reduce the levels of free amino acids in conifers [16], while long-term fumigations to slightly elevated 0, concentrations could increase foliar levels of free amino acids [ 17, 181. The observed increase of some individual amino acids after four weeks exposure to 0.15 ppm 0, in the present study could be due to accelerated senescence of older needles and reallocation of nutrients in the younger parts of the plant [ 183. Reduction of free amino acids at very high 0, levels may indicate reduction in the primary metabolism of plants. Exposures to SO, and NO, quite often result in better aphid performance on woody plants [19]. However, exposures to 0, have resulted in variable aphid performances on conifers depending on the temperature and length of the exposure period [19]. The increased densities of aphids in trees exposed to air pollution is usually associated with elevated levels of free amino acids in host plant (e.g. [ZO]), but S. pineti has shown the opposite trend [2l, 221. In the present study, the levels of essential amino acids in young shoots and the density of S. pineti responded mainly in the same direction in 0, treatments, but without significant effects. The levels of amino acids in the youngest parts of the shoot are possibly not the best indicator of the quality of host plant to S. pin&, because this aphid feeds on needle phloem preferably on older needles. Under pollution stress, old needles might become less nutritive due to translocation of nutrients to the younger parts. Carbohydrates
Ozone had no effect on the concentrations of glucose, fructose and sucrose. After two weeks of exposure, significantly reduced levels of starch were observed in O,-exposed shoots (P
reduced carbohydrate posure [32].
allocation to roots under O3 ex-
Total phenolics and catechins No significant changes in concentrations of total phenolics and catechins were observed, although at 0.1 ppm 0, total phenolic concentration was nearly significantly (P=O.O51 by t-teat) higher in Osexposed shoots. New shoots had less total phenolics than old needles. In earlier studies no effects [33], or higher 124,343 levels of phenols of pine foliage exposed to OJ have been found. The individual phenolic compounds, e.g. pinosylvin and pinosylvin methyl ether have a dose-dependent biochemical response to 0, [35]. Also, the amounts of some other phenolic compounds can change during 0, exposure C361. Monotmpenes and resin acids
Ozone did not significantly affect individual or total monoterpene and resin acid concentrations, which is consistent with the literature [37,38]. Combined OJ and acid mist treatment has generally diminished needle terpene concentrations of Norway spruce [33], but as in our study the difference was not significant. Monoterpenes appear to play a significant role in the chemistry of the forest atmosphere due to their reaction with other atmospheric constituents [39,4O]. Stangl et al. [41] have shown that SO, is oxidized to sulphuric acid faster in a mixture of OJ and terpenes than by 0, alone. Assuming that terpene emissions close to the needle surface are relatively high, terpenes might lower the 0, concentrations close to the plant. An application of /I-pinene to the leaf surface of Malus pumila reduced ozone injury [42]. In this study no changes in secondary metabolism were detected. Reduction of free amino acids and starch indicate a trend that may be a response to stress in pine seedlings. Because primary metabolism was affected, also secondary metabolism, e.g. tannin synthesis may change later. The impact of 0, may be cumulative and its effect should be characterized over several growing seasons. In any case, a short fumigation with 0, can affect carbohydrate and amino acid metabolism, but the consequences to aphid populations are not as distinct as the effects of SO1 and NO,. EXPWUMENTAL
P/ant material. One-year-old Scats pine (P. sylvestris L.) seedlings overwintered at 4” were obtained from a forest nursery (68” 38’ N, 27” 04’ E). In the first experiment, seedlings were planted in 1.3-l pots, 3 seedlings per pot, in a mixt. of peat (Vapo, Mettitaimiturve, N-P-K 155-l 1, with micronutrients) and vermiculite (2: 1, v/v). In expts 2 and 3, the seedlings were planted in 0.45-l pots, 1 seedling per pot, in a mixt. of peat and sand (1: 1, v/v). The seedlings were fertilized once a week with 0.1% soln 5Superex (N-P-K; 114-25). Before the initiation of new growth, the seedlings were randomized to 0, and control
Effect of ozone on Scats pine treatments. The new shoot developed during the expts in controlled growth chambers simulating the weather conditions of central Finland in early June: day/night temp. 19/12”, air humidity 65/85%. The light/dark cycle was 22/2 hr with highest daytime illumination of ca 260 PE m-’ s- ‘. Sampling was made at the same time of day in each expt to avoid diurnal fluctuation. After the exposure period, the shoot height of the seedlings was measured, and after oven-drying at 60” for 48 hr, shoots and roots were weighed. Ozone exposure. 0, fumigations performed in growth chambers [43] were 0.1 ppm O,, 6 hr day- I, dose 12 ppm hr-’ (expt 1); 0.15 ppm 0,, 7 hr day-‘, dose 21 ppm hr- ’ (expt 2) on weekdays for 4 weeks and 0.3 ppm 0,, 12 hr day-’ for 6 days, dose 21.6 ppm hr- ’ (expt 3). Ozone was produced from pure oxygen with an 0, generator (Fischer 500) and monitored by an O3 analyser (Dasibi 1008-RS). Infestation with aphids. Reproduction of the grey pine aphid, S. pineti (F.), that feeds on pine needles was monitored. In expt 1, 1 individual of 3rd or 4th instar of fundatrix female was transferred per seedling before the start of 0, fumigation, and the final aphid count was done 35 days later. In expt 2, 1 newly moulted fundatrix per seedling was transferred after 2 weeks from the start of the fumigation. Final aphid count was done 38 days later. In expt 3, 1 even-aged apterous viviparous female, taken from a laboratory culture, was transferred per seedling, 1 day before the start of a 6 day fumigation period, and aphids were counted 7 days later. Amino acid unulysis. At the end of the fumigation period, current year shoots were collected for amino acid analyses. Samples were extracted as described earlier [44] and stored before analysis at -25”. Analysis was performed with an automatic amino acid analyser (LKB Alpha Plus 4151). Carbohydrate analysis. Glucose, fructose and sucrose were measured from new shoots in expts 1 and 2, and also from roots in expt 2, and starch from shoots and roots in expts 1 and 2. Samples for carbohydrate analyses were frozen at -SO”, freeze-dried and milled. Carbohydrates were analysed using enzymatic techniques (Boehringer kits for food analysis). Total phenolics and cutechin analysis. Total phenol& and catechins were analysed from new shoots and the previous years needles in expts 1 and 2. Samples were dried in a circulating oven at 48”, milled to dust and stored in a desiccator at 4”. Needle powder was extracted with 80% (v/v) aq. Me&O [22]. Total phenolics and catechins were analysed as described in ref. [45]. Monoterpene and resin acid analysis. Monoterpenes (expts 1 and 2) and resin acids (expt 2) were analysed from new needles. Monoterpenes were extracted from fresh needles [46], and resin acid samples were treated and extracted as-in ref. [47]. Only abietane and pimarane diterpene acids were investigated. Extracts were analysed by GC-MS using capillary columns: J and W Scientific DP-5 (30 m x 0.25 mm i.d., 0.25 pm film thickness) and Hewlett Packard HP-5 (25 m x 0.2 mm i.d., 0.11 pm film thickness) in exps 1 and 2, respectively. Carrier gas: He;
41
temp. prog.: 50- 250” at 5” min - ’ for terpenes and 150-250” at 5” min- ’ for resin acids. Individual terpenes and resin acids were identified by their mass spectra and retention times. Peak areas were used to quantify the individual substances. For quantification, calibrations were made using known amounts of available pure terpenes and resin acids, and response factors were determined for each substance relative to known amounts of the int. standard. Acknowledgements-This study was supported by the Academy of Finland and the Foundation of Maj and Tor Nessling. We thank Mr Juhani Tarhanen for the help in terpene and resin acid analysis, Mrs Liisa Alakuijala for the help in amino acid analyses, and Miss Eila Kainulainen, Miss Heli Kinnunen and Mr Timo Oksanen for technical assistance. REFERENCES
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