Concentrations of secondary compounds in Scots pine needles at different stages of decomposition

Soil Biology & Biochemistry 34 (2002) 37±42

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Concentrations of secondary compounds in Scots pine needles at different stages of decomposition P. Kainulainen a,b,*, J.K. Holopainen a,b a

Department of Ecology and Environmental Science, University of Kuopio, PO Box 1627, 70211 Kuopio, Finland b MTTÐAgrifood Research Finland, Plant Protection, 31600 Jokioinen, Finland Received 24 October 2000; received in revised form 1 May 2001; accepted 23 July 2001

Abstract A litterbag experiment was conducted to study chemical changes in decomposition of Scots pine needle litter in central Finland. Concentration of secondary metabolites (terpenes, resin acids and total phenolics) and main nutrients (N, P, K, Ca and Mg) were analysed four times over a 19-month period. After 19 months decomposition, the mass loss by pine needle litter was about 22%. Initial concentrations of monoterpenes and total phenolics were 36 and 27% lower in needle litter than in green needles, respectively, while more (44%) resin acids were found in needle litter than in green needles. Concentrations of monoterpenes were 6%, resin acids 35% and total phenolics 17% of the initial concentration after 19 months decomposition. In green needles and senescent brown needles most common monoterpenes were a-pinene and 3-carene. During the decomposition monoterpenes, sabinene, myrcene, limonene 1 b-phellandrene, terpinolene and bornylacetat, were lost to a greater extent than a-pinene, camphene and tricyclene. At the end of decomposition experiment several oxygencontaining hydrocarbons were detected in terpene samples. The most commonly identi®ed compounds were verbenone and verbenol. Resin acid composition also changed substantially during decomposition, neoabietic acid decomposed faster than other resin acids. Dehydroabietic acid was the main resin acid in needle litter after 19 months decomposition. The concentrations of N, P and Ca signi®cantly increased during decomposition, while concentrations of Mg and K decreased. The results suggest that degradation of secondary organic compounds in needle litter is a slow process and these compounds might have effects on decomposer organisms for several years after needle abscision. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Pinus sylvestris; Litter decomposition; Secondary compounds; Monoterpenes; Resin acids; Total phenolics

1. Introduction Litter decomposition has long been recognised as an important process in forest ecosystems, both for nutrient recycling and humus formation. Decomposition processes are in¯uenced by macro- and microclimate, litter quality, and activity of decomposing organisms (CouÃteaux et al., 1995). A considerable number of studies have been conducted on conifer litter decay. In comparison, there are only a small number of studies to describe the process of decomposition of secondary organic compounds. Conifers produce large quantities of oleoresin, which is a complex mixture of volatile (mono- and sesquiterpenes) and non-volatile (resin acids) terpenoids (Phillips and Croteau, 1999). Many of these compounds serve in defence against herbivores and pathogens (Langenheim, 1994). Monoterpenes have been shown to have effects on litter decay * Corresponding author. Tel.: 1358-17-163193; fax: 1358-17-163230. E-mail address: pirjo.kainulainen@uku.® (P. Kainulainen).

and nutrient cycling (White, 1991, 1994). Monoterpenes may be regulators of microbial processes in nature (Amaral et al., 1998). If activity of microbial and/or invertebrate decomposers is inhibited by either individual monoterpenes or combinations of various monoterpenes, then decomposition and the release of ammonium (the process of N mineralization) would slow (White, 1994). On the other hand, monoterpenes in pine litter have allelopathic effects on growth of native understory grasses (Wilt et al., 1988). The decomposition of terpenoids in needle litter has been poorly studied. Compared to living materials monoterpene concentrations in pine litter and other plant detritus can have nearly equal concentrations (Wilt et al., 1993). Monoterpenes can be released from litter by volatilisation and microbiological degradation (Dyk et al., 1998). Water solubility of monoterpenes ranges from the low solubility of hydrocarbons to the rather high solubility of oxygenated monoterpenes (Weidenhamer et al., 1993). The monoterpenes in Scots pine needles are hydrocarbons, which had poor solubility in water (Weidenhamer et al., 1993).

0038-0717/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0038-071 7(01)00147-X

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P. Kainulainen, J.K. Holopainen / Soil Biology & Biochemistry 34 (2002) 37±42

Phenolic compounds are also important constituents of pine needle material. Within living plant tissue phenolics occur as free compounds or phenolic glycosides in vacuoles and cell wall components (Harborne, 1989). Phenolic compounds, as well as terpenoids, can act as toxins and deterrents to pathogens and herbivores (Dixon and Paiva, 1995) as well as litter decomposing animals (PoinsotBalaguer et al., 1993). Phenolic substances have effects on root symbionts and site quality through interference with decomposition, mineralization and humi®cation (Kuiters, 1990). Phenolics in plant litter are to a small extent watersoluble and are released by rainwater (Kuiters, 1990). Fresh litter, instead of commonly used air-dried litter, was used in this experiment, because earlier studies have shown air-drying depressing rates of leaf litter decomposition (Taylor, 1998). The aim of the present study was to measure concentrations of secondary compounds, monoterpenes and resin acids, and total phenolics, and major nutrients in Scots pine needle litter during natural decomposition in a typical pine forest of Finland. 2. Materials and methods 2.1. Site description and exposure of needle litter in the ®eld Senescent brown needles still remaining on the branches of about 20-year-old Scots pine (Pinus sylvestris L.) trees were collected. The trees were in Suonenjoki, central Finland in a naturally regenerated stand of Scots pine. The forest type according to Finnish classi®cation was Calluna (CT) (Cajander, 1949). Collected needle litter from each tree (12 trees) was immediately divided to six litterbags (16 £ 16 cm; 1 mm mesh). Litterbags were randomised and they were fastened to the top of the native litter layer with small wooden sticks on 2 October 1997 beneath Scots pine trees. 2.2. Sampling and chemical analysis During decomposition needles for analysis of secondary compounds (terpenes, resin acids, total phenolics) and major nutrients were collected from litterbags on 2 October 1997 (incubation time: 0 days), 20 May 1998 (231 days), 14 October 1998 (377 days) and 20 May 1999 (594 days). The mass loss of needles was monitored by weighting intact needles (two replicate samples containing 10 needles/litterbag). To compare concentrations between litter and living needles, needles were sampled from the third needle generation (C 1 2) on 2 October 1997. After collection litter samples were quickly (about 6 days) air-dried at room temperature (the litter from 2 litterbags was combined to form one composite sample). For terpene analyses needles were cut to small pieces and extracted with n-hexane for 2 h at room temperature as reported earlier (Kainulainen et al., 1992). Resin acids were extracted from powdered needles following the

procedures of Gref and Ericsson (1985). Only abietane and pimarane diterpene acids were investigated. Extracts were analysed by gas chromatography±mass spectrometry (Hewlett Packard GC type 6890, MSD 5973) using 30-mlong HP-5MS (0.25 mm ID, 0.25 mm ®lm thickness, Hewlett Packard) capillary column as described earlier by Kainulainen et al. (1993). For quantitation of some terpenoids, calibrations were made using known amounts of available pure terpenoids relative to known amounts of the internal standard (mono- and sesquiterpenes: 1-chloro-octane; resin acids: heptadecanoic acid). Results are expressed on an extracted air-dry weight basis. In order to express the concentrations of monoterpenes in green needles on an extracted dry weight basis, the water content was measured from a subsample (resin acids; freeze-dried). The identi®cation of unknown compounds was determined by comparing of spectra with those in Wiley and NBS Libraries. Total phenolics were analysed from the same needle powder as resin acids. About 50 mg of powdered needle material was extracted with 80% (v/v) aqueous acetone (Kainulainen et al., 1993), and total phenolics were analysed by the Folin-Ciocalteu technique as described by JulkunenTiitto (1985). Nutrients were analysed from pooled samples (one sample/tree). The ®nely ground needle material was wet digested (Allen, 1989) for N, K, Ca, Mg and P determinations. Nitrogen was analysed by the Kjeldahl method, P by spectrophotometer and K, Ca and Mg by atomic absorption spectrophotometer (Allen, 1989). 2.3. Statistics All data were ®rst checked for normal distribution. When necessary, data were normalised by log(x 1 1) transformations for statistical analysis. The data was analysed with one-way analysis of variance and the means were compared with Tukey-HSD procedure. 3. Results 3.1. Secondary compounds Concentrations of monoterpenes and total phenolics were initially at lower concentrations in needle litter than in green needles, while more resin acids were found in needle litter than green needles (Table 1). After 19 months decomposition concentrations of monoterpenes were about 6% of the initial concentration, while concentrations of resin acids were about 35% of the initial concentration. During the ®rst seven months concentrations of total monoterpenes decreased 68% and total resin acids 55% of the initial concentrations. There were huge differences in concentrations of secondary compounds between individual trees (data not shown). In green needles and senescent brown needles the most common compounds were a-pinene and 3-carene (Table 1).

P. Kainulainen, J.K. Holopainen / Soil Biology & Biochemistry 34 (2002) 37±42

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Table 1 Mean concentrations total phenolics (mg/g dw) of individual mono- and sesquiterpenes and resin acids in green needles (C 1 2) and decomposing needle litter (n ˆ 12). Standard errors are in parentheses. Different letters in the same row indicate signi®cant difference between samples according to Tukey's test (P , 0.05) Green needles

Monoterpenes Tricyclene a-Pinene Camphene Sabinene b-Pinene Myrcene 3-Carene Limonene 1 b-Phellandrene Terpinolene Bornylacetat Total Sesquiterpenes a-Copaene Longifolene b-Caryophyllene a-Humulene Resin acids Pimaric acid Sandaracopimaric acid Isopimaric acid Levopimaric 1 palustric acid Dehydroabietic acid Abietic acid Neoabietic acid Total Total phenolics

Decomposing needle litter (days) 0

231

377

594

132 (17)a 2398 (232)a 523 (61)a 49 (10)a 258 (49)a 170 (17)a 758 (203)a 204 (22)a 105 (15)a 303 (79)a 4900 (330)a

79 (10)b 1593 (165)b 295 (35)b 26 (5)b 156 (29)b 97 (10)b 534 (124)ab 141 (18)b 60 (9)b 143 (46)ab 3125 (277)b

37 (3)c 660 (60)c 108 (8)c 3 (0)c 24 (4)c 2 (0)c 142 (24)bc 17 (2)c 8 (1)c 7 (1)b 1007 (69)c

11 (1)c 69 (9)d 22 (3)c 0 (0)c 2 (0)c 0 (0)c 6 (2)c 2 (0)c 0 (0)c 1 (1)b 112 (11)d

20 (2)c 106 (11)d 38 (4)c 0 (0)c 3 (1)c 0 (0)c 19 (11)c 2 (0)c 0 (0)c 4 (1)b 192 (20)d

46 (8)a 7 (1)a 340 (58)a 58 (10)a

30 (5)ab 3 (0)b 222 (29)a 38 (5)a

19 (2)bc 1 (0)c 97 (10)b 16 (2)b

10 (1)b 0 (0)c 22 (3)b 3 (0)b

14 (2)bc 0 (0)c 17 (3)b 2 (0)b

13 (1)b 305 (38)ab 40 (5)a 633 (176)b 1811 (274)ab 1373 (219)a 762 (135)bc 4936 (782)b 67.4 (2.6)a

15 (2)ab 318 (33)a 44 (8)a 1590 (262)a 2550 (340)a 1428 (189)a 2880 (494)a 8825 (1170)a 49.0 (2.3)b

35 (6)a 245 (16)ab 69 (13)a 950 (90)ab 1438 (76)b 1023 (91)a 1052 (105)b 4812 (359)b 12.5 (0.6)c

33 (8)ab 197 (14)b 57 (14)a 332 (47)b 1426 (85)b 361 (38)b 209 (25)bc 2616 (201)b 8.4 (0.4)c

28 (4)ab 215 (14)ab 41 (4)a 336 (58)b 2031 (111)ab 400 (44)b 46 (6)c 3096 (215)b 8.8 (0.3)c

Proportional quantities of monoterpenes were similar in green and senescent needles. During decomposition monoterpenes, sabinene, myrcene, limonene 1 b-phellandrene, terpinolene and bornylacetat, were lost to a greater extent than other monoterpenes (Table 1). After 19 months decomposition a-pinene, camphene and tricyclene were still found in needle litter and they accounted for over 80% of the total monoterpene amount (Fig. 1).

Fig. 1. Proportional quantities of monoterpenes in pine needle litter during decomposition (n ˆ 12).

At the end of decomposition experiment several oxygencontaining hydrocarbons (C10H14O and C10H16O) were found in terpene samples. The most common compounds were verbenone and verbenol. Minor compounds were identi®ed as myrtenal, pinocarveol, pinocarvone and campholene aldehyde. Several unidenti®ed were also found. Proportional quantities of resin acids were different in green and senescent needles. Proportions of levopimaric 1 palustric and neoabietic acids were lower and sandaracompimaric and abietic acids higher in green than senescent needles. Resin acid composition changed also substantially during decomposition, neoabietic acid decomposed faster than other resin acids (Fig. 2). Dehydroabietic acid was the main resin acid in needle litter after 19 months decomposition. Total phenolic concentrations decreased 75% during the ®rst months. However, after that no signi®cant degradation was detected (Table 1). After 19 months decomposition concentration of total phenolics was about 17% of the initial concentration. 3.2. Mass loss of needles and nutrients After 19 months decomposition the accumulated mass

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Fig. 2. Proportional quantities of resin acids in pine needle litter during decomposition (n ˆ 12).

loss for pine needle litter was about 22% (data not shown). The concentration of nitrogen, phosphorus and calcium signi®cantly increased during decomposition, while concentrations of magnesium and potassium decreased (Table 2). 4. Discussion In general, decomposition of several chemical compounds in pine needle litter is a slow process. The complete decomposition of needle litter takes numerous years. Overall, litter mass loss rates in pine forests vary widely depending on climate and litter quality (Berg et al., 1993). Therefore, comparing mass loss to earlier studies is rather dif®cult. In the present study mass loss rate of needle litter was near the values reported earlier in a Finnish Scots pine forest (Mikola, 1960; Berg et al., 1993). Smolander et al. (1996) found higher accumulated mass loss values for Scots pine litter in central Finland, but litter bags were placed in the middle of the humus layer. Although highly volatile, monoterpenes can initially be present at relatively high concentrations in recently fallen litter as earlier studies have also shown (Wilt et al., 1993; Wood et al., 1995). Senescent leaves have similar monoterpene composition compared to green leaves, which suggest a poor catabolism of monoterpenes prior to leaf senescence. However, after 7 months decomposition, concentrations of total monoterpenes decreased over 60% of the

initial concentrations. Also the composition of monoterpene fraction changed rapidly. The monoterpenes, sabinene, myrcene, limonene 1 b-phellandrene, terpinolene and bornylacetat, were lost to a greater extent than a-pinene, camphene and tricyclene. At the end of decomposition experiment several oxygen containing hydrocarbons in terpene samples were found. White (1991) also found verbenone and a number of unidenti®ed probably oxidized monoterpenes from mineral soil samples of ponderosa pine forest. These compounds might be biotransformation products of monoterpenes. Dyk et al. (1998) observed that black yeast (Hormonema sp.), isolated from pine forest litter, converted a-pinene to mixture of verbenone and verbenol. In the present study, these compounds were the main oxygen containing hydrocarbons in pine needle litter, and as well as a-pinene was the main monoterpene hydrocarbon. Also myrtenal, found from terpene samples, is metabolically related to a-pinene (Hardie et al., 1994). These oxygenated monoterpene derivates might have also higher solubility in water, because monoterpenes containing oxygen had solubilities 10±100 times greater than hydrocarbon with comparable skeletons (Weidenhamer et al., 1993). Degradation of resin acids was rather slow. Gao et al. (1993) suggested that high degree of saturation in their composition make resin acid very stable and dif®cult to degrade. Resin acids are isomerized and oxidized rather easily (Enoki, 1976). Palustric and levopimaric acid yielded dehydroabietic acid. During decomposition resin acid composition changed substantially, which might indicate these chemical reactions were occurring. Concentration of dehydroabietic acid remained at high level during decomposition, while concentrations of other resin acids decreased. Overall, dehydroabietic acid is rather stable and studies have shown to be most common resin acid in sedimenting particles (LeppaÈnen et al., 2000). Concentrations phenolic compounds decreased faster than monoterpenes and resin acids during the initial phase of decomposition. This might partly re¯ect their chemical properties, because phenolic compounds might be more water-soluble and are leached by rainwater (Kuiters, 1990). However, after 19 months the concentration of total phenolics was still rather high, demonstrating that there might be phenolic compounds that are poorly decomposed. Overall, phenolic substances have effects on root

Table 2 Mean concentrations of nutrients (mg/g dw) in green needles (C 1 2) and needle litters during decomposition (n ˆ 12). Standard errors are in parentheses. Different letters in the same column indicate signi®cant difference between samples according to Tukey's test (P , 0.05) N Green needles (C 1 2) Litter 0 days Litter 231 days Litter 377 days Litter 594 days

9.1 3.6 4.0 4.9 5.2

(0.01)a (0.01)c (0.01)c (0.01)b (0.01)b

P

Ca

Mg

K

0.85 (0.02)a 0.34 (0.01)c 0.33 (0.01)c 0.44 (0.01)b 0.46 (0.01)b

4.30 (0.39)c 5.96 (0.27)b 5.17 (0.37)bc 7.53 (0.43)a 7.82 (0.48)a

0.50 (0.03)a 0.46 (0.04)ab 0.46 (0.04)ab 0.38 (0.03)ab 0.37 (0.03)b

3.45 (0.13)a 0.64 (0.08)b 0.34 (0.05)c 0.48 (0.03)bc 0.51 (0.04)bc

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symbionts and site quality through interference with decomposition, mineralization and humi®cation (Kuiters, 1990). It has been shown that Pinus muricata can strongly in¯uence release of dissolved organic nitrogen in soils through the production of polyphenols in leaf litter (Northup et al., 1995). P. muricata is capable of absorbing organic nitrogen from protein-tannin complexes by using some ectomycorrhizal fungi (Northup et al., 1995). Initial mass-loss rates of recently fallen litter appear to be stimulated by high concentrations of essential nutrients such as N and P (Berg et al., 1987; Berg and Ekbohm, 1991; Berg et al., 1996). In the present study initial N and P concentration tended to be quite low, which might lead to slow mass loss rate. The absolute amount of nitrogen and phosphorus in the needle litter increased during decomposition, which might due to the increase of fungal biomass (Berg and SoÈderstroÈm, 1979; CouÃteaux et al. 1998). Nitrogen can be transported by ingrowing fungal hyphae from the surroundings of the needles. After this accumulation phase, a release of nitrogen began. Potassium and magnesium were the elements, which more rapidly lost from the litter as earlier observed (Berg and Staaf, 1980). Because of rather low decomposition rate terpenoids and phenolic compounds have the potential to be important regulators of carbon and nutrient cycling in coniferous environments in northern latitudes. Secondary compounds are regulators of microbial processes (Amaral and Knowles, 1998; Amaral et al., 1998; Paavolainen et al., 1998) as well as litter decomposing animals (Poinsot-Balaguer et al., 1993) and in¯uence the pools and ¯uxes of inorganic and organic soil nutrients (HaÈttenschwiler and Vitousek, 2000). Acknowledgements This study has been supported by the Academy of Finland (Resource Council for the Environment and Natural Resources, project no. 39495 and 42650). We thank Jaana Rissanen, Leena Mettinen, Mirja Korhonen and Juhani Tarhanen for technical assistance. References Allen, S.E., 1989. Chemical Analysis of Ecological Materials. 2nd ed, Blackwell Scienti®c Publications. Amaral, J.A., Knowles, R., 1998. Inhibition of methane consumption in forest soils by monoterpenes. Journal of Chemical Ecology 24, 723± 734. Amaral, J.A., Ekins, A., Richards, R., Knowles, R., 1998. Effect of selected monoterpenes on methane oxidation, denitri®cation, and aerobic metabolism by bacteria in pure culture. Applied and Environmental Microbiology 64, 520±525. Berg, B., Ekbohm, G., 1991. Litter mass-loss and decomposition patterns in some needle and leaf litter types. Long-term decomposition in a Scots pine forest. Canadian Journal of Botany 69, 1449±1456. Berg, B., SoÈderstroÈm, B., 1979. Fungal biomass and nitrogen in decomposing Scots pine needle litter. Soil Biology & Biochemistry 11, 339±341. Berg, B., Staaf, H., 1980. Decomposition rate and chemical changes of

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