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Germination and post-germination response of Acacia seeds to smoke-water and butenolide, a smoke-derived compound
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Journal of Arid Environments 69 (2007) 177–187
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Germination and post-germination response of Acacia seeds to smoke-water and butenolide, a smoke-derived compound M.G. Kulkarni, S.G. Sparg, J. Van Staden Research Centre for Plant Growth and Development, School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa Received 10 February 2006; received in revised form 27 July 2006; accepted 11 September 2006 Available online 23 October 2006
Abstract Fire plays a critical role in breaking hard-seeded dormancy and establishing seedlings of several Acacia species in arid and semi-arid regions. Numerous studies have reported an increase in seedling densities of some Acacias after fire without identifying the exact cause. However, it is generally believed and speculated that the effect is largely physical and heat related. Recent studies have revealed that smoke generated from wildfires has the ability to stimulate seed germination and also to improve seedling vigour. This stimulatory characteristic of smoke has now been confirmed by the isolation of a butenolide (3-methyl-2H-furo[2,3-c]pyran-2-one) from plant smoke that actively promotes germination of many plant species. The species of Acacia investigated were A. hebeclada (deciduous shrub), A. mearnsii (invasive tree, native to Australia) and A. robusta (deciduous tree). Seeds of A. hebeclada germinated under different light conditions with smoke-derived butenolide solution (107 M), exhibited a significantly (po0.05) greater germination percentage than untreated seeds. Whereas A. mearnsii seeds exposed to constant dark conditions showed a significantly (po0.05) better germination percentage than the control. However, there was a non-significant improvement for A. robusta seeds. All three species responded positively to the butenolide treatment (107 M) after incubating for 10 days under constant dark conditions at 2570.5 1C, achieving a higher vigour index and seedling mass in comparison to the controls.
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E-mail address: [email protected] (J. Van Staden). 0140-1963/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2006.09.001
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Smoke-water (1:500) had an intermediate effect on these species. This study shows that the butenolide, isolated from smoke, may have a significant effect on the post-fire seedling ecology of Acacias. r 2006 Elsevier Ltd. All rights reserved. Keywords: Post Fire; Seedling emergence; Smoke; Stimulation
1. Introduction Species of Acacia are widely distributed in arid and semi-arid regions (Demel, 1996; Wilson and Witkowski, 1998; Aref et al., 2003), where fire plays an important role in breaking hard seed coat dormancy and stimulating seed germination (Harker, 1959). Particularly, temperature or heat shocks, generated from fires, are reported to play a role in releasing seed dormancy of legumes in arid and semi-arid regions (Pieterse and Cairns, 1986; Sabiiti and Wein, 1987; Oba, 1990; Cox et al., 1993; Mbalo and Witkowski, 1997). In addition, heat is a specific requirement for triggering germination of hard-seeded species of Fabaceae (Enright and Kintrup, 2001). It is also reported that seedling densities of Acacia species become dominant after fire (Cavanagh, 1980; Milton and Hall, 1981; Sabiiti and Wein, 1987; Pieterse and Boucher, 1997), with increasing stem density in some cases (Pratt and Knight, 1971). Some studies show that a reduction in grass cover allows the establishment of woody seedlings (Schultz et al., 1955; Kanz, 2001), whilst other studies disagree with these findings (O’Connor, 1995; Brown and Archer, 1999). In addition, some studies indicate that an increase in soil nutrient levels after fire (Allen et al., 1969; Anderson and Menges, 1997) may be responsible for the subsequent vigourous growth of plants (Brys et al., 2005). The effects of fire on Acacia species are inconsistent and usually not clear (Radford et al., 2001) therefore the exact cause of post-fire stimulation of Acacia seedling growth remains unknown. Smoke generated from fires plays an important role in re-establishing many plant communities in semi-arid regions such as chaparral in southern California (Keeley and Pizzorno, 1986), fynbos in South Africa (De Lange and Boucher, 1990), Kwongan in Australia (Dixon et al., 1995) and the Mediterranean basin (Crosti et al., 2006). Smoke is now widely recognized as a germination cue for fire-dependent as well as non fire-dependent plant species (Ja¨ger et al., 1996; Light and Van Staden, 2004; Light et al., 2005). Smoke treatments have also shown promise in the propagation of economically important wild plants, as germination percentage can be increased significantly (Brown and Van Staden, 1998; Brown and Botha, 2004). Thus smoke and smoke solutions have great potential for use in horticulture, agriculture, weed management, habitat restoration and conservation practices (Roche et al., 1997; Boucher and Meets, 2004; Light and Van Staden, 2004). The isolation of a new compound from smoke that stimulates seed germination of many plants is generating wide interest and will assist in unravelling certain processes of post-fire ecology. The compound has been characterized as the butenolide 3-methyl-2H-furo[2,3-c]pyran-2-one, isolated from plant-derived smoke (Van Staden et al., 2004), burned cellulose (Flematti et al., 2004), and also from products formed by heating combinations of carbohydrates and amino acids (Light et al., 2005). Until recently, smoke has been assessed for its ability to release seed dormancy and to improve germination, but a few studies have shown that the effects of smoke extend beyond post-germination events resulting in the stimulation of seedling vigour (Baxter and
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Van Staden, 1994; Sparg et al., 2005). In the past many studies on Acacia species have focused on physical aspects of fire-related cues for releasing dormancy and seed germination and little attention has been given to the chemical/physiological influence of smoke components at post-germination levels. This study was conducted to determine the effects of smoke-water and the smokeisolated butenolide on seedling vigour and growth of three different Acacia species. The species investigated were two endemic species A. hebeclada (candle thorn), A. robusta (splendid thorn) and one exotic A. mearnsii (black wattle) native to Australia. 1.1. Seed collection Mature pods of A. hebeclada DC. were collected in March 2004 from the University of KwaZulu-Natal Pietermaritzburg Botanical Garden, South Africa. Mature pods of A. robusta Burch. were obtained in July 2005 from areas surrounding Johannesburg. Dry pods of both species were opened in the laboratory and healthy seeds were then stored in plastic containers at room temperature before use. Seeds of A. mearnsii De Wild. collected in July 2005 were obtained from the Institute for Commercial Forestry Research, Pietermaritzburg. The germination test showed X 98% seed viability of all the three species examined. 1.2. Seed pretreatment Many Acacia species have a hard seed coat which makes it difficult to imbibe water unless some scarification pretreatment is adopted (Demel, 1996). In this study all three Acacia species examined had a hard seed coat requiring scarification for better germination. The seeds of A. hebeclada and A. robusta were acid scarified by immersing them in concentrated sulphuric acid (H2SO4) for 90 min and then carefully rinsed in distilled water before germination. The smaller seeds of A. mearnsii were acid scarified for 25 min only and then carefully rinsed with distilled water. 1.3. Smoke treatments An aqueous smoke extract was prepared by continuously bubbling smoke from the leaf material of a fire-climax grass, Themeda triandra Forssk. (Poaceae), through a column of water (500 ml) for 45 min (Baxter et al., 1994). Solutions of this smoke extract were prepared by diluting 1 ml of the concentrated solution in 500 ml of distilled water, and diluting this further, as required. The smoke compound, butenolide, was isolated from plant-derived smoke solution (Van Staden et al., 2004). A solution of 107 M of this compound was used to test germination and seedling growth as outlined below. 1.4. Germination and seedling vigour experiments Scarified seeds of all three Acacia species were germinated in 9 cm Petri dishes fitted with two layers of Whatman No.1 filter paper moistened initially with 4 ml distilled water or treatment solution. Each treatment consisted of four replicates with 20 seeds in each. Seeds were incubated under three light conditions for germination and seedling growth: alternate light (16:8 h light/dark), constant light and constant dark. For alternate and constant light
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conditions, Petri dishes were placed in incubators set at 2570.5 1C with a light intensity of 8075 mmol m2 s1 provided by cool-white fluorescent lamps. The Petri dishes for constant dark conditions were kept in a lightproof box at 2570.5 1C. Germination (2 mm protrusion of radicle) was scored daily and germinated seeds were allowed to grow for a further 10 days to evaluate post-germination responses. At the end of the experiment the seedling vigour was calculated as VI ¼ [average shoot length (mm)+average root length (mm)] percentage germination (Dhindwal et al., 1991). 1.5. Statistical analysis Statistical analysis was conducted using MINITABs statistical package, release 12.1 (MINITAB INC., PA 16801-3008, USA). The germination data in each treatment were arcsine transformed (Sokal and Rohlf, 1995) and non-parametric Mann–Whitney Test was conducted to compare the treatments with the control at 5% level of significance (po0.05). Seedling (10-day-old) growth data were subjected to one-way analysis of variance (ANOVA) and Tukey’s method was used for pair-wise comparison at a 5% level of significance (po0.05). The butenolide-treated seeds of A. hebeclada showed a significant increase in percentage germination under the three different light conditions compared to the control (Table 1). Whilst treating seeds with smoke marginally improved the percentage germination. Under constant dark conditions, seeds of A. mearnsii treated with butenolide showed significantly better percentage germination than the control. However, in spite of having higher percentage germination, smoke-water did not significantly enhance the germination. Similarly, seeds of A. robusta germinated with butenolide solution, under constant dark conditions, showed an increased germination percentage, but this was not statistically different from the control. Seedling vigour indices of butenolide-treated A. hebeclada seeds, incubated in alternate and constant light conditions, increased significantly, compared to the control and smoke-water treated seeds. Under constant dark conditions, the smokewater and butenolide treatments significantly improved the lengths of shoots (16.9 and 20.3 mm respectively) and roots (18.3 and 18.4 mm, respectively) in comparison to the Table 1 Influence of smoke-water (1:500) and a butenolide solution (107 M) on seed germination of Acacia species under different light conditions at 2570.5 1C Percentage germination Treatment A. hebeclada
A. mearnsii
A. robusta
Alternate light (16:8 h light/dark)
Control Smoke-water Butenolide
70.070.89 76.770.67 83.470.89*
10070 93.470.67 96.770.32
10070 93.470.32 9070.57
Constant light
Control Smoke-water Butenolide
70.070 73.470.67 86.770.32*
10070 93.470.32 10070
86.770.32 96.770.32 96.770.32
Constant dark
Control Smoke-water Butenolide
63.470.32 73.470.32 83.470.32*
83.470.67 96.770.32 10070*
93.470.67 93.470.32 10070
Condition
*Mean value7SE with an asterix is significantly (po0.05) different from the control.
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Fig. 1. Influence of smoke-water (SW, 1:500) and butenolide solution (BS, 107 M) on shoot and root lengths of 10-day-old seedlings of Acacia species incubated under three different light regimes at 2570.5 1C. Control ¼ C. Bars within the same light condition with the same letter(s) are not significantly (po0.05) different.
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control (Fig. 1). In the other two light conditions, butenolide-treated-seedlings showed a significant increase in root length, but shoot length was not significantly affected under alternating light conditions. Although seedlings of A. hebeclada grown under constant light with smoke-water and butenolide solution exhibited increased seedling mass, these values were not significantly different from the control. However, A. hebeclada seedlings grown under alternate light and constant dark conditions with smoke-water and butenolide solutions had a significantly higher mass than the control seedlings (Table 2). Overall, the smoke-water and butenolide-treated-seedlings of A. hebeclada grown under constant dark conditions obtained significantly higher seedling mass and vigour indices compared to the control seedlings (Table 2). Growth of A. mearnsii seedlings was stimulated in response to the butenolide treatment. With the exception of the root length and seedling mass for seedlings grown under alternate light conditions, all other growth parameters for the butenolide treatment were significantly improved (Fig. 1 and Table 2). The smoke-water- and butenolide-treatedseedlings had significantly higher vigour indices under constant dark conditions (Table 2). Smoke-water did not have a promotive effect on the growth of A. robusta seedlings under alternate and constant light conditions. However, butenolide-treated seedlings grown under alternate light conditions had significantly improved root and shoot lengths (30.3 and 5.9 mm, respectively) in comparison to the control (24.7 and 5.2 mm, respectively) (Fig. 1). Although the vigour index, under alternate light conditions was greater in butenolide-treated seedlings, this value was not significantly different from the control. Under alternate light conditions, seedling mass was reduced, but with constant Table 2 Influence of smoke-water (1:500) and a butenolide solution (107 M) on vigour and mass of 10-day-old seedlings of Acacia species incubated under three different light conditions at 2570.5 1C Condition
Treatment
Species A. hebeclada
Alternate light (16:8 h light/dark)
Constant light
Constant dark
A. mearnsii
Vigour index
Seedling mass (mg)
Vigour index
Control
1631791 b
143.677.60 b
4283793 b
Smokewater Butenolide
1626779 b
170.477.56 a
2393795 a
Control
A. robusta Seedling mass (mg)
Vigour index
Seedling mass (mg)
24.770.65 a
29937110 ab
47078.87 a
37267155 c 23.970.90 a
29237108 b
405713.2 a
175.876.32 a
48357131 a 24.370.62 a
32677137 a
435714.7 a
1510784 b
143.577.30 a
3780794 b
21.370.41 c
27577114 b
420713.5 b
Smokewater Butenolide
1742787 b
159.477.60 a
39977122 b 23.970.70 b
2910794 ab
449710.1 ab
2306780 a
164.075.89 a
4453779 a
26.270.75 a
30177101 a
47277.21 a
Control
1623798 c
152.279.13 b
73237262 c 39.871.57 b
82567206 b
606714.5 ab
Smokewater Butenolide
25837153 b
191.979.46 a
92547239 b 43.770.95 b
72297239 c
574713.8 b
32277146 a
214.178.35 a 107307262 a 52.771.39 a
96507245 a
64479.08 a
Mean values7SE of each light condition in a column with different letter(s) are significantly (po0.05) different.
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light there was a significant increase in mass with butenolide treatment (Table 2). The seedlings grown with butenolide solution under constant light and dark conditions had significantly greater vigour indices than the control (Table 2). Soil seed bank and germination responses to fire-related cues are important factors which influence post-fire seedling emergence (Auld et al., 2000). Plant-derived smoke, one of the fire-related cues generated during wildfires, has been shown to stimulate the germination of diverse plant communities (Van Staden et al., 2000; Witkowski and Mbalo, 2003). Smoke, however, not only influences germination but importantly it also stimulates seedling vigour (Baxter and Van Staden, 1994; Sparg et al., 2005). A smoke-derived butenolide significantly increased germination percentages of A. hebeclada under all three light conditions. Seeds of A. mearnsii achieved significantly better percentage germination when they were treated with butenolide and exposed to constant dark conditions. While A. robusta seeds showed a non-significant improvement in percentage germination under these conditions. Therefore, the findings from these experiments indicate that the butenolide would be more effective under dark conditions. This suggests that the interaction of light and smoke could be an important factor for smoke-related germination studies. A study on the interactive effects of heat shock and smoke has shown an increase in germination of number of plant species (Thomas et al., 2003). However, interactive effects of heat shock and butenolide on hard-seeded Acacia species requires further investigation. This study showed that smoke-water or butenolide treatment of A. hebeclada and A. mearnsii seedlings improved the vigour index and seedling mass under constant dark conditions. The enhanced response of seedlings to butenolide in the dark suggests that the butenolide may have some interaction with light, similar to auxins (Kulkarni et al., 2006). In the case of A. robusta, smoke-water treatment under dark conditions resulted in an inhibitory effect on seedling growth, with a lower vigour index and seedling mass. This could be attributed to the presence of soluble inhibitor(s) at high smoke-water concentrations, as reported by Light et al. (2002). It is suggested that in natural environments this inhibitor(s) may be leached out by rain without losing the stimulatory effect of smoke (Baldwin et al., 1994). In contrast, the butenolide-treated seedlings of A. robusta had longer shoot and root lengths under dark conditions, resulting in a better vigour index and seedling mass (Table 2). Of the three light conditions examined, the Acacia species studied generally preferred constant dark conditions for germination and seedling growth. This suggests that under natural conditions the seeds of these species which are buried below the surface will show higher germination than those seeds scattered on the surface of the soil. It is reported that smoke treatment increased germination in buried seeds of Stylidium affine, S. crossocephalum and Hibbertia commutata (Tieu et al., 2001). To germinate, these hard seeds require natural scarification in the wild, where fire is reported to be one of the scarifying agents to break the hard seed coat dormancy of several Acacia species (Dell, 1980; Radford et al., 2001; Rinco´n-Rosales et al., 2003). Post-fire climatic conditions also influence seed germination (Whelan, 1995), and therefore the emergence of seedlings in many fire-prone communities occurs mostly in the first wet season following fire (Williams et al., 2005). Studies on Acacia sieberiana have shown that the emergence of seedlings on burnt plots, a week after the first rains, was much greater than in the unburnt plots (Sabiiti and Wein, 1987). A similar situation was noted in Acacia longifolia seedlings (Pieterse and Cairns, 1986). Studies on Acacia nilotica also suggest that seedlings emerging after fire, following
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Fig. 2. Response of 10-day-old seedlings of (A) Acacia hebeclada, (B) A. mearnsii and (C) A. robusta to smokewater (SW, 1:500) or butenolide solution (BS, 107 M) under constant dark conditions at 2570.5 1C. Bar ¼ 10 mm.
wet years, may be larger and more vigourous (Radford et al., 2001). In this study, seeds of all three Acacia species which were artificially scarified and subjected to butenolide treatment showed improved seedling size, indicating a stimulatory response (Fig. 2). Besides fire, the Acacia seed banks are also exposed to different natural scarifying agents. Once these seeds are scarified, either through fire or other modes, the butenolide from wildfires present on the soil surface would penetrate into the soil with the onset of rains and come into contact with buried Acacia seeds. The butenolide is reported to be heat stable, long lasting, water soluble and remains highly active at a wide range of concentrations (Flematti et al., 2004; Van Staden et al., 2004) making penetration into the soil effective. Consequently, smoke from wildfires is likely to stimulate seed germination and vigour, thereby increasing densities of Acacia seedlings. Furthermore, smoke and butenolide are fire constituents that should be seriously considered in the regeneration ecology of Acacias. Acknowledgements The financial support of the National Research Foundation (NRF), Pretoria, and the University of KwaZulu-Natal Research Fund is gratefully acknowledged. Dr Sascha Beck
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from the Institute for Commercial Forestry Research (ICFR), Pietermaritzburg, is thanked for providing seeds of Acacia mearnsii. Dr Nicolette Taylor and Miss Marnie Light are gratefully acknowledged for their assistance in preparing this manuscript. References Allen, S.E., Evans, C.C., Grimshaw, H.M., 1969. The distribution of mineral nutrients in the soil after heather burning. Oikos 20, 16–25. Anderson, R.C., Menges, E.S., 1997. Effects of fire on sandhill herbs: nutrients, mycorrhizae, and biomass allocation. American Journal of Botany 84, 938–948. Aref, I.M., El-Juhany, L.I., Hegazy, S.S., 2003. Comparison of the growth and biomass production of six Acacia species in Riyadh, Saudi Arabia after 4 years of irrigated cultivation. Journal of Arid Environments 54, 783–792. Auld, T.D., Keith, D.A., Bradstock, R.A., 2000. Patterns in longevity of soil seedbanks in fire-prone communities of south-eastern Australia. Australian Journal of Botany 48, 539–548. Baldwin, I.T., Staszak-Kozinski, L., Davidson, R., 1994. Up in smoke. I. Smoke-derived germination cues for post-fire annual, Nicotiana attenuata Torr ex Watson. Journal of Chemical Ecology 20, 2345–2371. Baxter, B.J.M., Van Staden, J., 1994. Plant-derived smoke: an effective seed pre-treatment. Plant Growth Regulation 14, 279–282. Baxter, B.J.M., Van Staden, J., Granger, J.E., Brown, N.A.C., 1994. Plant-derived smoke and smoke extracts stimulate seed germination of the fire-climax grass Themeda triandra Forssk. Environmental and Experimental Botany 34, 217–223. Boucher, C., Meets, M., 2004. Determination of the relative activity of aqueous plant-derived smoke solution used in seed germination. South African Journal of Botany 70, 313–318. Brown, J.R., Archer, S., 1999. Shrub invasion of grassland: recruitment is continuous and not regulated by herbaceous biomass or density. Ecology 80, 2385–2396. Brown, N.A.C., Botha, P.A., 2004. Smoke seed germination studies and a guide to seed propagation of plants from the major families of the Cape Floristic Region, South Africa. South African Journal of Botany 70, 559–581. Brown, N.A.C., Van Staden, J., 1998. Plant-derived smoke: an effective seed pre-soaking treatment for wildflower species and with potential for horticultural and vegetable crops. Seed Science and Technology 26, 669–673. Brys, R., Jacquemyn, H., De Blust, G., 2005. Fire increases aboveground biomass, seed production and recruitment success of Molinia caerulea in dry heathland. Acta Oecologica 28, 299–305. Cavanagh, A.K., 1980. A review of some aspects of the germination of Acacias. Proceedings of the Royal Society of Victoria 91, 161–180. Cox, J.R., Alba-Avila, A., Rice, R.W., Cox, J.N., 1993. Biological and physical factors influencing Acacia constricta and Prosopis velutina establishment in the Sonoran Desert. Journal of Range Management 46, 43–48. Crosti, R., Ladd, P.G., Dixon, K.W., Piotto, B., 2006. Post-fire germination: The effect of smoke on seeds of selected species from the central Mediterranean basin. Forest Ecology and Management 221, 306–312. De Lange, J.H., Boucher, C., 1990. Autecological studies on Audouinia capitata (Bruniaceae). I. Plant-derived smoke as a seed germination cue. South African Journal of Botany 56, 700–703. Dell, B., 1980. Structure and function of the strophiolar plug in seeds of Albizia lophantha. American Journal of Botany 67, 556–563. Demel, T., 1996. Germination ecology of twelve indigenous and eight exotic multipurpose leguminous species from Ethiopia. Forest Ecology and Management 80, 209–223. Dhindwal, A.S., Lather, B.P.S., Singh, J., 1991. Efficacy of seed treatment on germination, seedling emergence and vigour of cotton (Gossypium hirsutum) genotypes. Seed Research 19, 59–61. Dixon, K.W., Roche, S., Pate, J.S., 1995. The promotive effect of smoke derived from burnt native vegetation on seed germination of Western Australian plants. Oecologia 101, 185–192. Enright, N., Kintrup, A., 2001. Effects of smoke, heat and charred wood on the germination of dormant soilstored seeds from a Eucalyptus baxteri heathy-woodland in Victoria, SE Australia. Austral Ecology 26, 132–141. Flematti, G.R., Ghisalberti, E.L., Dixon, K.W., Trengove, R.D., 2004. A compound from smoke that promotes seed germination. Science 305, 977.
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Williams, P.R., Congdon, R.A., Grice, A.C., Clarke, P.J., 2005. Effect of season of burning and removal of herbaceous cover on seedling emergence in a eucalypt savanna of north-eastern Australia. Austral Ecology 30, 491–496. Wilson, T.B., Witkowski, T.F., 1998. Water requirements for germination and early seedling establishment in four African savanna woody plant species. Journal of Arid Environments 38, 541–550. Witkowski, E.T.F., Mbalo, B.A., 2003. Effects of plant-derived smoke and light on germination of six savanna woody plant species. South African Journal of Botany 69, 267–268.
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Journal of Arid Environments www.elsevier.com/locate/jaridenv
Short communication
Germination and post-germination response of Acacia seeds to smoke-water and butenolide, a smoke-derived compound M.G. Kulkarni, S.G. Sparg, J. Van Staden Research Centre for Plant Growth and Development, School of Biological and Conservation Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa Received 10 February 2006; received in revised form 27 July 2006; accepted 11 September 2006 Available online 23 October 2006
Abstract Fire plays a critical role in breaking hard-seeded dormancy and establishing seedlings of several Acacia species in arid and semi-arid regions. Numerous studies have reported an increase in seedling densities of some Acacias after fire without identifying the exact cause. However, it is generally believed and speculated that the effect is largely physical and heat related. Recent studies have revealed that smoke generated from wildfires has the ability to stimulate seed germination and also to improve seedling vigour. This stimulatory characteristic of smoke has now been confirmed by the isolation of a butenolide (3-methyl-2H-furo[2,3-c]pyran-2-one) from plant smoke that actively promotes germination of many plant species. The species of Acacia investigated were A. hebeclada (deciduous shrub), A. mearnsii (invasive tree, native to Australia) and A. robusta (deciduous tree). Seeds of A. hebeclada germinated under different light conditions with smoke-derived butenolide solution (107 M), exhibited a significantly (po0.05) greater germination percentage than untreated seeds. Whereas A. mearnsii seeds exposed to constant dark conditions showed a significantly (po0.05) better germination percentage than the control. However, there was a non-significant improvement for A. robusta seeds. All three species responded positively to the butenolide treatment (107 M) after incubating for 10 days under constant dark conditions at 2570.5 1C, achieving a higher vigour index and seedling mass in comparison to the controls.
Corresponding author. Tel.: +27 33 260 5130; fax: +27 33 260 5897.
E-mail address: [email protected] (J. Van Staden). 0140-1963/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaridenv.2006.09.001
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Smoke-water (1:500) had an intermediate effect on these species. This study shows that the butenolide, isolated from smoke, may have a significant effect on the post-fire seedling ecology of Acacias. r 2006 Elsevier Ltd. All rights reserved. Keywords: Post Fire; Seedling emergence; Smoke; Stimulation
1. Introduction Species of Acacia are widely distributed in arid and semi-arid regions (Demel, 1996; Wilson and Witkowski, 1998; Aref et al., 2003), where fire plays an important role in breaking hard seed coat dormancy and stimulating seed germination (Harker, 1959). Particularly, temperature or heat shocks, generated from fires, are reported to play a role in releasing seed dormancy of legumes in arid and semi-arid regions (Pieterse and Cairns, 1986; Sabiiti and Wein, 1987; Oba, 1990; Cox et al., 1993; Mbalo and Witkowski, 1997). In addition, heat is a specific requirement for triggering germination of hard-seeded species of Fabaceae (Enright and Kintrup, 2001). It is also reported that seedling densities of Acacia species become dominant after fire (Cavanagh, 1980; Milton and Hall, 1981; Sabiiti and Wein, 1987; Pieterse and Boucher, 1997), with increasing stem density in some cases (Pratt and Knight, 1971). Some studies show that a reduction in grass cover allows the establishment of woody seedlings (Schultz et al., 1955; Kanz, 2001), whilst other studies disagree with these findings (O’Connor, 1995; Brown and Archer, 1999). In addition, some studies indicate that an increase in soil nutrient levels after fire (Allen et al., 1969; Anderson and Menges, 1997) may be responsible for the subsequent vigourous growth of plants (Brys et al., 2005). The effects of fire on Acacia species are inconsistent and usually not clear (Radford et al., 2001) therefore the exact cause of post-fire stimulation of Acacia seedling growth remains unknown. Smoke generated from fires plays an important role in re-establishing many plant communities in semi-arid regions such as chaparral in southern California (Keeley and Pizzorno, 1986), fynbos in South Africa (De Lange and Boucher, 1990), Kwongan in Australia (Dixon et al., 1995) and the Mediterranean basin (Crosti et al., 2006). Smoke is now widely recognized as a germination cue for fire-dependent as well as non fire-dependent plant species (Ja¨ger et al., 1996; Light and Van Staden, 2004; Light et al., 2005). Smoke treatments have also shown promise in the propagation of economically important wild plants, as germination percentage can be increased significantly (Brown and Van Staden, 1998; Brown and Botha, 2004). Thus smoke and smoke solutions have great potential for use in horticulture, agriculture, weed management, habitat restoration and conservation practices (Roche et al., 1997; Boucher and Meets, 2004; Light and Van Staden, 2004). The isolation of a new compound from smoke that stimulates seed germination of many plants is generating wide interest and will assist in unravelling certain processes of post-fire ecology. The compound has been characterized as the butenolide 3-methyl-2H-furo[2,3-c]pyran-2-one, isolated from plant-derived smoke (Van Staden et al., 2004), burned cellulose (Flematti et al., 2004), and also from products formed by heating combinations of carbohydrates and amino acids (Light et al., 2005). Until recently, smoke has been assessed for its ability to release seed dormancy and to improve germination, but a few studies have shown that the effects of smoke extend beyond post-germination events resulting in the stimulation of seedling vigour (Baxter and
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Van Staden, 1994; Sparg et al., 2005). In the past many studies on Acacia species have focused on physical aspects of fire-related cues for releasing dormancy and seed germination and little attention has been given to the chemical/physiological influence of smoke components at post-germination levels. This study was conducted to determine the effects of smoke-water and the smokeisolated butenolide on seedling vigour and growth of three different Acacia species. The species investigated were two endemic species A. hebeclada (candle thorn), A. robusta (splendid thorn) and one exotic A. mearnsii (black wattle) native to Australia. 1.1. Seed collection Mature pods of A. hebeclada DC. were collected in March 2004 from the University of KwaZulu-Natal Pietermaritzburg Botanical Garden, South Africa. Mature pods of A. robusta Burch. were obtained in July 2005 from areas surrounding Johannesburg. Dry pods of both species were opened in the laboratory and healthy seeds were then stored in plastic containers at room temperature before use. Seeds of A. mearnsii De Wild. collected in July 2005 were obtained from the Institute for Commercial Forestry Research, Pietermaritzburg. The germination test showed X 98% seed viability of all the three species examined. 1.2. Seed pretreatment Many Acacia species have a hard seed coat which makes it difficult to imbibe water unless some scarification pretreatment is adopted (Demel, 1996). In this study all three Acacia species examined had a hard seed coat requiring scarification for better germination. The seeds of A. hebeclada and A. robusta were acid scarified by immersing them in concentrated sulphuric acid (H2SO4) for 90 min and then carefully rinsed in distilled water before germination. The smaller seeds of A. mearnsii were acid scarified for 25 min only and then carefully rinsed with distilled water. 1.3. Smoke treatments An aqueous smoke extract was prepared by continuously bubbling smoke from the leaf material of a fire-climax grass, Themeda triandra Forssk. (Poaceae), through a column of water (500 ml) for 45 min (Baxter et al., 1994). Solutions of this smoke extract were prepared by diluting 1 ml of the concentrated solution in 500 ml of distilled water, and diluting this further, as required. The smoke compound, butenolide, was isolated from plant-derived smoke solution (Van Staden et al., 2004). A solution of 107 M of this compound was used to test germination and seedling growth as outlined below. 1.4. Germination and seedling vigour experiments Scarified seeds of all three Acacia species were germinated in 9 cm Petri dishes fitted with two layers of Whatman No.1 filter paper moistened initially with 4 ml distilled water or treatment solution. Each treatment consisted of four replicates with 20 seeds in each. Seeds were incubated under three light conditions for germination and seedling growth: alternate light (16:8 h light/dark), constant light and constant dark. For alternate and constant light
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conditions, Petri dishes were placed in incubators set at 2570.5 1C with a light intensity of 8075 mmol m2 s1 provided by cool-white fluorescent lamps. The Petri dishes for constant dark conditions were kept in a lightproof box at 2570.5 1C. Germination (2 mm protrusion of radicle) was scored daily and germinated seeds were allowed to grow for a further 10 days to evaluate post-germination responses. At the end of the experiment the seedling vigour was calculated as VI ¼ [average shoot length (mm)+average root length (mm)] percentage germination (Dhindwal et al., 1991). 1.5. Statistical analysis Statistical analysis was conducted using MINITABs statistical package, release 12.1 (MINITAB INC., PA 16801-3008, USA). The germination data in each treatment were arcsine transformed (Sokal and Rohlf, 1995) and non-parametric Mann–Whitney Test was conducted to compare the treatments with the control at 5% level of significance (po0.05). Seedling (10-day-old) growth data were subjected to one-way analysis of variance (ANOVA) and Tukey’s method was used for pair-wise comparison at a 5% level of significance (po0.05). The butenolide-treated seeds of A. hebeclada showed a significant increase in percentage germination under the three different light conditions compared to the control (Table 1). Whilst treating seeds with smoke marginally improved the percentage germination. Under constant dark conditions, seeds of A. mearnsii treated with butenolide showed significantly better percentage germination than the control. However, in spite of having higher percentage germination, smoke-water did not significantly enhance the germination. Similarly, seeds of A. robusta germinated with butenolide solution, under constant dark conditions, showed an increased germination percentage, but this was not statistically different from the control. Seedling vigour indices of butenolide-treated A. hebeclada seeds, incubated in alternate and constant light conditions, increased significantly, compared to the control and smoke-water treated seeds. Under constant dark conditions, the smokewater and butenolide treatments significantly improved the lengths of shoots (16.9 and 20.3 mm respectively) and roots (18.3 and 18.4 mm, respectively) in comparison to the Table 1 Influence of smoke-water (1:500) and a butenolide solution (107 M) on seed germination of Acacia species under different light conditions at 2570.5 1C Percentage germination Treatment A. hebeclada
A. mearnsii
A. robusta
Alternate light (16:8 h light/dark)
Control Smoke-water Butenolide
70.070.89 76.770.67 83.470.89*
10070 93.470.67 96.770.32
10070 93.470.32 9070.57
Constant light
Control Smoke-water Butenolide
70.070 73.470.67 86.770.32*
10070 93.470.32 10070
86.770.32 96.770.32 96.770.32
Constant dark
Control Smoke-water Butenolide
63.470.32 73.470.32 83.470.32*
83.470.67 96.770.32 10070*
93.470.67 93.470.32 10070
Condition
*Mean value7SE with an asterix is significantly (po0.05) different from the control.
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Fig. 1. Influence of smoke-water (SW, 1:500) and butenolide solution (BS, 107 M) on shoot and root lengths of 10-day-old seedlings of Acacia species incubated under three different light regimes at 2570.5 1C. Control ¼ C. Bars within the same light condition with the same letter(s) are not significantly (po0.05) different.
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control (Fig. 1). In the other two light conditions, butenolide-treated-seedlings showed a significant increase in root length, but shoot length was not significantly affected under alternating light conditions. Although seedlings of A. hebeclada grown under constant light with smoke-water and butenolide solution exhibited increased seedling mass, these values were not significantly different from the control. However, A. hebeclada seedlings grown under alternate light and constant dark conditions with smoke-water and butenolide solutions had a significantly higher mass than the control seedlings (Table 2). Overall, the smoke-water and butenolide-treated-seedlings of A. hebeclada grown under constant dark conditions obtained significantly higher seedling mass and vigour indices compared to the control seedlings (Table 2). Growth of A. mearnsii seedlings was stimulated in response to the butenolide treatment. With the exception of the root length and seedling mass for seedlings grown under alternate light conditions, all other growth parameters for the butenolide treatment were significantly improved (Fig. 1 and Table 2). The smoke-water- and butenolide-treatedseedlings had significantly higher vigour indices under constant dark conditions (Table 2). Smoke-water did not have a promotive effect on the growth of A. robusta seedlings under alternate and constant light conditions. However, butenolide-treated seedlings grown under alternate light conditions had significantly improved root and shoot lengths (30.3 and 5.9 mm, respectively) in comparison to the control (24.7 and 5.2 mm, respectively) (Fig. 1). Although the vigour index, under alternate light conditions was greater in butenolide-treated seedlings, this value was not significantly different from the control. Under alternate light conditions, seedling mass was reduced, but with constant Table 2 Influence of smoke-water (1:500) and a butenolide solution (107 M) on vigour and mass of 10-day-old seedlings of Acacia species incubated under three different light conditions at 2570.5 1C Condition
Treatment
Species A. hebeclada
Alternate light (16:8 h light/dark)
Constant light
Constant dark
A. mearnsii
Vigour index
Seedling mass (mg)
Vigour index
Control
1631791 b
143.677.60 b
4283793 b
Smokewater Butenolide
1626779 b
170.477.56 a
2393795 a
Control
A. robusta Seedling mass (mg)
Vigour index
Seedling mass (mg)
24.770.65 a
29937110 ab
47078.87 a
37267155 c 23.970.90 a
29237108 b
405713.2 a
175.876.32 a
48357131 a 24.370.62 a
32677137 a
435714.7 a
1510784 b
143.577.30 a
3780794 b
21.370.41 c
27577114 b
420713.5 b
Smokewater Butenolide
1742787 b
159.477.60 a
39977122 b 23.970.70 b
2910794 ab
449710.1 ab
2306780 a
164.075.89 a
4453779 a
26.270.75 a
30177101 a
47277.21 a
Control
1623798 c
152.279.13 b
73237262 c 39.871.57 b
82567206 b
606714.5 ab
Smokewater Butenolide
25837153 b
191.979.46 a
92547239 b 43.770.95 b
72297239 c
574713.8 b
32277146 a
214.178.35 a 107307262 a 52.771.39 a
96507245 a
64479.08 a
Mean values7SE of each light condition in a column with different letter(s) are significantly (po0.05) different.
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light there was a significant increase in mass with butenolide treatment (Table 2). The seedlings grown with butenolide solution under constant light and dark conditions had significantly greater vigour indices than the control (Table 2). Soil seed bank and germination responses to fire-related cues are important factors which influence post-fire seedling emergence (Auld et al., 2000). Plant-derived smoke, one of the fire-related cues generated during wildfires, has been shown to stimulate the germination of diverse plant communities (Van Staden et al., 2000; Witkowski and Mbalo, 2003). Smoke, however, not only influences germination but importantly it also stimulates seedling vigour (Baxter and Van Staden, 1994; Sparg et al., 2005). A smoke-derived butenolide significantly increased germination percentages of A. hebeclada under all three light conditions. Seeds of A. mearnsii achieved significantly better percentage germination when they were treated with butenolide and exposed to constant dark conditions. While A. robusta seeds showed a non-significant improvement in percentage germination under these conditions. Therefore, the findings from these experiments indicate that the butenolide would be more effective under dark conditions. This suggests that the interaction of light and smoke could be an important factor for smoke-related germination studies. A study on the interactive effects of heat shock and smoke has shown an increase in germination of number of plant species (Thomas et al., 2003). However, interactive effects of heat shock and butenolide on hard-seeded Acacia species requires further investigation. This study showed that smoke-water or butenolide treatment of A. hebeclada and A. mearnsii seedlings improved the vigour index and seedling mass under constant dark conditions. The enhanced response of seedlings to butenolide in the dark suggests that the butenolide may have some interaction with light, similar to auxins (Kulkarni et al., 2006). In the case of A. robusta, smoke-water treatment under dark conditions resulted in an inhibitory effect on seedling growth, with a lower vigour index and seedling mass. This could be attributed to the presence of soluble inhibitor(s) at high smoke-water concentrations, as reported by Light et al. (2002). It is suggested that in natural environments this inhibitor(s) may be leached out by rain without losing the stimulatory effect of smoke (Baldwin et al., 1994). In contrast, the butenolide-treated seedlings of A. robusta had longer shoot and root lengths under dark conditions, resulting in a better vigour index and seedling mass (Table 2). Of the three light conditions examined, the Acacia species studied generally preferred constant dark conditions for germination and seedling growth. This suggests that under natural conditions the seeds of these species which are buried below the surface will show higher germination than those seeds scattered on the surface of the soil. It is reported that smoke treatment increased germination in buried seeds of Stylidium affine, S. crossocephalum and Hibbertia commutata (Tieu et al., 2001). To germinate, these hard seeds require natural scarification in the wild, where fire is reported to be one of the scarifying agents to break the hard seed coat dormancy of several Acacia species (Dell, 1980; Radford et al., 2001; Rinco´n-Rosales et al., 2003). Post-fire climatic conditions also influence seed germination (Whelan, 1995), and therefore the emergence of seedlings in many fire-prone communities occurs mostly in the first wet season following fire (Williams et al., 2005). Studies on Acacia sieberiana have shown that the emergence of seedlings on burnt plots, a week after the first rains, was much greater than in the unburnt plots (Sabiiti and Wein, 1987). A similar situation was noted in Acacia longifolia seedlings (Pieterse and Cairns, 1986). Studies on Acacia nilotica also suggest that seedlings emerging after fire, following
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Fig. 2. Response of 10-day-old seedlings of (A) Acacia hebeclada, (B) A. mearnsii and (C) A. robusta to smokewater (SW, 1:500) or butenolide solution (BS, 107 M) under constant dark conditions at 2570.5 1C. Bar ¼ 10 mm.
wet years, may be larger and more vigourous (Radford et al., 2001). In this study, seeds of all three Acacia species which were artificially scarified and subjected to butenolide treatment showed improved seedling size, indicating a stimulatory response (Fig. 2). Besides fire, the Acacia seed banks are also exposed to different natural scarifying agents. Once these seeds are scarified, either through fire or other modes, the butenolide from wildfires present on the soil surface would penetrate into the soil with the onset of rains and come into contact with buried Acacia seeds. The butenolide is reported to be heat stable, long lasting, water soluble and remains highly active at a wide range of concentrations (Flematti et al., 2004; Van Staden et al., 2004) making penetration into the soil effective. Consequently, smoke from wildfires is likely to stimulate seed germination and vigour, thereby increasing densities of Acacia seedlings. Furthermore, smoke and butenolide are fire constituents that should be seriously considered in the regeneration ecology of Acacias. Acknowledgements The financial support of the National Research Foundation (NRF), Pretoria, and the University of KwaZulu-Natal Research Fund is gratefully acknowledged. Dr Sascha Beck
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