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Keywords:

  • germination;
  • seedbank;
  • seeds;
  • temperature;
  • woody weeds;
  • smoke

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Physiological dormancy in weed species has significant implications for weed management, as viable seeds may persist in soil seedbanks for many years. The major stimulatory compound in smoke, karrikinolide (KAR1), promotes germination in a range of physiologically dormant weed species allowing targeted eradication methods to be employed. Control of Chrysanthemoides monilifera ssp. monilifera (boneseed), a Weed of National Significance in Australia, may benefit from adopting such an approach. In this study, we hypothesised that seeds of C. monilifera ssp. monilifera exhibit physiological dormancy, germinate more rapidly as dormancy is alleviated, show fluctuations in sensitivity to KAR1 and form a persistent soil seedbank. Seeds responded to 1 μM KAR1 (40–60% germination) even during months (i.e. March, April, July, August) when seeds were observed to be more deeply dormant (control germination: 7–20%). Seeds germinated readily over a range of cooler temperatures (i.e. 10, 15, 20, 20/10 and 25/15°C) and were responsive to KAR2 (~50% germination) as well. Eradication efforts for C. monilifera ssp. monilifera may benefit from use of karrikins to achieve synchronised germination from soil seedbanks, even at times of the year when C. monilifera ssp. monilifera seeds would be less likely to germinate, allowing more rapid depletion of the soil seedbank and targeted control of young plants.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Weeds impose significant threats to Australia's environment, economy and agricultural business (Sinden et al., 2004). To effectively control weeds, a thorough understanding of germination requirements, seed dormancy alleviation and the roles of various environmental factors in eliciting germination are required. For a species to be eradicated, all the viable seeds must either die or germinate and be subsequently controlled (Panetta et al., 2011).

Dormancy is an adaptation of many species existing in areas where environmental conditions immediately following seed dispersal are unfavourable for seedling establishment. Thus, seeds do not germinate in spite of conditions that would otherwise seem favourable (Baskin & Baskin, 2004). The most common type of dormancy, physiological dormancy, is characterised by a water-permeable seed/fruit coat and a fully developed embryo that is unable to germinate, due to a hormone-related mechanism suppressing radicle emergence (Baskin & Baskin, 2004). Physiological dormancy in weed species may have significant implications for weed management, with dormant seeds having the potential to persist in the soil seedbank for many years, germinating sporadically in response to environmental changes (Roberts & Potter, 1980).

Over multiple seasons, seeds may cycle through depths of dormancy in response to temperature, moisture and light, causing changes in the requirements for germination (Finch Savage & Leubner Metzger, 2006). With progressive loss of dormancy, species are able to germinate more quickly over a progressively wider range of temperatures (Baskin & Baskin, 2004). If dormancy is alleviated and yet seeds do not germinate in a given time, the onset of a new season that is less favourable for germination may induce secondary dormancy (Baskin & Baskin, 1973). Thus, seasonal temperature and moisture conditions cause annual dormancy cycling in many physiologically dormant species (Finch Savage & Leubner Metzger, 2006).

While physiological dormancy can be alleviated naturally in response to seasonal fluctuations, it can also be bypassed using germination-promoting chemicals, such as smoke and smoke products (De Lange & Boucher, 1990). The major stimulatory compound in smoke, termed karrikinolide (KAR1, 3-methyl-2H-furo[2,3-c]pyran-2-one), promotes germination in a wide range of species native to Australia and South Africa (Flematti et al., 2004) and has also been found to promote germination in a range of weed species as well, including Brassica tournefortii Gouan. and Raphanus raphanistrum L. (Stevens et al., 2007; Long et al., 2011). With the use of stimulants such as karrikins to synchronise germination, eradication methods can be more precise and targeted, thus reducing the time taken to exhaust the soil seedbank and reducing costs of control and eradication.

One species that could benefit from novel management approaches to enhance eradication efforts is Chrysanthemoides monilifera ssp. monilifera (L.) Norl. (boneseed – hereon simply referred to as Cmonilifera), an Asteraceae that originates from the cape region of South Africa. It is listed as a Weed of National Significance in Australia, as it imposes significant economic and environmental impacts (Brougham et al., 2006). Few studies have investigated fundamental aspects of its seed biology, with the majority of publications focusing on management (Cherry, 2008; Downey et al., 2008; Melland & Preston, 2008) and biological control methods (Ireson et al., 2002; Morley, 2004; Roberts, 2008). Schoeman et al. (2010) suggested a level of physiological dormancy was present in C. monilifera seeds due to low germination and the presence of the indehiscent fruit (endocarp) tissues acting as a mechanical barrier, although preliminary investigations by R.L. Long (unpublished results) observed that significantly higher percentages of C. monilifera seeds germinated in the presence of KAR1.

The aims of the current study were to develop an understanding of the seed biology and ecology of C. monilifera through a series of laboratory and field experiments and to investigate the effects of KAR1 under a range of conditions. We hypothesised that (i) C. monilifera seeds exhibit physiological dormancy, with the endocarp acting as a mechanical barrier restricting germination; (ii) seeds germinate more rapidly over a wide range of conditions, as dormancy is alleviated naturally or bypassed using KAR1; (iii) C. monilifera seeds show seasonal fluctuations in sensitivity to germination conditions; and (iv) C. monilifera seeds form a persistent soil seedbank.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Endocarps and seeds

Chrysanthemoides monilifera (five collections) drupes (pericarp + seed) were collected between November 2009 and January 2011 (Table 1). The number of drupes from each collection ranged from c. 900 to 3000 (based on weight). Drupes were collected from a number of locations throughout South Australia (SA) and one location in Victoria (Vic). Only mature drupes were collected from parental plants; these were readily identifiable by the distinctive dark red/brown outer papery exocarp layer. The exocarp along with the thin desiccated mesocarp readily flaked off upon drying and handling, leaving behind the hard inner indehiscent endocarp (+ seed) that is the natural germination unit in this species. All collections were transported via mail to Kings Park within several days of collection, where they were placed in a seed storage (drying) room at 15°C and 15% RH or, for the 2009 Ironbark Basin collection, dried (as previously described) for 4 weeks, then frozen at −20°C until used in this study. Between experiments, unused drupes (hereon referred to as endocarps as the outer layers readily exfoliated during handling) were maintained in the drying room (Table 1).

Table 1. Seed collection and storage information for each seed lot
Seed lot nameCollection locationGPSCollection dateType of storageWeight of 1000 seeds (g) (mean ± SE)Seed fill (%) (mean ± SE)Experiment/s
Ironbark BasinIronbark Basin, Great Otway National Park, Victoria38° 46′24″S 143° 33′27″E17 November 2009Frozen (−20°C)144.0Storage experiment
Long RidgeLong Ridge in Cleland National Park, South Australia34° 57′22″S 138° 40′58″E24 January 2011Seed storage room(15%RH/15°C)103.9 ± 1.837.2 ± 3.6Temperature trials
Mount MuirheadMount Muirhead, Mount Burr Forest Reserve about 6 km north-east of Millicent, South Australia37° 33′50″S 140° 24′44″E19 January 2011Seed storage room (15%RH/15°C)123.2 ± 2.244.2 ± 4.7Initial germination and burial
Naracoorte5 km north-east of Naracoorte, South Australia36° 56′27″S 140° 45′54″E19 January 2011Seed storage room (15%RH/15°C)112.3 ± 2.134.8 ± 3.9Initial germination and burial
WaikerieWaikerie irrigation area, South Australia34° 10′56″S 139°57′12″E18 January 2011Seed storage room (15%RH/15°C)50.0 ± 2.9Concentration curves

Seed viability

Endocarp fill of each collection was determined by X-ray imaging using a calibrated Faxitron Specimen Radiography System (MX-20 Cabinet X-ray Unit; Faxitron, Wheeling, IL, USA). Images were visually assessed to identify empty endocarps, endocarps with damaged seeds and filled endocarps with fully developed seeds.

Seed characteristics

Basic endocarp and seed structures were examined using a binocular microscope (Nikon SMZ800 Model C-BD230, Tokyo, Japan). To investigate whether water uptake was occurring and to determine the time taken for endocarps and seeds to fully imbibe and re-dry, several imbibition tests were conducted using intact endocarps (with seeds), cracked endocarps (with seeds) and extracted seeds (n = 20 per sample). Dry weights were recorded before endocarps, and seeds were placed into Petri dishes with irrigated germination papers (10 mL tap water). Endocarps and seeds were initially moistened for 5 min, blotted dry and reweighed (time 0) before placing them back in the Petri dishes in order to account for water attached to the outside of each germination unit. Endocarps and seeds were reweighed after 0.5, 1, 2, 3, 4, 6, 8, 24, 48 h. The percentage increase in mass was calculated using the following formula:

  • display math(1)

where Wt = weight at any given time and W0 = initial wet weight.

Seeds were then either dried at 103°C for 17 h to determine water content gravimetrically (g H2O/g dw) (ISTA, 1999) and seed:endocarp ratio (seed weight/endocarp weight), or placed in Petri dishes with dry germination papers under standard room conditions (~23°C and ~50% RH) and weighed after 0.5, 1, 2, 3, 4, 6, 8, 24, 48 h to determine the rate at which seeds desiccate in a dry environment. Percentage decrease in mass was calculated using the following formula:

  • display math(2)

where Wt = weight at any given time and D0 = initial dry weight.

Chemicals

Karrikinolide (KAR1) and 2H-furo[2,3-c]pyran-2-one (KAR2) were synthesised according to the methods of Goddard-Borger et al. (2007).

Germination trials

Germination trials were conducted to investigate the effects of various temperatures, lighting regimes, physical states (i.e. intact endocarp or extracted seed) and stimulants (KNO3, KAR1, KAR2). For all experiments, three replicates of 22 naked seeds or intact endocarps were plated onto 0.8% (w/v) water agar in 9-cm Petri dishes. Initial germination trials (using Naracoorte and Mount Muirhead seed lots) were conducted using a factorial design, with either a 12-hourly alternating 20/10, 35/20°C or constant 20°C. Within each temperature regime, diaspores (intact endocarps or extracted seeds) were exposed to either a 12-h light/dark photo regime (white fluorescent light – irradiance of 30 μmol m−2 s−1, 400–700 nm wavelength) or incubated in constant darkness for the first 3 weeks (double wrapped in aluminium foil and only opened after this time). When exposed to these conditions, extracted seeds and endocarps were incubated with or without the presence of 1 μM KAR1. Diaspores were scored for germination (when the radicle had emerged by at least 1 mm) and checked for bacterial and fungal contamination on a weekly basis for 10 weeks and transferred to clean dishes as required. Follow-up germination trials used intact endocarps (due to their ecological significance, being the natural germination unit) incubated under the 12-h light/dark photoperiod and 20/10°C temperature regime, as was found to be optimal in the initial test. The germination response (using Long Ridge seed lot) to a wider range of temperatures (10, 15, 20, 25, 30, 35, 20/10, 25/15, 35/20°C) was then tested using intact endocarps. Mean germination time was calculated using the following formula:

  • display math(3)

where si is the number of germinants on a given day, i, since the start of the germination test, and Stot is the total number of germinants observed. Finally, to assess whether other stimulants may also be effective at promoting C. monilifera germination, solutions of 1 μM KAR2 and KNO3 (1 μM, 10 μM, 100 μM, 1 M) were also assessed using the Waikerie seed lot.

Burial trials

To investigate whether the dormancy state of C. monilifera seeds fluctuated seasonally, filled intact endocarps were buried in the field and retrieved monthly to assess their germination characteristics. Twenty mesh bags (10 × Naracoorte, 10 × Mount Muirhead), each containing 50 endocarps, were buried 10 mm deep in a completely randomised design at the University of Western Australia's Shenton Park Research Station, Perth, Western Australia (31°57′S, 115°48′E). Two bags from each population were retrieved every month for 5 months (April, May, June, July, August 2011) from the field site. Seeds that germinated in the field were removed from the samples, and the viability of remaining units was determined via X-ray analysis, as described previously. Filled endocarps were plated onto 0.8% agar with and without 1 μM KAR1 at alternating 20/10°C with a 12-h light/dark photoperiod. Soil temperature (°C) and soil volumetric water content (%) were monitored for the duration of the seed burial trial (Fig. 1) using an ECH2O EC-20 probe (Decagon Devices, supplied by Monitor Sensors, Caboolture, Qld, Australia). Estimates of soil water content (in the range 0–40% volumetric water content, converted from 0.0 to 1.0 V measurements of capacitance) and temperature were recorded at 1 h intervals.

image

Figure 1. Soil temperature (max. and min. °C) and soil volumetric water content (% SVWC) for the duration of the burial experiment measured at the seed burial site, UWA Shenton Park Research Station, Perth Western Australia, from 22 March to 22 August 2011. Tick marks on the x-axis represent the beginning of a month, and arrows indicate time points when endocarps were retrieved from the ground.

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Soil seedbank persistence

To investigate the extent of seed persistence after the removal of mature plants (2008), soil samples were taken from an eradication site (a ~5 ha area in which all known mature plants of C. monilifera have been located and removed) in Henley Brook ~25 km north-east of Perth in September 2011, and all endocarps from C. monilifera were recovered for analyses. Soil samples (50 × 50 cm, depth = 5–10 cm) were randomly collected from six quadrats within this site, and viable endocarp numbers were determined by identifying filled endocarps via X-ray analysis, as described above. Additionally, germinability was assessed by placing endocarps onto 0.8% agar with and without 1 μM KAR1 at alternating 20/10°C with a 12-h light/dark photoperiod.

Data analysis

Generalised linear modelling was used to analyse germination results, with germination proportions analysed as binomial data (with a logit link function) and mean germination time analysed as time-based data (with identity link function) (GenStat v12.1; [McCullagh & Nelder, 1989]). The significance of factors for each experiment was assessed by stepwise addition to the model. Two-sample one-tailed binomial t-tests were also conducted where appropriate. The significance level used was = 0.05. However, where multiple comparisons were made, a Bonferroni-adjusted significance level was used by dividing 0.05 by the number of comparisons made.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Endocarp and seed characteristics

The natural germination unit for C. monilifera is a thick indehiscent endocarp that encases the seed/embryo. However, this structure is noticeably thinner near the radicle of the seed (Fig. 2). Upon germination, the endocarp breaks into three distinctive sections, thus freeing the developing seedling.

image

Figure 2. Endocarp (after the surrounding exocarp and mesocarp layers have exfoliated) and seed morphology. Intact indehiscent endocarp (left-hand side). Longitudinally cracked endocarp (middle) with seed sitting within (radicle end pointing upward). Extracted intact seed (right-hand side). Line in lower right hand corner = 1.4 mm.

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Imbibition assessment found that mass increase by C. monilifera seeds within intact endocarps reached a plateau after 48 h (Fig. 3). Mass increase was greatest and most rapid in those seeds removed from the woody endocarp, followed by seeds within intact endocarps and seeds within cracked endocarps (Fig. 3). Seeds without endocarps did not show a plateau in mass increase, as germination started to occur after 24 h (Fig. 3). Extracted seeds dried at a much faster rate than seeds within endocarps. Seeds within both cracked and intact endocarps dried out at the same rate and to a similar degree (Fig. 3).

image

Figure 3. Imbibition (above) and reverse imbibition (below) curves for extracted seeds, intact endocarps and cracked endocarps over 96 h. Points show mean percentage increase/decrease in mass ± standard error bars (n = 4 for imbibition and n = 2 for desiccation) and are fitted with exponential curves.

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The water content of imbibed seeds was similar between seeds derived from intact endocarps (extracted after imbibing), seeds imbibed without endocarps and seeds derived from cracked endocarps (extracted after imbibing) (> 0.05; Table 2). Surrounding endocarps also took up a significant amount of water, with similar water contents measured between intact and cracked endocarps (> 0.05; Table 2). The seed:endocarp ratio was 0.15 ± 0.01.

Table 2. Water content (g H2O/g dw) of seeds and endocarps derived from seeds that imbibed water while within (intact or cracked) and without endocarps. Values show means ± standard errors (n = 2 replicates of five seeds)
 Without endocarpWithin intact endocarpWithin cracked endocarp
Endocarp26.2 ± 1.926.3 ± 0.7
Embryo41.1 ± 0.240.9 ± 1.340.1 ± 0.5

Germination trials

Application of 1 μM KAR1 promoted germination both when the endocarp was intact and when it was removed (< 0.001 and = 0.022 respectively; Fig. 4). More germination occurred at the 20/10°C alternating temperature regime than the constant 20°C, which also had more seeds germinate than the 35/20°C alternating temperature regime after 10 weeks (< 0.001; see Fig. S1). The responses of populations differed, with more seeds germinating from the Naracoorte population than Mount Muirhead population (< 0.001; Fig. 4). Similar numbers of seeds germinated at the low alternating temperature (20/10°C) with and without intact endocarps when exposed to KAR1 in the Naracoorte population, However, more seeds germinated when endocarps were intact in the Mount Muirhead population (> 0.05 and < 0.001 respectively; Fig. 4). The majority of germination was seen in the first 3 weeks; however, germination continued after this time at the two lower temperatures, especially with the endocarps present (Fig. S1). Seeds that had their endocarps removed showed a higher initial rate of germination within the first week than seeds with intact endocarps (15.8% of the total seeds that germinated with endocarps in the first week compared with 67.6% of the total seeds that germinated without endocarp in the first week) (P < 0.001; Fig. S1). Similar numbers of seeds germinated in the different lighting regimes (> 0.05; Fig. 4). Even with the 3-week exposure to darkness, initial exposure to light did not result in higher germination than those seeds exposed to alternating light/dark for the duration of the experiment (> 0.05; results not shown).

image

Figure 4. Primary germination trial with a full factorial design for two different populations (Naracoorte and Mount Muirhead). Bars represent mean total germination after 3 weeks with 95% confidence intervals shown.

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To determine the effects of KAR1 under a wider range of temperatures, seeds with endocarps intact were subjected to incubation temperatures ranging from 10 to 35°C. Generally, more seeds germinated with the application of 1 μM KAR1 (< 0.001), except for seeds incubated at constant 10°C, where similar germination was observed both with and without 1 μM KAR1 (> 0.05; Fig. 5). Lower temperatures (≤25°C) were favourable for germination, with temperatures 30°C and over resulting in reduced germination (< 0.001; Fig. 5). Similar numbers of seeds germinated at low constant (10, 15, 20°C) and low alternating temperatures (20/10, 25/15°C), with the application of 1 μM KAR1 (> 0.05), However, more seeds germinated at the 25/15°C temperature regime without 1 μM KAR1 compared with the two constant temperatures 25 and 15°C (< 0.001; Fig. 5). Application of 1 μM KAR1 increased the rate of germination (< 0.001), with the rate of germination gradually increasing with increasing temperature (Fig. 5). Lower temperatures (10, 15 and 20°C) resulted in similar rates of germination (> 0.05), whereas the few seeds that germinated at temperatures ≥25°C germinated in the first three or 4 weeks, leading to a faster rate of germination (Fig. 5). Although the lower alternating temperature regimes (20/10 and 25/15°C) resulted in similar germination overall (> 0.05), 25/15°C elicited faster germination (= 0.028; Fig. 5).

image

Figure 5. Germination for Long Ridge population over a range of temperatures with and without 1 μM KAR1. Bars represent mean total germination after 10 weeks with 95% confidence intervals. Triangles and squares represent mean germination time with standard error bars.

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The use of 1 μM KAR1 and KAR2 resulted in more and faster germination than both the control and nitrate treatments (< 0.001; Fig. 6). Similar numbers of seeds germinated with nitrates as in the control (> 0.05) (Fig. 6). Although 1 μM KAR1 and KAR2 caused similar numbers of seeds to germinate (> 0.05), KAR1 elicited a faster rate of germination, with mean germination time being around 5 days shorter than for KAR2 (= 0.01; Fig. 6).

image

Figure 6. Germination for Waikerie population with the application of 1 μM KAR1, 1 μM KAR2 and 1 μM, 10 μM, 100 μM and 1 M KNO3. Bars represent mean total germination after 10 weeks with 95% confidence intervals. Diamonds represent mean germination time with standard error bars.

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Seed burial trials

Application of 1 μM KAR1 upon retrieval of seeds resulted in germination percentages between 40% and 75% within 5 weeks of soil burial (Fig. 7). The application of 1 μM KAR1 achieved similar germination throughout the seasons, although the Naracoorte population responded best to KAR1 in August (late summer, differing from April < 0.001 and May = 0.021) and the Mount Muirhead population exhibited a reduced response in April (autumn, differing from the control = 0.006, May = 0.028, June, July and August < 0.001; Fig. 7).

image

Figure 7. Germination of seeds prior to burial (March) and following burial at 1 cm depth, at the UWA Shenton Park Research Station. Seeds were retrieved monthly for 5 months and assessed for their germination with and without 1 μM KAR1. Data points represent total germination percentage after 5 weeks with 95% confidence intervals (upper graphs) or mean germination time in days with standard errors (lower graphs). Tick marks on the x-axis represent the start of each month.

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Without KAR1, few dormant seeds germinated prior to burial and after the first retrieval in April, but seeds of both populations became more germinable in late autumn and early winter [May and June for the Naracoorte population (< 0.001); in June for the Mount Muirhead population (< 0.001); Fig. 7]. Germination without KAR1 was lower in July for both populations (< 0.001; Fig. 7). These changes in germinability of seeds correspond to changes in soil moisture and temperature (Fig. 1). Seeds were less germinable when temperatures were high and soil moisture was low, and were more germinable when temperatures fell and soil moisture increased with sporadic rainfall events (Figs. 1 and 7). Fewer seeds germinated as temperatures stabilised at a lower range and soil moisture remained high in late winter (Figs. 1 and 7). Germination of seeds in the field increased in June (< 0.001) but did not increase further during the trial (> 0.05; Fig. 7).

Soil seedbank persistence

Significant numbers of intact endocarps were found in soil samples. However, only a small proportion of these endocarps were filled with viable seeds (Table 3). High germinability of seeds was observed with the majority of seeds germinating in the control treatments (Table 3). Application of 1 μM KAR1 increased the total number of retrieved seeds that germinated (Table 3).

Table 3. Total intact endocarp numbers, fill and germination (with and without 1 μM KAR1) derived from six soil samples collected from the Henley Brook boneseed eradication site
Soil sampleNumber of intact seedsFilled seedsWeight of soil (kg)Filled seeds per kg of soilGermination (%)
WaterKar1
Number% t
  1. n.t (not tested) refers to situations where no testing was conducted due to insufficient numbers of seeds, and n = x (Germination (%) refers to the number of seeds assessed for germination.

18955.6100 (n = 3)100 (n = 2)
253000n.tn.t
34003.60n.tn.t
48112.54.20.2100 (n = 1)n.t
5164116.74.02.840 (n = 6)50 (n = 5)
6248208.15.23.910 (n = 10)100 (n = 10)
Mean94.36.25.54.21.762.583.3
SE39.13.32.00.30.822.516.7

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

By studying the fundamental seed biology of C. monilifera, this study has revealed that this species can form a persistent seedbank of physiologically dormant seeds, highlighting the need for tools to hasten the depletion of the soil seedbank of this invasive weed. The study has also shed light on conditions under which C. monilifera germination can be promoted, thus allowing control and eradication efforts to be targeted. Viable seeds persisted at the Henley Brook eradication site for at least 3 years, supporting findings by Schoeman et al. (2010) who suggested that seeds of this species were long-lived and likely to survive for several years in situ. Although some ‘eradication’ sites appear to be under control, the fact that this species can exhibit a long-lived soil seedbank means that careful monitoring is essential to ensure that new germinants are destroyed as they appear. With the targeted use of karrikins to speed up the seedbank exhaustion process, eradication may be realised in the near future.

Our hypothesis that C. monilifera has physiological dormancy was supported, as intact endocarps and seeds readily absorbed water (Fig. 3), thus ruling out physical dormancy, that is, a water impermeable barrier as to the cause of low germination as reported for the endocarps (+seeds) of Rhus aromatica L. (Baskin & Baskin, 2004). Morphological and morphophysiological dormancy (i.e. the possession of tiny underdeveloped embryos) can be excluded as well, as seeds possessed a large well-developed cotyledonous embryo (Fig. 2) as reported for other Compositae (=Asteraceae) (Martin, 1946). Physiological dormancy is clearly evident, as the initial germination trials using intact endocarps found that these did not readily germinate within 4 weeks without the presence of KAR1 (Fig. S1). This supports the definition of physiological dormancy as described by Baskin and Baskin (2004). Indeed, the fact that extracted seeds germinated to similar percentages and as rapidly as intact endocarps exposed to KAR1 suggests that the surrounding tissues, that is, the endocarp, is probably acting as a mechanical suppressant to germination, although another possibility is that the endocarp possesses some form of germination inhibitor as demonstrated for the endocarps of Eremophila maculata (Ker Gawl.) F.Muell. (aromatic glycosides – Richmond & Ghisalberti, 1994). Studies on Raphanus raphanistrum, a species that exhibits physiological dormancy and which has analogous fruit morphology, also found limited germination without the removal of surrounding structures (Cousens et al., 2009). To overcome the restriction imposed by the endocarp in C. monilifera, seeds need to be stimulated through application of karrikins, or through natural dormancy loss, so that they may push through the surrounding endocarp and complete the germination process.

Chrysanthemoides monilifera seeds germinated more quickly and under a wider range of conditions with the application of karrikins, showing that certain stimulants can promote germination of this species. KAR1 and KAR2 were the most effective chemicals for promoting germination within the scope of this study (Fig. 6). Indeed, as was found in the current study, KAR1 has been previously shown to widen the range of conditions suitable for the germination of other Asteraceae species (Merritt et al., 2006). Although a low concentration of KAR1 (1 μM) was effective under laboratory conditions, higher concentrations may be needed for effective field application, due to dilution when KAR1 washes through the soil profile with rainfall events (Stevens et al., 2007). Nitrate (KNO3) did not appear to provide an effective option for promoting germination (Fig. 6), so eradication efforts should focus on the use of karrikins instead. Indeed, these results highlight the effectiveness of the use of karrikins in promoting germination over a wide range of conditions, allowing synchronised germination and facilitating targeted eradication efforts.

Chrysanthemoides monilifera seeds showed dormancy cycling over the course of the burial trial, coming out of dormancy at the end of autumn and start of winter (May to June), before secondary dormancy was induced in July (Merritt et al., 2007). This supports our hypothesis that seeds would show seasonal fluctuations in sensitivity to germination conditions, being most responsive to the broadest range of conditions during late autumn and relatively less responsive during late winter and early spring (Figs. 1 and 7). This type of dormancy cycling is common in seeds with physiological dormancy and has been found in other weed species such as R. raphanistrum and Sisymbrium orientale L. (Long et al., 2011).

In this study, we found that applying KAR1 while seeds are dormant resulted in germination levels similar to those seen with flushes of germination occurring in late autumn, when seeds are relatively non-dormant. Studies on a number of Brassicaceae species have also found that KAR1 can be used to promote germination in seasons when seeds are more deeply dormant (Long et al., 2011). Given that applying KAR1 to non-dormant seeds failed to increase the germination rate or the total number of seeds germinating in this study (Fig. 7), standard eradication efforts (without the use of KAR1) could occur during late autumn/early winter when seeds are cycling out of dormancy and are readily germinable. Additional eradication efforts could then continue in late winter and spring with the application of KAR1 to promote synchronised germination of the remaining seedbank, when seeds are relatively more dormant and consequently less likely to germinate. This proposed management scenario needs further testing, as our burial trial continued for only 5 months and a longer-term burial trial may provide additional insight into the changes in dormancy state experienced over a number of years. Still, with application of KAR1 in our laboratory-based experiments appearing to be less effective at higher temperatures, it may not be practical to apply KAR1 to eradication sites during summer months in Mediterranean climates, as the chemical's effectiveness would be greatly reduced due to high temperatures (>25°C) and low soil moisture levels (Fig. 5). Eradication efforts should thus be directed towards autumn, winter and spring, when temperatures are cooler and soil moisture levels higher. With the use of KAR1, eradication efforts could continue beyond times when peak germination would naturally occur, thus allowing more rapid depletion of the soil seedbank over a wider time frame.

Differences in seed dormancy and germination characteristics were observed for different C. monilifera populations, highlighting the importance of considering how seeds from different locations respond to germination stimulants and environmental conditions. With large differences in the number of seeds that germinated in the control treatments from different seed lots, it is evident that seeds exist at various depths of dormancy. Differences in dormancy states may be due to the age of the seeds, with seeds collected in the peak of the season being more deeply dormant than those collected late in the season, as the seeds would have aged or after-ripened on the plant prior to collection. Differences in parental environment during seed development may also have imparted different seed dormancy levels as well (Gorecki et al., 2012).

In summary, the use of KAR1 and KAR2 may provide an effective means of synchronising germination of C. monilifera seeds under a range of conditions, thus reducing the time needed to deplete the persistent soil seedbank. Although the majority of the seed populations tested showed more germination with the application of KAR1, within-species variations in the degree of responsiveness to KAR1 needs to be understood to maximise its effectiveness as a germination stimulant for the control of C. monilifera populations. With the current study investigating germination under controlled laboratory conditions, the next logical step is to explore the responses of C. monilifera seeds under field conditions to facilitate translation of results into field eradication practices.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We would like to acknowledge Perth Region NRM for providing a student scholarship to support this research. RLL was supported by the ARC (LP0776951) and a RIRDC Grant (PRJ 006918). GRF was supported by the ARC (DP0880484), and SRT was supported by Grange Resources. We would also like to say thanks to Phil Cramon, Susan Ivory, Peter Tucker, Richard Fossett and Trevor Wynniat for providing seed for this study and Luke McMillan from the Perth Region NRM for assistance.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
wre12051-sup-0001-FigS1.docxWord document2758KFigure S1 Cumulative germination over 10 weeks for Naracoorte and Mount Muirhead populations of Chrysanthemoides monilifera ssp. monilifera.
wre12051-sup-0002-FigS1.tifimage/tif2746K 

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