The response of Nassella trichotoma (serrated tussock) seeds and seedlings to different levels of fire intensity

Seed collection and preparation

Seeds were collected from mature N. trichotoma plants in Mambourin, Victoria, Australia (37°55′18.12″S, 144°32′43.079″E), located within the Greater Western Grasslands in central Victoria, Australia (Fig. 1), in December 2016, 2017, 2018 and 2019. The seeds were stored within their panicles in zip-locked bags at room temperature until use. When the study was about to commence, seeds were removed from the panicles and gently squeezed with forceps to test for seed fill. Seeds that collapsed easily were discarded. Seed mass was determined for each age class by weighing 100 randomly selected seeds from each age group.

Fig. 1. 

Map showing seed-collection areas located within the Greater Western Grasslands in central Victoria, Australia.

Effect of radiant heat on seed germinability

For each treatment combination, 25 seeds were counted and placed into aluminium bowls (10 cm in diameter), and were then exposed to temperatures of either 80°C, 100°C, 120°C or 140°C for 1, 3, 6 or 9 min inside a temperature-controlled oven (Memmert, Type No. ULE500). This multifactor experiment contained four seed ages, four heat treatments and four exposure durations for a total of 64 treatment combinations, plus a control for each seed-collection year. Each treatment combination was replicated six times.

Immediately following heat exposure, seeds were placed into Petri dishes lined with sterilised filter paper and moistened with sterilised reverse osmosis (RO) water, with parafilm being used to prevent drying. The treatments were placed into incubation chambers set to alternating 25°C/15°C, 12-h light/12-h dark conditions, which have been pre-determined as the optimal growth conditions for N. trichotoma (Humphries et al. 2018). The treatments were checked twice a week for 35 days and germination was determined by protrusion of the radicle.

Effect of moisture content and radiant heat on seed germinability

It was observed that the seed collected in 2016 provided the highest germination across all tested temperatures, and they were therefore preferentially selected for the rehydration treatments. To prepare for the experiments, nylon mesh bags containing filled seeds (determined by the squeeze test described previously) were secured within an airtight polycarbonate electrical enclosure box (28 × 28 × 14 cm) containing LiCl solutions adjusted to either 15% RH (740 g/L), 50% RH (364 g/L), or 95% RH (48 g/L). The relative humidity (RH) levels were selected because these moisture contents are associated with the three distinct seed hydration levels that have previously been shown to promote different physiological activity within the seeds (Walters et al. 2005). The boxes were stored at 20°C for 2 weeks, and seed hydration was assessed at the end of this period with a digital hygrometer. After the rehydration period, the seeds were exposed to the methods previously described for heat exposure and subsequent germination monitoring.

Effect of radiant heat on established plants

To assess the effect of radiant heat on established plants, 100 plastic garden pots (140 mm in diameter and 140 mm in height) were lined with absorbent towelling, filled with a fine layer of river sand and then topped with commercially purchased soil. Five viable N. trichotoma seeds were placed into each pot and lightly covered with soil. The pots were placed into watertight plastic trays (28 cm × 44 cm × 5.5 cm), and were placed into a glasshouse at ambient temperature on 24 February 2022. Pots were bottom-watered at least once a week and seedlings were thinned to one plant per pot after the first month of growth. The final plants were grown under glasshouse conditions for 3 months.

The heat treatments were conducted on 22 May 2022. To prepare the plants for the heat-exposure experiment, the plant and soil were together carefully removed from the pots and placed into heat-safe aluminium trays. Five plants were randomly selected and exposed to radiant heat levels of 20°C, 60°C, 80°C, 100°C or 120°C for 3, 6 or 9 min. The plants were then re-potted and monitored for a further 2 weeks under glasshouse conditions, when any signs of wilting or recovery were visually observed.

Data analysis

Climate conditions for each collection year

Variations in the seed mass were observed among the collections made across the different years, with the highest seed mass being recorded for the seeds collected in 2016, which correlated with optimal germination parameters (Table 1). In 2016, the average spring maximum monthly temperature was lower than in the following years, and the total rainfall was much higher during the same period, and these factors may have contributed to the higher seed mass than for the seeds collected in 2017–2019. The higher average spring temperature may have been a leading cause for the reduced seed mass for those collected in 2017, whereas the low total spring rainfall may have contributed to the low seed mass in the seeds collected in 2019.

Effect of radiant heat on seed germinability

The effect of temperature (P < 0.001), duration of exposure (P < 0.001), and seed-collection year (P < 0.001) had a significant impact on N. trichotoma’s total seed germination (%), as did the interactions of these factors (Table 2). The control experiment (no heat) demonstrated that seeds collected in 2016 had a significantly higher total germination percentage than did the seeds collected in more recent years, and the germinability of the seeds appeared to increase with the age of collection, with the total germination percentage increasing with the age of the seeds (Table 3).

Total germination percentage and GI decreased as the temperature gradient increased, whereas the effect of the duration of the heat exposure had a lesser effect on these factors (Table 2). A similar observation was made for mean germination time (MGT) and time to 50% germination (T₅₀), with these parameters increasing when exposed to the higher temperature gradients. The two older seed collections, namely from 2016 and 2017, reached 50% germination sooner than the more recently collected seeds in 2018 and 2019. In most cases, the heat treatments increased N. trichotoma germination percentage compared with the germination percentage recorded for the control treatment. For all the seed-collection times, the 140°C temperature was significant at reducing total seed germination percentage, with the exception of only 0.64% of the seeds germinating in the 6- and 9-min treatments for the seeds collected in 2018. The seeds collected in 2019 had significantly reduced viability when exposed to 120°C for 9 min; however, this decline was not observed for the other treatments. There are variations in total germination percentage for the 80°C, 100°C and 120°C treatments for all the collected seeds, with 2016 having the highest consistency.

Effect of moisture content and radiant heat on seed germinability

As the seeds collected in 2016 provided the highest total germination percentage under controlled testing, as well as providing the most uniformed germination percentage across all temperatures, these seeds were selected for the subsequent tests.

The seed moisture content (P < 0.001), temperature (P = 0.002), and their interaction (P < 0.001) were significant factors affecting N. trichotoma total seed germination percentage (Table 4). Seeds hydrated to 15% had no significant difference in germination percentage when exposed to 80°C, 100°C or 120°C, and the first two temperatures increased germination percentage when compared with the control (Table 5). The seeds hydrated to 50% experienced a significant decline in total germination percentage when the seeds were exposed to 120°C for 9 min (P = 0.002). The exposure to heat was lethal to seeds with moisture contents of 90%, with only 4.6% of the seeds germinating after 1 min of exposure to 80°C. No further germination occurred for seeds hydrated to 90%.

Effect of radiant heat on seedlings

All the plants exposed to the temperatures of 20°C and 60°C were undamaged, and the leaves remained a vibrant green from the base to the tip. At the higher temperatures tested, namely 80°C, 100°C, and 120°C, notable drying of the leaves was observed, but the base of the plant remained undamaged. Four plants exposed to the 120°C treatment (two exposed for 6 min, and two exposed for 9 min) suffered extensive drying, but these plants resprouted from the base. Fig. 2 shows the visual differences among the damage scores of 1 (undamaged), 2 (leaf drying) and 3 (extensive leaf drying and resprouting). No plants were completely killed from the trialled heat treatments.

Fig. 2. 

(L → R) Visual differences among the damage scores of 1 (undamaged), 2 (leaf drying) and 3 (extensive leaf drying and resprouting).

Seed germinability

Our results observed a significant increase in germination percentage for the seeds collected in 2016 compared with those from the following collection years, with the most recent seeds, namely those collected in 2019, demonstrating the lowest germination percentage. In many cases, seed viability and seedling vigour decline as time in dry storage increases (De Vitis et al. 2020), making the results of the present study controversial. Studies into N. tricotoma’s seed longevity under field conditions suggest that the majority of this species seedbank will germinate within the first year of seed set, with only small proportion (<10%) demonstrating short-term persistence of up to 3 years (Ruttledge et al. 2020; Humphries and Florentine 2022). However, under stored conditions, 20-year-old seeds of this species have been observed to germinate successfully (Taylor 1987). This suggests that dry storage does not have a significant impact on N. trichotoma seed viability.

The complex interactions of various biotic and abiotic pressures experienced by the maternal environment during seed development can result in interannual variations in seed traits (Franzese and Ghermandi 2011; Pearse et al. 2017). Various environmental factors, such as temperature and rainfall that a plant experiences during its seed-development stage, can directly influence the progeny seeds dormancy and germination potential (Penfield and MacGregor 2017; Souza et al. 2019). For example, the combination of high temperature and osmotic stress reduce the production of viable seeds of the invasive annual grass Avena fatua (Peters 1982). Plants exposed to higher rainfall and soil nutrients will often allocate more resources to reproduction, resulting in higher seed output and seeds of greater mass and size (Salisbury 1943; Lazaro-Nogal et al. 2015; Souza et al. 2019). The substantially higher spring rainfall experienced in 2016 than that in the following years, may have been one of the factors contributing to the higher seed mass observed for this collection year. Increased seed mass can often result in more vigorous germination and seedling emergence, providing these seeds with a competitive advantage in this critical life stage (Vaughton and Ramsey 2001; Paz and Martinez-Ramos 2003).

The seeds collected in 2016 had greater average seed mass than did those collected in the subsequent years, which was a significant issue for the investigation because increased seed mass can improve the total germination percentage and germination time, together with improving the success of the seedling survival (Bonfil 1998; Cordazzo 2002). The results of this study support the contention that greater seed mass increases total gemination percentage and germination time compared with seeds with a reduced mass. Additionally, an inverse relationship in seed size and seed longevity has been observed for several grassland species, including Ambrosia trifida (Schutte et al. 2008), whereby the smaller seeds persist longer in the seedbank than do larger, denser seeds. It is possible that N. trichotoma has adopted a similar strategy in the present study, and the lighter seeds collected in 2019 may have a higher proportion of dormant seeds, therefore reducing the germinability of this sample.

Understanding the variations in the degree of dormancy a population displays has important implications on the populations ability to successfully establish and persist (Baloch et al. 2001). It is known that N. trichotoma experiences a primary dormancy period that extends for approximately 3 months, which prevents the seeds from germinating in the water-limited summer months (Osmond et al. 2008). This species has also demonstrated the capacity to undergo secondary dormancy, which is induced when the seeds are exposed to prolonged unfavourable conditions, even after the primary dormancy period has ended (Buijs 2020). Intraspecific variations in secondary dormancy are important for ensuring survival in disturbance-prone ecosystems, such as the grassland communities that N. trichotoma has successfully invaded, because this strategy protects the population from failed establishment in times where multiple disturbances are present, such as multiple fire events or prolonged drought (Liyanage and Ooi 2015).

Effect of radiant heat on seed age

N. trichotoma seeds demonstrated a high level of resistance to the tested radiant-heat treatments, with seeds collected in 3 of the 4 years maintaining a high total germination percentage at 120°C. The only exception was seen in the seeds collected in 2019, where the seed germination was significantly (P = 0.002) reduced when exposed for 9 min at 120°C treatment. For the seeds collected in 2016, exposure to 80°C and 100°C increased the total germination percentage compared with the control. The seeds collected in 2017–2019 also evidenced an increased total germination percentage compared with the control.

Mean germination time indicates the lag period between seed imbibition and radicle protrusion where the seed undergoes repairs from damages caused by the environment or seed aging (Mavi et al. 2010). In the present study, N. trichotoma seeds decreased their mean germination time (MGT) after exposure to the increasing temperature gradient, particularly when the exposure time was short. Longer duration of exposure to the temperature gradient resulted in an increase in MGT, indicating these seeds required time to repair any damage prior to germination.

The ability of a species to germinate with greater speed and uniformity increases its competitiveness following fire (Hodges et al. 2022). Mean germination time can be used as an indicator in determining field emergence, with an increased MGT being correlated to lower and less vigorous field emergence (Matthews and Khajeh-Hosseini 2006). Although this could indicate that a high-intensity fire that heats the soil to 120°C or above could damage the seeds enough to reduce competitive emergence in field, this would, however, be subject to the parallel fire response of the native species in the same corresponding seedbank area (Hodges et al. 2022). Time to 50% germination followed the same pattern as did MGT, whereby germination rate slowed as the temperature gradients and duration of exposure increased.

The 140°C treatment was shown to significantly devitalise the seeds, with the exception of one seed germinating in each of the 6- and 9-min-exposure treatments (0.64%) for the seeds collected in 2018. This temperature has been observed to be lethal in other awned grass seeds, such as Piptochaetium napostaense (Kin et al. 2016). Seed awns are an important adaptive trait for seed survival in fire-prone ecosystems, and increased fire intensity can be a selection mechanism for awns with an increased length, because this allows for the seed to burrow further into the soil to escape lethal temperatures (Garnier and Dajoz 2001). The hygroscopic awn on N. trichotoma seeds allows it to burrow into the soil profile, with the majority of this species seedbank found buried at an approximately 2.5-cm depth (Joubert 1984). Shallow seed burial can alleviate the severity of heat shock from fire events, and temperatures 2 cm below the soil surface rarely exceed 80°C (Penman and Towerton 2008). Indeed, a controlled study demonstrated that for surface temperatures that reached 500°C, these conditions were reduced to approximately 70°C at 2 cm depth (Girona-Garcia et al. 2015). Another study showed a significant reduction in temperature (approximately 60%) at a depth of 1 cm, and increased soil moisture further reduced the transfer of heat into the soil profile (Valette et al. 1994). It is therefore unlikely that the majority of N. trichotoma seeds that have burrowed into the soil will be exposed to temperatures that exceed their lethal temperature threshold.

Effect of radiant heat on hydrated seeds

The moisture levels of vegetation and the soil have been shown to influence the intensity of prescribed fire, with the highest-intensity fires thus occuring under low moisture levels. Soils with a high moisture content decrease the thermal energy of the fire (Valette et al. 1994), and this is due to (1) a reduced rate of fuel consumption (Marino et al. 2012), and (2) water in the soil dissipating heat as steam (Stoof et al. 2013). The soil moisture levels strongly influence the moisture level of the seeds that reside within the subsequent soil seedbank, particularly those lacking physical dormancy structures (Dasberg 1971; Josuah et al. 1994). However, N. trichtoma seeds possess no such structures, but and can readily take up water when it is available in the soil (Campbell and Nicol 1999).

Germination percentages for the seeds hydrated to 15% and 50% experienced no significant changes in germination compared with the control treatment. The seeds hydrated to 95% experienced a significant decline in germination percentage at all tested temperatures. The literature suggests that seeds hydrated to 95% are killed by much lower temperatures than those with lower moisture contents (Ruprecht et al. 2016; Tangney et al. 2018), and in the present study, this was confirmed by comparison with levels of 15% and 50%. As the seed moisture content increases, so too does the seeds physiological activity (Walters et al. 2005). When the seeds are hydrated above 90%, there is high free-water content within the seeds, and this can be heated to a temperature that damages cellular processes, subsequently stopping the physiological processes that initiate the germination process (Tangney et al. 2018). Also, proteins associated with protecting the seed while it is in a dormant state, including the synthesis of small heat-shock proteins, cease to provide adequate protection at high seed moisture levels as they do when the seed is in a dry and dormant state (Wehmeyer et al. 1996).

Effect of radiant heat on seedlings

It has been reported that prescribed burning has been implemented effectively for weed-control programs and ecological restoration of grasslands (Mainardis et al. 2020; Assis et al. 2021). However, it is known that adult N. trichotoma plants are able to resist high-intensity fire (Joubert 1984; CRC 2003), and our results suggested that this resistance is also demonstrated in 3-month-old seedlings. In our work, the N. trichotoma seedlings resisted all the temperatures and duration of exposure times tested in this study, and no plants were killed up to 2 weeks post-heat exposure. Exposure to the 120°C treatment did cause extensive drying to the leaves; however, in some cases, the plant was seen to resprout from the base. The ability to resprout rapidly following a fire is critical for maintaining competitive dominance, and ensuring survival of the population, particularly when the seedbank may have been depleted following a fire through either increased seed germination or devitalisation (Tangney et al. 2020; Hodges et al. 2022).

This study has thus demonstrated that N. trichotoma can withstand high temperatures related to fire. Because this species produces unpalatable, sclerophyllous leaves, it allows it to contribute a large volume of dry biomass to the grasslands that it invades (Distel 2013). This further allows for this species to contribute to increases in the intensity of fire regimes, which it can withstand, and thus successfully displace native plants, which cannot survive such high temperatures. This suggests that for control of N. trichotoma, it would be recommended that managers use fire-integrated techniques, using control actions such as target spraying this species with herbicide prior to burning, then broadcasting competing seeds and introducing controlled grazing post-fire (Distel 2013) in grassland ecosystems.