Seedling establishment and early growth in Calobota sericea subjected to moisture stress

The South African perennial legume Calobota sericea (Thunb.) Boatwr. & B-E van Wyk has been shown to be drought tolerant as mature plants, but information on drought tolerance of seedlings is lacking. This study evaluated the impact of moisture stress on seedling emergence, survival and growth in C. sericea. In the first trial, pre-germinated seeds were planted at 100, 70, 50 and 40% of soil moisture holding capacity without additional watering. Seedling emergence and mortality was recorded daily for 14 days. In the second trial, seeds were allowed to grow under well-watered conditions for one month, after which moisture stress was imposed for 15 and 30 days. Thereafter, the seedlings were uprooted, for shoot and root measurements. Results from these trials show that C. sericea seedlings will establish even at severely reduced water-availabilities, but without subsequent watering, significant seedling mortality will occur. Calobota sericea seedlings displayed a range of morphological adaptive strategies to moisture stress including the minimisation of water loss and optimisation of water uptake. Further research into the impacts of regular cycles of moisture stress is needed to determine if changes in morphology due to prior moisture stress will result in improved adaptation to subsequent moisture stress.

Forage production under dryland conditions depends on adequate rainfall (Liu et al. 2018).This has become problematic in the semi-arid agro-ecological areas of the Mediterranean-like climatic region of South Africa, where local bioclimatic conditions result in increased summer droughts and variable rainfall during the wet winter months (Kim et al. 2019).This unpredictability in rainfall often means that these agro-ecological areas suffer from insufficient supply of water which significantly affects forage production, resulting in poor pasture establishment and decreasing yields, depending on the severity and duration of the moisture stress (Farooq et al. 2009;Jaleel et al. 2009).Within the Mediterranean-like regions of South Africa, climate change has resulted in an intensification of extreme climatic events causing a decrease in winter rainfall, increases in out of season rainfall events, maximum winter temperatures, and durations between rainfall events, as well as a delay in the start of the winter rainfall season.Under future climate change conditions, further increases in aridity can also be expected (Roffe et al. 2020;Engelbrecht and Monteiro 2021).This, in turn, means that when establishing pastures within these areas, longer periods of water-limitation and increased temperatures will result in soils drying faster and remaining dry for longer.A renewed interest in perennial forage resources that can persist in these drought prone environments has resulted in the prioritisation of Calobota sericea (Thunb.)Boatwr.and B-E van Wyk (Müller et al. 2017).
Calobota sericea is a small seeded (thousand seed mass 4.2 ± 0.4 g), deciduous, indigenous, perennial legume species native to the winter rainfall regions of South Africa (Boatwright et al. 2018).This plant could be used as a cut-and-carry forage or directly browsed by livestock (Müller et al. 2021a;Britz et al. 2022).The species have been found to cope well with extended periods of moisture stress through a range of morphological and physiological adjustments (Müller et al. 2021b).However, information regarding the impacts of moisture stress on early seedling growth and development of C. sericea is lacking.
Insufficient soil moisture content can result in significant seedling mortality if seedlings are not well adapted to establish under moisture stress conditions.For some known drought tolerant forages like tedera (Bituminaria bituminosa (L.) CH Stirt.var.albomarginata) and lucerne (Medicago sativa), seedlings that establish under these adverse conditions, avoid moisture stress by rapidly developing an elongated taproot system to access subsoil moisture (Foster et al. 2012).Furthermore, it was reported that seedlings of tedera also display mechanisms of reducing water loss such as improved stomatal control and paraheliotropic leaf movements, as well as improved resource allocation under moisture stress conditions, thus allocating more resources to root growth and reducing shoot growth (Foster et al. 2012).Similar mechanisms of drought tolerance were also found in mature C. sericea plants subjected to moisture stress (Müller et al. 2021b).

Introduction
Unfortunately, the mechanisms of drought resistance at seedling stage in C. sericea, and in forage legumes in general, have generally not been well characterised (Foster et al. 2012).However, with the increased unpredictability in rainfall under climate change conditions (Kim et al. 2019), there is a need to better understand seedling establishment of forages under adverse conditions.This study aimed to determine the impacts of short-term moisture stress on the establishment and survival of C. sericea seedlings.

Seedling emergence and mortality under moisture stress
Seedling emergence in C. sericea were evaluated under greenhouse conditions (natural light and average temperature of 24 ± 2 °C).Prior to planting, four pots (15 cm wide and 17 cm deep) filled with a sandy loam soil (sand 69.8%, silt 10.0% and clay 20.2%) were irrigated until water started draining from the bottom of the pots.No additional nutrients were applied to the soil.Draining of excess water from the pots was allowed for 12 hours to reach field capacity after which the gravimetric water content (θ g g g −1 ) was determined.The soil moisture content of these pots was regarded as pot capacity (100%).Thereafter, the experimental pots were watered to soil moisture contents of approximately 100%, 70%, 50% and 40% of capacity and expressed as a percentage of the initial field capacity.At each of these soil moisture treatments four replicates of 25 pre-germinated seeds (radicle ≥ 0.3 cm) were planted at a depth of 1 cm and arranged randomly on the greenhouse benches.Pre-germinated seeds were used because seeds from native populations were used in the trial, and therefore their germination potential may vary.Using the pre-germinated seeds means that all seeds planted had the potential of establishing under the trial conditions.Seedling emergence (two expanded cotyledons visible) and mortality (indicated by the decrease in seedlings after maximum seedling emergence) was counted daily for 14 days after planting, and the number of emerged seedlings as well as the number of seedlings that eventually died in each pot were recorded.The date of first seedling emergence and last seedling emergence was recorded, and at the end of the trial the rate of seedling mortality was calculated from the maximum seedling emergence per pot.

Early growth responses in Calobota sericea seedlings to water-limitation
A greenhouse study (natural light and average temperature of 24 ± 2 °C) was conducted to determine how C. sericea seedlings would respond to moisture stress.The experiment consisted of two treatments, the amount of water (well-watered or moisture stressed) and the time of harvest (15 or 30 days after moisture stress) arranged randomly on the greenhouse benches.Before planting, the seeds were pre-germinated in 90 mm petri-dishes on two layers of moist filter paper.After radicle emergence (≥ 0.3 cm), five pre-germinated seeds were transplanted into 15 cm tall and 10 cm wide plastic planting bags filled with a sandy loam soil (sand 69.8%, silt 10.0% and clay 20.2%).After seedling emergence, C. sericea sprouts were thinned to one plant per bag.The bags were watered to capacity (until water started draining from planting bags) once a week until 30 days after establishment, after which watering was withheld for the 30-day moisture stress test.Watering continued up to 45 days for the 15-day moisture stress test, after which water was withheld.Water-limitation was staggered so that all plants could be harvested at the same time and the same age for comparative purposes (Supplementary Figure 1).
At each harvesting time, leaf angle inclination was recorded by taking a picture of the plants and using a desktop protractor to measure leaf angle from the pulvinus of the petiole for four leaves per plant.Thereafter, the complete seedling was carefully removed from the bag.Roots of the seedlings were carefully washed and blotted dry, and the total shoot height (measured as the height from the soil surface to the highest tip of the shoots) and total root length (measured from the soil surface to the tip of the longest root) were measured using a digital caliper.Thereafter, seedlings were separated into roots and shoots, oven dried, and dry mass determined.

Statistical analyses
Statistical analyses were performed in IBM SPSS v. 22 (SPSS Inc., Chicago, IL, USA).All data was tested for normality using a Shapiro-Wilks test.A one-way ANOVA was performed on all variables to determine whether significant differences were found between the different treatments.Where significant differences were observed, a Fisher's Least Significant Difference (LSD) post-hoc test was performed to determine between which treatments these differences occurred.For the seedling emergence trial, all vigour indices derived from the data were analysed against soil moisture content at planting using regression analyses and plotted with a polynomial trend line.

Seedling emergence under moisture stress
The results from the current study showed that at soil moisture contents of 100% and 70% field capacity, no difference in the total number of emerged seedlings were recorded, with more than 95% of planted seeds successfully emerging (Table 1).However, when soil moisture content decreased to 50% and 40% of field capacity at the time of planting, the maximum number of emerged seedlings significantly decreased (Table 1).When seeds were planted at soil moisture content of 50% capacity, nearly 80% of seedlings successfully emerged.These results indicate a positive correlation in the total number of seedlings emerging and the soil moisture holding capacity at which the seeds were planted (Figure 1a).When planted at 100% and 70% field capacity, no differences in seedling emergence were observed for up to five days after planting, indicating that at these soil moisture levels the rate of seedling emergence is not significantly influenced (Table 1).However, the number of seedlings emerged on these days were always significantly less (p < 0.05) when seeds were planted at 50% and 40% of field capacity.When planted at 100% and 70% field capacity, seedling emergence occurred the day after planting.However, when the seeds were planted at 50% and 40% field capacity it took 2 (± 0.25) and 4 (± 0.25), respectively, for the Table 1: Cumulative Calobota sericea seedling numbers over 14 days in relation to soil moisture content (%) at planting (mean of 4 replicates).Statistically significant (p < 0.05) differences in seedling emergence within each soil moisture content at planting are indicated by superscripts of different lowercase letters.Comparisons were made between soil moisture content treatments at each day of counting as well as for maximum seedling emergence seedlings to start emerging (Table 1).This shows that as soil moisture capacity at planting decreases, the time to first seedling emergence was significantly prolonged (Figure 1b); and, may account for the rapid seedling mortality after emergence at both initial and 100% mortality at the lower soil moisture contents (Table 1).Ergo, the days to first mortality happened sooner at 40% and 50% soil moisture content (Figure 1c).Similarly, the days to 100% seedling mortality decreased as moisture content at planting decreased, i.e. mortality was more rapid at 40% and 50% soil moisture content (Figure 1d).Both relationships between the soil moisture content at emergence and seedling mortality were statistically significant (p < 0.05).

Early growth responses in Calobota sericea seedlings to moisture stress
Under moisture stressed conditions, seedlings of C. sericea were found to significantly adjust their leaf inclination, with well-watered plants having leaves angled at approximately 45 ± 3.6º from horizontal, and stressed plants having leaves that were angled at approximately 78 ± 4.7º from horizontal (Figure 2a).Furthermore, under moisture stress conditions, a preferential allocation of resources to root growth and a reduction in shoot growth was observed.Root length increased under moisture stressed conditions, but no differences were observed in root length in plants harvested between 15 and 30 days of moisture stress (Figure 2b).Plants that were harvested after 30 days of moisture stress had a root mass which was significantly less than those of plants that were grown under well-watered conditions.Under early moisture stress conditions, i.e. plants harvested after 15 days of moisture stress, shoot length (Figure 2c) did not differ from plants that were grown under well-watered conditions.However, shoot length significantly decreased further when plants were harvested after 30 days of moisture stress.Root mass (Figure 2e) in plants that were harvested after 15 days of moisture stress did not differ significantly from plants grown under well-watered conditions.Shoot mass, however, was already negatively affected under early moisture stress conditions, i.e. plants harvested after 15 days of moisture stress, and decreased further as stress was prolonged, i.e. when plants were harvested after 30 days of moisture stress conditions (Figure 2f).Significantly more resources were allocated to root growth (length and mass) under moisture stress conditions (Figure 2d and 2g), with root growth increasing significantly between plants harvested at 15 days and those harvested at 30 days of moisture stress.

Discussion
This study aimed to determine the impact of moisture stress on seedling establishment and early seedling growth in C. sericea.With climate change, rainfall has become more unpredictable.Consequently, in these Mediterranean-like agro-ecological areas the establishment of sown seeds are often restricted to sporadic rainfall periods where subsequent water-limitation and rapid soil drying are the primary factors that limit their establishment (Moles and Westoby 2004;Pugnaire et al. 2006;Padilla and Pugnaire 2007).Furthermore, at different stages of development, plants are often exposed to different degrees of a particular stress, impacting differently on their survival.This is due to the ecological niche requirements of plants varying as they develop, resulting in changes in the plant traits that determine the success with which a plant can survive a specific environment (Grubb 1977).Results from the current study indicated that C. sericea seedling establishment is significantly impacted by moisture stress, resulting in a reduced number of seedlings establishing, and faster mortality in those seedlings that were able to initially establish when planted under moisture stress conditions.It is therefore indicated that when considering to plant C. sericea pastures under dry-land conditions, farmers have to choose the appropriate time to plant the forages to ensure that enough moisture is in the soil to stimulate germination and initial seedling emergence, but also taking into consideration when follow-up rains will occur for the forages to successfully establish and grow.
When considering the early growth responses in C. sericea seedlings to moisture stress, results showed that established C. sericea seedlings displayed several adaptive responses to moisture stress.One of the key mechanisms observed in C. sericea seedlings when growing under moisture stress conditions is leaf movements or heliotropism.Heliotropism is known to improve plant production under different stress conditions by optimising the leaf energy balance (Chaves et al. 2003;Raeini-Sarjaz and Chalavi 2008).In C. sericea seedlings, paraheliotropic leaf movements, or light-avoiding leaf movements, were observed in water-limited plants.Paraheliotropism has been described as a stress avoidance mechanism used by plants to avoid direct sunlight and heat, and thus reduce transpirational water loss (Pastenes et al. 2005;Puglielli et al. 2017).In comparison to horizontally angled leaves, this type of leaf movement minimises light interception and avoids excessive heating of the leaves.Several researchers have indicated that paraheliotropic leaf movements are also an enhancing factor for increasing plant water-use efficiency (Bielenberg et al. 2003;Raeini-Sarjaz and Chalavi 2008), and has been reported as an important adaptive strategy in several drought tolerant forage species in the Fabaceae, including species in the genera Medicago (Gamon and Pearcy 1989), Phaseolus (Bielenberg et al. 2003), and Bituminaria (Foster et al. 2012(Foster et al. , 2015)).
With regard to resource allocation, results from this trial are consistent with what was found in older C. sericea plants subjected to moisture stress (Müller et al. 2021b), as well as for other species (Poorter et al. 2012;Eziz et al. 2017).Calobota sericea seedlings were found to allocate more resources to deeper root growth under early moisture stress conditions, but even though root length increased from well-watered conditions to moisture stressed conditions, under extended periods of moisture stress C. sericea roots still suffered desiccation.The increased root:shoot ratio observed under moisture stress conditions can be explained by greater inhibition of shoot growth rather than root growth under moisture stressed conditions.This is in accordance with the optimal partitioning theory, which states that plants will allocate more resources to the structures by which the limiting resources are captured to optimise the plants performance, and therefore improve the fitness and the success of those plants (Beebe et al. 2013).Deeper roots are able access deeper water resources as the topsoil dries and, thus, the development of deeper roots by plants such as beans (Beebe et al. 2013;Fenta et al. 2014;Polania et al. 2017), has been shown to improve drought tolerance under moisture stress conditions (Comas et al. 2013).Additional research into the drought tolerance of C. sericea seedlings following prior droughts needs to be investigated.This is due to the improved root traits developed by C. sericea plants under early stages of moisture stress which may result in better adaptation to subsequent water-limited conditions.Under dryland conditions, C. sericea pastures may be subjected to short but regular periods of moisture stress, and therefore further research is needed to determine how regular cycles of water-limitation and re-watering may influence C. sericea seedling survival and growth.
In conclusion, the results from this study show that C. sericea seedlings display a range of adaptive strategies under moisture stress conditions, including traits involving the minimisation of water loss (leaf movements) and optimisation of water uptake (development of longer roots).Further research is needed to determine how regular cycles of moisture stress and the subsequent adjustments in resource allocation between roots and shoots will influence drought tolerance, forage biomass production and quality.The seedling emergence results presented in this work is also based on pre-germinated seeds, and therefore the impacts of moisture stress on imbibition and subsequent germination and seedling emergence are also needed.

Figure 1 :
Figure 1: Relationship (R 2 ) between the maximum seedling emergence (a), days to first seedling emergence (b), days to initial seedling mortality (c), days to 100% seedling mortality (d) and soil moisture content at planting.Polynomial regression lines fitted to indicate trends Soil moisture (%)