Environmental factors effecting the germination and seedling emergence of two populations of an aggressive agricultural weed; Nassella trichotoma
Environmental factors effecting the germination and seedling emergence of two populations of an aggressive agricultural weed; Nassella trichotoma
Talia Humphries 0 1
Bhagirath S. Chauhan 1
Singarayer K. Florentine 0 1
0 Centre for Environmental Management, Faculty of Science and Technology, Federation University Australia , Mount Helen, Victoria , Australia , 2 Centre for Plant Science, Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland , Toowoomba, Queensland , Australia
1 Editor: David A. Lightfoot, College of Agricultural Sciences , UNITED STATES
Nassella trichotoma (Nees) Hack. ex Arechav. (Serrated tussock) is an aggressive globally significant weed to agricultural and natural ecosystems. Herbicide resistant populations of this C3 perennial weed have emerged, increasing the need for effective wide-scale cultural control strategies. A thorough seed ecology study on two spatially distinct populations of N. trichotoma was conducted on this weed to identify differences in important environmental factors (drought, salinity, alternating temperature, photoperiod, burial depth, soil pH, artificial seed aging, and radiant heat) which influence seed dormancy. Seeds were collected from two spatially distinct populations; Gnarwarre (38 O 9' 8.892'' S, 144 O 7' 38.784'' E) and Ingliston (37O 40' 4.44'' S, 144 O 18' 39.24'' E) in December 2016 and February 2017, respectively. Twenty sterilized seeds were placed into Petri dishes lined with a single Whatman® No. 10 filter paper dampened with the relevant treatments solution and then incubated under the identified optimal alternating temperature and photoperiod regime of 25ÊC/ 15ÊC (light/dark, 12h/12h). For the burial depth treatment, 20 seeds were placed into plastic containers (10cm in diameter and 6cm in depth) and buried to the relevant depth in sterilized soil. All trials were monitored for 30 days and germination was indicated by 5mm exposure of the radicle and emergence was indicated by the exposure of the cotyledon. Each treatment had three replicates for each population, and each treatment was repeated to give a total of six replicates per treatment, per population. Nassella trichotoma was identified to be non-photoblastic, with germination (%) being similar under alternating light and dark and complete darkness conditions. With an increase of osmotic potential and salinity, a significant decline in germination was observed. There was no effect of pH on germination. Exposure to a radiant heat of 120ÊC for 9 minutes resulted in the lowest germination in the Ingliston population (33%) and the Gnarwarre population (60%). In the burial depth treatment, the Ingliston population and the Gnarwarre population had highest emergence of 75% and 80%, respectively at a depth of 1cm. Variation between the two populations was observed for the burial depth treatments; Gnarwarre had greater emergence than Ingliston
Data Availability Statement: All relevant data are
in the paper and its Supporting Information files.
Funding: The authors received no specific funding
for this work.
Competing interests: The authors have declared
that no competing interests exist.
from the 4cm burial depth, while Ingliston had greater emergence at the soil surface than
Gnarwarre. The Gnarwarre population had greater overall germination than Ingliston, which
could be attributed to the greater seed mass (0.86mg compared to 0.76mg, respectively).
This study identifies that spatial variations in N. trichotoma's seed ecology are present
between spatially distinct populations.
The ability for aggressive weeds to germinate and emerge vigorously allows them to dominate
and displace desirable species. Therefore, an understanding of seed ecology is essential for
developing effective management programs for problematic weed species. Weeds are usually
most susceptible to control methods, making control strategies targeted to early life stages
highly effective [
]. Abiotic factors such as drought, light, salinity, seed burial depth, soil pH,
and temperature as well as disturbance events such as a fire, flooding or tillage can play an
important role in initiating or inhibiting seed germination [3±4]. Therefore, in order to
develop smarter and more effective control strategies for aggressive weeds like Nassella
trichotoma (Nees) Hack. ex Arechav., a comprehensive study into the requirements for their
successful germination, seedling emergence and subsequent establishment should be investigated [
]. By identifying the parameters which positively or negatively influence seed germination
and seedling vigour, suitable management strategies can be developed to reduce the successful
establishment of seedlings and deplete the soil stored seedbank .
High reproductive output is a key trait of successful weeds. A high density of seeds in the
soil seedbank can give a weed a competitive advantage over crops or native plant species,
particularly if the weed species is faster growing than desirable species. Dense seedbanks can
cause persistent management challenges. Nassella trichotoma of the Poaceae family is
problematic weed can produce over 140,000 seeds per plant on an annual basis, allowing it to quickly
dominate the soil seedbank [9±10]. It has been identified that between 74% to 91% of N.
trichotoma seeds will germinate within their first six to twelve months, with some seeds remaining
dormant in the soil for up to three years before losing their viability [
]. Dormancy is an
internal feature of a seed that prevents germination, even when environmental conditions are
]. Once a seed has initiated the germination process it, it cannot be stopped.
Therefore, to ensure the best chance of successful growth and survival, dormancy break is
strongly linked with specific environmental cues. Understanding dormancy patterns for
invasive weeds has important implications for their management [
]. Many weed species,
including N. trichotoma undergo a brief period of non-deep physiological dormancy [
type of dormancy is strongly associated with seasonality, particularly alternating temperatures.
Non-deep physiological dormancy is caused by a physiological mechanism within the seeds
embryo that requires specific stimulation, before the radical will emerge [
]. This trait allows
N. trichotoma to avoid germinating in summer after seed drop and allows it to wait for more
suitable wetter and cooler conditions [
Studies have shown that light and alternating temperature regimes have been identified as
two of the most important environmental factors in triggering seed germination [
4, 15, 16
Photochromes within an imbibed seed allow identification of the intensity of competition
within its environment [17±18]. The ability to detect competition prior to germination may
improve seedling survival rates [
]. Ratios of far-red to red light are higher in environments
with intense competition, as the more favourable red light is absorbed by established plants,
2 / 25
therefore less red light reaches the soil surface [
]. In an environment where competition is
low, red light will be detected in higher ratios than far-red light by the seed, promoting the
germination process [
]. Seeds which germinate under completely dark conditions may have an
abundance of far-red phytochromes within their embryonic tissue, helping them to identify
intense competition [
]. Researchers have identified that light can promote significantly
higher germination in many plants species including Halocnemum strobilaceum ,
Leptochloa chinensis [
], Carduus nutans [
], and Echinochloa colona [
]. By identifying if a weed
is positive photoblastic, light restrictive management strategies such as mulching using crop
residue [24±25] or developing dense perennial competition [
] can be introduced for effective
control. Despite the implications of light sensitivity on successful recruitment, there are also
many plants that exhibit light independent germination [
]. Light independent germination
is closely linked to other environmental triggers, particularly temperature [
breaks dormancy by altering seed physiology and has been observed to influence the rate and
percentage of germination, although this effect varies greatly by species [
]. For example,
optimal alternating temperature regimes were found to break the dormancy of, and hence
significantly increase germination in Moehringia trinervia seeds, in contrast to this, it had
minimal effect on Stellaria nemorum . Therefore, while temperature is an important trigger for
breaking seed dormancy in some species, like M. trinervia, different environmental factors
such as rainfall and soil type can also play an important role in triggering the germination
Seeds buried deeper into the soil profile often have lower success rates of emergence and
establishment due to the amount of energy required to reach the soil surface. The size of a seed
may determine the depth from which it emerges; large seeds may have greater energy reserves,
allowing emergence from greater depths than smaller seeds. The effect of seed size on
emergence was observed in four species of Amaranthus, with the lightest species (Amaranthus
spinosus) having significantly shallower optimal burial depth compared to the denser species
(Amaranthus viridis) [
]. Germination of photoblastic seeds decrease with an increase in
burial depth. Seed burial has been observed to significantly reduce seedling emergence in light
dependent weeds such Eclipta prostrata [
], L. chinensis [
], and Murdannia nudiflora [
Depending on the vigour of the seed, those species that germinate independent of light can
also be restricted by increased burial, as observed in the desert weed Marrubium vulgare [
By identifying the burial depth from which weed seedlings cannot emerge, recommendations
in tillage depths for control can be proposed.
Seed germination can be linked to other environmental factors. Low moisture availability
can prolong dormancy as soil moisture levels may be insufficient for imbibition and
competitive emergence of seedlings [
]. This may prove problematic for N. trichotoma as mass
germination events have been strongly linked to periods of heavy rainfall [
]. In saline
environments, the salt ions in the soil can reverse the natural osmotic flow of moisture into the
dry seed and rather force water out of the seed, preventing imbibition. Increased salinity has
been observed to significantly reduce seed germination in many weed species including
Cardaria draba  and Eragrostis plana [
]. However, it generally does not affect their viability
when these seeds were alleviated from the salinity stress, and normal germination was
observed. It is common for weeds to tolerate a wide range of soil pH levels [
4, 28, 33, 34
is a key trait of an invasive generalist species. However, by identifying if particular soil factors,
such as pH and salinity enhance germination, regions at risk will be easier to identify.
Fire can also play an important role in breaking seed dormancy and triggering germination
events. Fire can remove established competition, allowing for greater light penetration to the
soil surface, which can trigger germination in light sensitive seeds. The reduced competition
allows for higher nutrient and moisture availability for seedling establishment. As weeds are
3 / 25
generally good, fast-growing coloniser species, they can have a competitive advantage over
desirable species. Due to fire being an important ecological management tool, it is important
to understand how weeds like N. trichotoma respond to burn temperatures and durations. Fire
may be a useful tool to decrease the seedbank if seed viability or establishment can be reduced
. On the contrary, fire can also act as a germination trigger, as observed for N. trichotoma,
and utilized to promote a flush of seed germination from the seedbank before herbicide
10, 32, 33, 36, 37
Understanding how these environmental cues influence the germination of weeds may not
be sufficient alone for developing wide scale strategic management plans. Weeds are
considered to be pioneer species, which contributes to their wide dispersal and fast adaptability to a
variety of ecosystems. The selective pressures exerted by these different ecosystems can,
overtime, lead to in local adaptions between geographically distinct populations [
]. This can
result in one species responding differently to the same environmental cues based on the
selective pressures acting on the population. Germination rates varied significantly between two
spatially distinct populations of Poa annua in response to photoperiod, temperature and the
fungicide, fenarimol . Differences in temperature tolerance were observed in different
populations of the widespread crop weeds Galinsoga quadriradiata and G. parviflora, with
optimal germination under controlled conditions reflecting that of the given populations
Nassella trichotoma has adapted to a range of managed and natural ecosystems across the
world, which may have resulted in spatially distinct populations exhibiting some variability in
their seed ecology [
]. Phenotypic variations have been observed in the size and height of
Australian populations with Victorian populations being notably smaller than those in New
South Wales and Tasmanian [
]. In Victoria, some populations have been identified to
exhibit resistance to flupropanate herbicide, requiring four times the recommended dose,
which can be harmful to native plants and therefore reducing competition [
identifying any local adaptions, more specialised, ecosystem-specific management practices can be
The objective of this study was to identify how the environmental factors of light,
temperature, heat, salinity, drought, soil pH, and seed burial influence germination and seedling
emergence of two N. trichotoma populations.
Seed collection and storage
Mature N. trichotoma seeds were collected from over 100 plants from two populations in
Victoria, Australia; Ingliston (37O 40' 4.44'' S, 144O 18' 39.24'' E) and Gnarwarre (38 O 9' 8.892'' S,
144 O 7' 38.784'' E) during February 2017 and December 2016, respectively. Seeds were
collected on private properties, and both landholders gave us a permission to collect seeds. Given
that is a weed species and seeds were used for research purpose no further permission or
approval required. These two populations are separated by approximately 100 km. The seeds
were placed in labelled plastic, zip-lock bags and transported to Federation University
Australia's seed ecology lab. Seeds were stored within labelled plastic zip-lock bags at room
temperature until the trials began in March 2017.
The Ingliston site is located on a privately owned eucalypt bushland within a valley, which
offers the vegetation some protection from harsh winds. Aside from the established trees, this
site was heavily infested with almost a monoculture of N. trichotoma. A soil analysis was
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conducted to identify pH and salinity. The soil pH of 4.5 was identified by using a Manutec
soil pH test kit. The soil salinity was measured using the 1:5 soil:water ratio methods described
by Slavich & Patterson [
], and was identified to be 3.8dS/m, which is considered to be
slightly saline [
]. Ingliston receives its highest rainfall throughout August (49ml), and the
average temperature ranges from a maximum of 25ÊC in summer to a minimum of 3ÊC during
the winter (Fig 1A) [
The Gnarwarre site is located on a privately owned pastoral field for grazing sheep. The site
is located on an open hill, with little shelter from the elements. Pastural grasses provide intense
competition for this population of N. trichotoma, resulting in the population size being smaller
than that at Ingliston. The same soil analysis techniques used for the Ingliston site were applied
to Gnarwarre, and identified the soil to have a pH of 6 and soil salinity of 4.3dS/m, which is
considered to be moderately saline [
]. Gnarwarre receives its highest rainfall throughout
August (49ml), and the average temperature ranges from 26ÊC in summer to 6ÊC during the
winter (Fig 1B) [
Seeds were assumed to be viable when they had a plump appearance and a soft ªclinkº was
heard when the seed was dropped into the petri dish. All the trials had three replicates with 20
randomly selected seeds in each, which were repeated to give a total of six replicates (120
seeds) per treatment. All seeds were sterilized using 1% sodium hypochlorite for 5 minutes
and then were thoroughly rinsed with sterilized reverse osmosis (RO) water. All trials had 20
sterilized seeds placed into each plastic Petri dish lined with a single layer of sterilized
Whatman1 No. 10 filter paper and then moistened with 10ml of the relevant solution. The Petri
dishes were wrapped with a strip of parafilm to maintain moisture, and germination was
counted weekly for 30 days. Germination was determined when approximately 2mm of the
radicle was visible and the cotyledon had emerged from the seed coat [
]. At the conclusion
of the treatments, any un-germinated seeds were tested for their viability using
2,3,5-triphenyltetrazolium chloride (TTC) test [
The effect of photoperiod and alternating temperature
Determination of the photoperiod and temperature range that generates the highest
germination percentage for N. trichotoma is essential for the success of all subsequent experiments.
Replicates were placed into one of four incubation cabinets (Thermoline Scientific and
Humidity Cabinet, TRISLH-495-1-SD, Vol. 240, Australia) fitted with cool-white fluorescent
lamps that provided a photosynthetic photon flux of 40μmol m-2 s-1 set at various temperature
regimes: 17/7, 25/15, 30/20 and 40/30ÊC, each alternating 12 hours light and 12 hours dark. To
prevent excessive water loss, the dishes exposed to the light and dark treatments had a strip of
parafilm wrapped around the outside of each Petri dish, and the 24-hour dark replicates were
covered in a double layer of aluminum foil, which also blocked out light. To ensure
appropriate conditions for the 24-hour dark treatment, seeds were not subjected to any white light,
which was assured by the practice of examining Petri dishes containing these seeds under a
green safe light. The dishes exposed to the 40/30ÊC treatments also had an additional strip of
cling wrap placed over the parafilm as an added precaution, as the parafilm was observed to
melt at this temperature regime.
The effect of drought
To identify the effects of drought on germination, polyethylene glycol 80001 (PEG,
Aldrich Co., 3050, Spruce St., MO 63103 in sterilized distilled water) was dissolved into
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Fig 1. a: The average monthly rainfall and maximum and minimum temperature collected from the closest weather stations with relevant and recent data to the
Ingliston site. The rainfall data was collected from the Pykes Creek station (37Ê36'40"S, 144Ê18'0"E) located approximately 10 km from the Ingliston site, and the data
was averaged from November 1956 to August 2017, and the temperature data was collected from the Ballarat Aerodrome station (37Ê30'46"S, 143Ê47'28"E) located
approximately 50 km from the Ingliston site, with the data averaged from January 1908 to August 2017. The information was sourced from the Bureau of Meteorology
. b: The average monthly rainfall and maximum and minimum temperature collected from the closest weather stations with relevant and recent data to the
Gnarwarre site. The rainfall data was collected from the Gnarwarre station (38Ê8'37"S, 144Ê11'14"E) located approximately 5 km from the Gnarwarre site, and the data
6 / 25
was averaged from October 1996 to August 2017, and the temperature data was collected from the Geelong Racecourse station (38Ê10'25"S, 144Ê22'35"E) located
approximately 25 km from the Gnarwarre site, with the data averaged from June 2011 to August 2017. The information was sourced from the Bureau of Meteorology
sterilized RO water to make aqueous osmotic potential solutions of 0 (sterilized RO water for
the control), -0.1, -0.2, -0.4, -0.6, -0.8, and -1.0MPa. To make 500ml of each solutions for the
average temperature of 20ÊC, PEG was weighed out using an electric scale and added to a flask
containing 500ml of RO water and stirred automatically until dissolved. The solutions were
placed into a labelled bottle wrapped in aluminium foil and stored in a fridge until use. The
concentrations used for each solution was 46.8, 66.175, 93.575, 114.6, 132.35, and 147.95g to
make the -0.1, -0.2, -0.4, -0.6, -0.8, and -1MPa solutions, respectively. The filter papers were
dampened with 10ml of the relevant solution, and the dishes were incubated under alternating
temperatures of 25/15ÊC, 12 hours light and 12 hours dark.
The effect of salinity
The effect of salinity on N. trichotoma germination was examined by using sodium chloride
(NaCl) solutions of 0 (sterilized RO water for the control), 25, 50, 100, 150, 200, and 250mM.
This range of NaCl concentrations was selected to reflect the level of salinity occurring in
typical Australian disturbed soil [
]. Approximately 10ml of the relevant saline solution was used
to dampen the filter paper, and the petri dishes were incubated under alternating temperatures
of 25/15ÊC, 12 hours light and 12 hours dark.
The effect of seed burial
To test the impact of seed burial on germination and subsequent seedling emergence, the
seeds were placed at depths of 0 (surface), 1, 2, 3 and 4cm in sterilized soil. Soil was collected
from the Ingliston site (37O 40' 4.44'' S, 144O 18' 39.24'' E) and sterilized in an autoclave at
Federation University (Victoria), to kill other seeds and propagules. Soil was sieved using a 2cm
sieve and stored in a sealed 100L plastic tub until use. Round plastic containers 10cm in
diameter and 6cm in depth were prepared by drilling small holes into the bottom of each to allow the
percolation of water into the soil. Each container had a single layer of cleaning cloth placed at
the base prior to being filled with soil and burying the seeds. The trials were placed into large
white trays (28cm x 44cm x 5.5cm), which were lined with two sheets of cleaning cloth. The
trays were initially filled with 500ml of RO water, and this amount was added on every
alternating day. The trials were housed in the incubation cabinets under alternating temperatures
of 25/15ÊC, 12 hours light and 12 hours dark. Seedling emergence was monitored on
alternating days. Emergence was indicated by the cotyledon protruding from the soils surface.
Seed longevity under field conditions
In order to determine the effect of burial depth on seed viability under field conditions, 120
viable seeds from the Ingliston population were randomly selected and placed into a 5cm X
5cm semi-permeable bag made of 0.5mm aluminium mesh that allowed for the natural flow of
water and micro pathogens, while keeping the seeds contained. A total of 24 bags containing
120 seeds each were made in total and they were sealed using a hot glue gun. The mesh bags
were then buried at a randomly selected site within the location of seed collection at Ingliston,
Victoria (37O 40' 4.44'' S, 144 O 18' 39.24'' E). The bags were buried at depths of 0 (surface), 1,
2 and 4cm. One bag from each depth was collected each month and returned to Federation
University Australia, seed ecology lab where germinated seeds were counted and removed
7 / 25
from the mesh bag. The remaining seeds had excess dirt removed using tap water and up to 20
seeds were plated into Petri dishes lined with a single layer of sterilized Whatman1 No. 10
filter paper and then moistened with 10ml of sterilized RO water. The Petri dishes were placed
into an incubation cabinet at alternating temperatures of 25/15ÊC, 12 hours light and 12 hours
The effect of heat shock
The effect of heat on seed germination and viability was examined by exposing the seeds to
five temperatures; 40, 60, 80, 100, and 120ÊC. Furthermore, at each temperature, seeds were
exposed to the heat for three durations; 3, 6 or 9 minutes. Seeds were placed circular into
aluminum trays (8cm diameter and 3cm depth) and then placed into a digital oven (Memmert,
Type No. ULE500) at the relevant temperature for the required duration. Once removed, they
were immediately plated on plastic Petri dish lined with a single layer of sterilized Whatman1
No. 10 filter paper and then moistened with sterilized RO water and placed into an incubation
cabinet under alternating temperatures of 25/15ÊC, 12 hours light and 12 hours dark.
The effect of pH
The effect of pH on seed germination was determined by dampening the filter papers with
relevant buffer solutions ranging from pH 4 through to pH 10, prepared according to the method
described by Chachalis and Reddy [
]. Potassium hydrogen phthalate was adjusted to pH 4
using 1 N of hydrogen chloride (HCl). The buffer solutions of pH 5 and 6 were prepared by
altering 2mM of MES [2-(N-morpholino) ethanesulfonic acid] with 1 N of sodium hydroxide
(NaOH). To make the buffer solutions of pH 7 and 8, 2mM of HEPES [N-(2-hydroxymethyl)
piperazine±N±(2- ethane sulfonic acid)] was adjusted using 1 N of NaOH. The buffer solutions
of pH 9 and 10 were created by adjusting a 2mM solution of Tricine [N-Tris (hydroxymethyl)
methyl glycine] with 1 N of NaOH. The dishes were incubated at an alternating light and
temperature regime of 25/15ÊC, 12 hours light and 12 hours dark.
The final germination percentage (FG%) was calculated dividing the sum of germinated seeds
(SG) by the total number (TS) of seeds placed into each Petri dish:
G% y0 a
The average germination percentage (G%) and standard error was calculated for each
treatment, and these values for all the treatments except for the rate of germination data, were
entered into the statistical software SigmaPlot 13 (Systat Software, Inc., Point Richmond, CA,
USA) for analysis. The rate of germination was analysed using Microsoft Excel. The effect of
drought on germination percentage was fitted with a polynomial linear model:
-where, G% is the averaged germination (%) at the osmotic potential of x and a indicates the
The effect of salinity on germination percentage was fitted with a three-parameter sigmoid
G% a= 1 e
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-where, G% is the total germination (%) at the NaCl concentration of x and b indicates the
slope, a is the maximum emergence (%) and x0 is defined as the concentration for 50%
inhibition of the maximum germination (%) as a result of the treatment.
The effect of burial depth on seedling emergence was fitted with a three-parameter peak
-where, E% indicates the emergence (%), a is the maximum emergence (%), b indicates the
slope, and x0 is defined as the concentration for 50% inhibition of the maximum germination
(%) as a result of the treatment.
A two-way ANOVA was generated for each treatment by using a general linear model on
with the statistical program Minitab.
Results and discussion
The effect of photoperiod and alternating temperature on germination (%)
Both N. trichotoma populations had the highest germination (%) at the alternating
temperature of 25/15ÊC (Fig 2A and 2B). Under the alternating photoperiod of 12 hours light and 12
hours dark at this temperature, the Ingliston population had 82.5% germination and
Gnarwarre had 90.8%, and similar counts were obtained in complete darkness with 80% and 92.5%
germination for the Ingliston and Gnarwarre populations, respectively. Both populations also
demonstrated high germination (%) under the alternating temperature of 17/7ÊC under both
alternating light and dark (75% and 75.8% for Ingliston and Gnarwarre, respectively) and
complete darkness photoperiods (74.16% and 77.5% for Ingliston and Gnarwarre,
respectively). For both populations, germination (%) was significantly reduced under the alternating
temperature of 40/30ÊC (p = 0.000), with a total germination (%) for the Ingliston population
of 34.2% (alternating) and 9.2% (complete darkness), and was even further reduced for the
Gnarwarre population, which had a germination (%) of only 6.7% (alternating) and 0.8%
(complete darkness). At the alternating temperature of 30/20ÊC, the 12 hours light and 12
hours dark photoperiod significantly enhanced the germination (%) in the Gnarwarre
population (p = 0.002), with a total germination of 80.8% for the alternating photoperiod and only
60.8% under complete darkness. At this temperature, germination (%) was significantly
reduced compared to the 17/7ÊC and 25/15ÊC treatments within the Ingliston population,
having a total germination (%) of only 34.1% for both photoperiods. The germination (%) for
Ingliston in the 30/20ÊC alternating photoperiod treatment was also lower compared to the
Gnarwarre population at the same light and temperature regime (p = 0.028). Temperature was
a more influential factor on germination (%) than photoperiod (Table 1). The high R-squared
value of 89% shows that the results obtained are strongly associated with the treatment
The effect of photoperiod and alternating temperature on rate of germination
Germination rate was steady, with both populations taking two weeks before 50% germination
or higher was observed (Fig 3A and 3B). For the Ingliston population, 53.3% germination was
observed for both the 17/7ÊC and 25/15ÊC temperature regimes under complete darkness after
two weeks of incubation, which was higher than the germination (%) observed for the
alternating photoperiod at the same temperature, being only 36.6% and 25.8%, respectively. A similar
result was also observed in the Gnarwarre population for the 25/15ÊC temperature regime
9 / 25
Fig 2. a: The effect of alternating temperature and photoperiod regimes on the germination (%) of Nassella trichotoma seeds for the Ingliston and b:
Gnarwarre populations after incubation in a growth chamber for 30 days. Vertical bars represent standard error of the mean.
under both photoperiod treatments, with 55.8% and 64.2% germination being observed for
the alternating and complete darkness photoperiods respectively. Unlike the Ingliston
population, Gnarwarre had a lower germination (%) at the 17/7ÊC temperature treatment, with only
29.2% germination observed under alternating and 5.8% germination in complete darkness
after two weeks of incubation. However, at the two-week mark, the Gnarwarre population had
50.8% germination in the 30/20ÊC alternating photoperiod treatment, which was much higher
than the 23.3% germination observed in the Ingliston population at the same point in time.
These results show that for the Ingliston population, the temperatures of 17/7ÊC and 25/15ÊC
under complete darkness favours more rapid germination rates, while the Gnarwarre
population demonstrated a faster germination rate under the temperature regimes of 25/15ÊC and
30/20ÊC, with light being an irrelevant factor. After three-weeks of incubation, the germination
(%) rate declined for all the tested treatments.
For many plants, light plays an important role in allowing a seed to gauge its position within
the soil profile, identify existing competition, and detect any soil disturbance events [
]. For N.
trichotoma, light was not observed to be an important factor for regulating germination when
alternating temperatures were favourable. Photoperiod as a singular factor was only significant
in the 30/20ÊC alternating temperature for the Gnarwarre population with only 60% of the
seeds germinating compared with 80% in the alternating light and dark trials. Germination in
complete darkness indicates that N. trichotoma is non-photoblastic; rather, other
environmental factors may be more closely linked with breaking its dormancy [
1, 16, 52
]. The germinated
seedlings from the complete darkness treatment exhibited etiolated growth, while those
seedlings from the alternating photoperiod treatment were observed to be larger and a vibrant
green colour. Light also had little influence on the rate of germination, with both tested
photoperiods producing similar weekly germination yields. Germination was highest in both
populations at the alternating temperatures of 17/7ÊC (approximately 75% for Ingliston and 76%
for Gnarwarre) and 25/15ÊC (81% for Ingliston and 91% for Gnarwarre). The in situ average
maximum temperature of the two populations is approximately 25ÊC and 15ÊC, respectively,
in the spring and summer months, and 15ÊC and 5ÊC, respectively, throughout winter, which
is fitting with this optimal temperature result [
10 / 25
Temperature X Population
Temperature X Photoperiod
Population X Photoperiod
Temperature X Population X Photoperiod
pH X Population
NaCl X Population
Duration of Exposure
Temperature X Duration of Exposure
Temperature X Population
Duration of Exposure X Population
Temperature X Duration of Exposure X Population
Depth X Population
PEG Concentration X Population
There was a significant difference between the two populations when exposed to the higher
two alternating temperature regimes. The Ingliston population experience significantly higher
germination at the 40/30ÊC regime than that of the Gnarwarre population. In the 30/20ÊC and
the 40/30ÊC alternating photoperiod treatments, germination was reduced to a similar level
for the Ingliston population, indicating that if moisture levels are adequate, approximately
34% of this population's seeds will still germinate at these unfavourable temperatures. The
Gnarwarre population experienced an exponential reduction in the 40/30ÊC treatment, with
only 6% of the seeds germinating under alternating light and dark conditions, and no seeds
germinating under complete darkness at this temperature. The Gnarwarre population had
significantly higher germination in the 30/20ÊC treatment (80% in alternating light and 60% in
complete darkness) compared to the Ingliston population. The average seasonal temperatures
are slightly warmer across all seasons at Gnarwarre compared to Ingliston, which may have
contributed to this population's higher optimal germination temperatures [
]. These results
observed subtle variations in germination response to alternating temperature regimes, and it
is possible that these differences could be stronger between more spatially distinct populations.
11 / 25
Fig 3. a The germination (%) for the Ingliston and b: Gnarwarre populations of Nassella trichotoma seeds tallied at the end of
every week from day 0 for the alternating temperature and photoperiod regime treatment.
12 / 25
Effect of drought
For the drought treatment, germination was highest in the control for both populations with
the Ingliston population having 70% germination and Gnarwarre having 93.3% germination
(Fig 4). There was little variation in germination (%) for the osmotic potential of 0.1MPa with
Ingliston having 65.8% germination and Gnarwarre having 92.5% germination. Exposure of
the seeds to an osmotic potential of 0.2MPa resulted in a decline in germination within each
population, as compared to 0.1MPa, with the germination (%) in the Ingliston population
declining to 46.6% and 75.8% for the Gnarwarre population. Both populations had
significantly reduced germination at the osmotic potential of 0.4MPa and above, with zero
germination being observed from this concentration onwards (p = 0.000). The Gnarwarre population
had significantly higher germination compared to the Ingliston population in the control,
0.1MPa and 0.2Mpa treatments (p = 0.000), suggesting that the Gnarwarre population was
able to germinate better under the effect of drought than the Ingliston population (p = 0.000)
(Table 1). The r-squared value of 96% demonstrates that the effect of this osmotic potential
treatment strongly inhibited N. trichotoma’s seed germination at concentrations of 0.4MPa
Effect of salinity
For the salinity treatment, the highest germination (%) for the Ingliston population of 64.1%
was obtained in the 25mM treatment, and the highest germination (%) for the Gnarwarre
population of 85% was obtained in the control treatment (Fig 5). Significantly higher germination
in the Gnarwarre population compared to the Ingliston population in the control and 25mM
treatments (p = 0.001), and this was independent of the salinity treatment (p = 0.031)
(Table 1). A NaCl concentration of 150mM significantly reduced N. trichotoma’s germination
in both populations (p = 0.000), with only 9.1% germination being observed for the Ingliston
population and 10% for the Gnarwarre. The model suggests that the Gnarwarre population
could tolerate up to 71.63mM of the NaCl solution before germination was inhibited by 50%,
while the Ingliston population germination was reduced to 50% with a NaCl concentration of
55.99mM. Germination continued to decline as the concentration of NaCl increased, and zero
germination was observed in both populations in the 250mM treatment. The r-squared value
of 89% confirms that the salinity treatment was the main factor reducing seed germination
Drought and salinity are environmental factors that impose osmotic stress on seeds,
preventing the natural flow of water into the seed from its surrounding environment. Under
osmotically stressful conditions, seeds may be unable to achieve the critical moisture levels
required for imbibition, and therefore unable to prepare for germination. The results of this
study demonstrated that water availability was a highly influential factor for triggering N.
trichotoma seed germination. In the drought treatments, both populations demonstrated
reasonable germination rates under the osmotic stress of -0.2MPa (46.6% for Ingliston and 75.8% for
Gnarwarre), but doubling this stress to -0.4MPa completely inhibited germination in both
populations. The effect of osmotic stress had a similar effect on C. nutans, where germination
was observed to be somewhat unaffected by osmotic stress between 0 and 0.2MPa, but was
almost completely inhibited by -0.4MPa [
]. A tetrazolium test identified a high proportion
of the un-germinated N. trichotoma seeds were still viable at the conclusion of the trials,
indicating that these seeds may germinate if osmotic conditions became favourable [
The Gnarwarre population had a higher tolerance to osmotic stress than the Ingliston
population. At an osmotic potential of -0.2MPa, Gnarwarre had significantly higher germination
than that of the Ingliston population. The home ranges of the two populations have substantial
13 / 25
Fig 4. The effect of osmotic potential (-MPa) on the germination (%) of Nassella trichotoma for Ingliston (white dot) and Gnarwarre
(black dot) after incubation in a growth chamber at 25/15ÊC 12 hours light/12 hours dark for 30 days. The line for Ingliston represents a
linear polynomial model fitted to the data with the equation G% = 77.671+83.09 X. The osmotic potential for 50% inhibition of maximum
germination for Ingliston is estimated as -0.15MPa. The same model was fitted to the Gnarwarre data with the equation G% = 108.67
+24.43 X. The osmotic potential for 50% inhibition of maximum germination for Gnarwarre is estimated as -0.24MPa. The vertical bars
represent standard error of the mean.
variations in the volume of rainfall, with Ingliston having an average yearly rainfall of 654mm,
while the average for Gnarwarre is only 437mm, in addition to this the pattern of rainfall for
Ingliston is higher than Gnarwarre across all seasons, particularly in the autumn months
which is when N. trichotoma’s non-deep dormancy begins to break [
36, 46, 53
]. In addition to
this, the average maximum temperature is lower at Ingliston compared to Gnarwarre,
meaning this site is likely to have higher soil moisture conditions and exert less osmotic stress on
seeds and mature seed producing plants. The Gnarwarre population is subjected to lower
rainfall and warmer maximum temperatures, therefore the osmotic pressures of this environment
is selecting for those plants that are more tolerant of dryer conditions compared to Ingliston.
The results suggest that the different osmotic selective pressures of these two environments
have resulted in variations in the seeds sensitivity to osmotic stress.
Salinity exerts a similar osmotic stress as drought, however as a result of the increased ion
concentrations, saline conditions can have a more profound inhibiting effect on seed
4, 54, 55
]. While salinity often has a dormancy inducing effect on seeds, some salt
tolerant species, like Vicia faba [
], Atriplex lentiformis [
] and Juncas ranarius [
] have been
observed to germinate under higher salinity stress, however the rate and vigour of germination
14 / 25
Fig 5. The effect of NaCl (mM) on the average germination (%) of Nassella trichotoma for Ingliston (black dot) and Gnarwarre
(white dot) after incubation at 25/15ÊC 12 hours light/12 hours dark for 30 days. The Ingliston population was fitted with a
threeparameter sigmoid model with the equation G% = 62.45/(1+e(-x-109.6/27.22). The Gnarwarre population was also fitted with a
threeparameter sigmoid model with the equation G% = 86.02/(1+e(-x-98.6/29.79). Germination was reduced to 50% at a NaCl concentration of
109.6mM for the Ingliston population and 98.6mM for the Gnarwarre population. The vertical bars represent standard error of the mean.
is considerably reduced. In addition to this, salinity can reduce a seedlings ability to take up
nutrients, such as potassium ions, and accumulate higher proportions of sodium and chloride
ions, reducing the seedlings growth potential [
]. The germination inhibiting effect of
increasing salinity concentrations was similar in both populations. The Gnarwarre population
proved to have greater germination (%) than the Ingliston population, particularly in the
control and 25mM treatments. The 100mM treatment reduced germination to 47% in the
Gnarwarre population and to 39% in the Ingliston population, which was significantly lower than
the control treatments. Germination was reduced to 9.2% and 10% for Ingliston and
Gnarwarre respectively in the 150mM treatment, and the 200mM treatment reduced germination
of the Ingliston population to only 4.2%, and no germination occurred for the Gnarwarre
population at this concentration. No further germination occurred beyond 150mM indicating that
high salinity concentrations have an inhibiting effect on the germination volume of N.
trichotoma seeds. The Gnarwarre collection site had moderately soil salinity (4.3dS m-1) compared
the Ingliston site's soil being only slightly saline soil (3.8dS m-1), therefore the greater
germination observed in the Gnarwarre population could be attributed to this environmental
selective pressure. Similar responses to salinity have been observed in other noxious weeds,
including Amaranthus spinosus , Croton setigerus [
], and Emex australis [
15 / 25
inhibiting effect of salinity on seed germination can explain why mature N. trichotoma plants
are rarely seen growing in saline affected regions of Australia, and the small proportion of the
population that do germinate in these regions are outcompeted with more salt tolerant plants
14, 19, 36, 53
Effect of burial depth on seedling emergence
The burial depth treatment obtained the highest emergence (%)at the 1cm burial depth
treatment for both populations, with the Ingliston population having 75% seedling emergence and
the Gnarwarre population having 80% emergence (Fig 6). The Ingliston population had the
same proportion of seedlings emerge at the 2cm burial treatment. A variation was observed
between the two populations in the surface treatment and the 4cm burial treatment
(p = 0.045). In the surface treatments, the Ingliston population had an emergence (%) of 51.6%
compared to only 30.8% for the Gnarwarre population. Contrastingly, in the 4cm burial
treatment, Gnarwarre had 50.8% emergence, while Ingliston had only 20.8%. Despite an
identifiable bell-curve response to the effect of seed burial in both populations, the r-squared value of
only 49% suggests that other factors may have also been influencing the results of this
Effect of burial depth on seed germination and viability under field conditions
The results of the seed germination under field conditions treatment, shows that N. trichotoma
demonstrated similar germination (%) at 1, 2 and 4cm burial depth under field conditions (Fig
7). The germination (%) observed at these depths were significantly higher than the
germination on the soil surface (0cm) (p = 0.000). The seeds viability remained consistent throughout
the six-month collection span, which suggests that seeds have the ability to remain viable
under field conditions for at least 170 days (Fig 8). Burial of 1cm or deeper appeared to have a
protective effect on the seeds, as these seeds had higher viability compared to those exposed to
The effect of burial depth influenced the emergence (%) of seedlings slightly differently
between the two populations. The depth of 1cm was optimal for seedling emergence with 75%
and 80% germination for Ingliston and Gnarwarre respectively, with the Ingliston population
also having the same proportion of seeds germinate at a burial depth of 2cm. A burial depth of
4cm had significantly different proportion of emergence between the two populations, with
Gnarwarre having 50% emergence at this depth, while Ingliston's emergence was reduced to
only 20%. As it was identified in the photoperiod treatment, N. trichotoma does not require
light for germination, therefore it is likely that the significant difference is related to seedling
vigour. Lighter seed weight was observed to increase sensitivity to burial depth interspecifically
amongst 13 different annual species collected from a Spanish grassland [
variations in the seed size was observed in two spatially distinct populations of Caucalis platycarpos
] and Ambrosia artemisiifolia [
] as a response to different environmental pressures,
allowing the population with larger, denser seeds to have higher emergence from greater burial
]. A similar variation in the seed density was observed between the two N.
trichotoma populations studied, which may have influenced the difference in emergence at the 4cm
depth. The average individual seed weight of the Gnarwarre seeds were heavier (0.86mg) than
the Ingliston seeds (0.76mg), which could explain the significant difference in emergence at
this depth. It was identified in the artificial aging under field conditions treatment that
germination of more than 20% does occur at a burial of 4cm in the Ingliston population, in fact, an
16 / 25
Fig 6. The effect of seed burial (cm) on the average seedling emergence (%) of Nassella trichotoma for Ingliston (black dot) and Gnarwarre
(white dot) after incubation at 25/15ÊC 12 hours light/12 hours dark for 30 days. The Ingliston population was fitted with a three-parameter peak
Gaussian model with the equation E(%) = 79.53 e(-0.5 X-1.49/1.59)2. The Gnarwarre population was also fitted with a three-parameter sigmoid
model with the equation E(%) = 77.68 e(-0.5 X-2.15/1.88)2. Maximum emergence occurred at a burial of 1.49cm for Ingliston and 2.15cm for
Gnarwarre. The vertical bars represent standard error of the mean.
average of 65% of the seeds germinated at this depth. Therefore, the lighter density of the
Ingliston population seeds is the likely factor decreasing emergence.
Nassella trichotoma seeds experienced a significant reduction in germination and seedling
emergence at the surface treatments compared to 1cm burial in both populations, with
Ingliston being reduced to 50% and Gnarwarre to 30% germination. Under field conditions, only
one seed germinated on the soil surface across the 6 months tested, and the total viability
results indicated that surface conditions reduce seed viability compared to a burial of 1cm or
greater. This is somewhat uncommon in species that germinate well in alternating light and
dark regimes, as surface conditions have been identified to be favourable for optimal
germination in a magnitude of weed species inclusive of: Chromolaena odorata [
], Galinsoga quadriradiata and Galinsoga parviflora [
]. Germination and emergence
of the noxious grass weed E. colona, was significantly reduced from 97% at the soil surface to
12% with a burial depth of only 0.5cm [
]. A possible reason for the reduced germination in
the surface burial treatment may be related to a defensive response of the seeds far-red
phytochromes, as these play an important role in identifying the optimal time for germination by
17 / 25
Fig 7. The effect of seed burial (cm) under field conditions on seed germination (%) for Nassella trichotoma. Each month 120
seeds were collected from each depth and this graph shows the proportion (%) of seeds that had germinated within the field.
Fig 8. The effect of seed burial (cm) under field conditions on total seed viability (%) for Nassella trichotoma collected from
Ingliston. Each month 120 seeds were collected from each depth and then incubated for up to 30 days and then had a TTC viability
test conducted on the seeds. This graph shows the total number (%) of seeds that had germinated within the field, within the
incubation period, and responded positively to the viability test.
18 / 25
not only sensing the intensity of competition, but also excessive light associated with soil surface
]. This mechanism is known as high irradiance response sensitivity and it protects
the seed from germinating under intense sunlight as these factors can indicate harsh and
unfavourable temperatures and dry conditions [
]. As it was identified in the photoperiod trials, N.
trichotoma is non-photoblastic and can germinate well with alternating light and dark
conditions and in complete darkness, furthermore the results of the drought treatment highlighted
that N. trichotoma germination is highly dependent on ample water availability. The yearly
average solar exposure for Ingliston and Gnarwarre is 15.1 MJ/m2 and 15.2 MJ/m2 respectively,
which indicates that these sites experience predominantly overcast conditions, and tolerating
full sunlight would not be a selective pressure of these environments [
]. Throughout the
burial depth experiment, it was observed that the surface conditions experienced loss of soil
moisture quicker that the soil layers just below, despite regular watering. Therefore, it is likely
that in addition to the far-red phytochromes preventing germination under full sunlight,
difference in soil moisture between the surface and 1cm burial treatments also may have influenced
the significant difference observed in germination and emergence at these depths.
Effect of exposure to radiant heat under increasing time durations
Pre-exposure to radiant heat had a somewhat positive influence on germination (%) of both N.
trichotoma populations (Fig 9A and 9B). Exposure to the 120ÊC treatment reduced Ingliston's,
germination (%) to 35.8%, 37.5% and 33.3% for the 3, 6 and 9 minutes durations, respectively.
This reduction was significantly lower than the 40ÊC treatments for this population
(p = 0.000). None of the temperatures or exposure durations resulted in germination (%) of
less than 50% for Gnarwarre, and this population experienced significantly higher germination
than the Ingliston population at all tested treatments (p = 0.000). The lowest germination for
the Gnarwarre population of 60% was obtained when seeds were exposed to 120ÊC for 9
The Gnarwarre population responded positively to radiant heat, particularly in the 40, 80
and 100ÊC treatments, which produced higher germination (%) than the optimal photoperiod
and temperature treatment. For this population, germination was only reduced to 60% when
exposed to 120ÊC for nine minutes. The Ingliston population showed greater sensitivity to
radiant heat, with no heat treatments producing better germination than the optimal
photoperiod and temperature regimes. Germination was reduced to approximately 50% in the 60 and
80ÊC treatments, and to only 35% in the 120ÊC treatments. Despite this, germination
proportions between 76 and 50% in newly burnt areas could still give the Ingliston population a
decent competitive advantage. There was no significant difference to germination for either
population as a result of the duration of heat exposure. These results highlight the importance
for integrating fire management with weed management, as fire has been observed to enhance
weed invasion, particularly in areas with poor nutrient availability such as roadsides [
was observed to enhance the rate and volume of germination in the invasive pastoral grass
Hyparrhenia rufa, despite it killing most of the established population [
]. The seeds of N.
trichotoma are fire tolerant to temperatures of at least 120ÊC and germination and seedling
recruitment is enhanced by heat. Furthermore, the reduced competition associated with
burning will likely promote the recruitment of this opportunistic weed.
Effect of pH on germination and variations in germination
Tthe range of pH levels tested did not have a significant effect on the germination (%) within
either population (Fig 10A and 10B). The germination (%) was significantly higher in the
Gnarwarre population compared to the Ingliston population across all the tested treatments
19 / 25
Fig 9. a: The effect of exposing Nassella trichotoma seeds to radiant heat (OC) at increasing time durations (minutes) on
germination (%) of for the Ingliston, b: Gnarwarre populations after incubation in a growth chamber at an alternating
temperature of 25/15ÊC 12 hours light/12 hours dark for 30 days. Vertical bars represent standard error of the mean.
(p = 0.000), however this was not linked to the pH level (p = 0.244). The r-squared value of
36.6% suggests that the pH treatment was not the dominant factor influencing these
20 / 25
Fig 10. a: The effect of pH on the germination (%) of Nassella trichotoma seeds collected from Ingliston and Gnarwarre,
b: after incubation in a growth chamber at an alternating temperature of 25/15ÊC 12 hours light/12 hours dark for 30
days. Vertical bars represent standard error of the mean.
21 / 25
differences. Despite this variation, both populations responded with a similar trend to the
range of pH levels treated.
This study highlighted that N. trichotoma does not have a significant preference for a
particular pH level, and both populations were able to germinate well across the tested range of pH 4
to 10. Despite both populations being collected from sites with acidic soils, the lower pH levels
tested were not favoured any more than the higher levels, suggesting that soil pH is not an
active selective pressure on either population. The generalist attributes of most weeds allows
them to take advantage of a wide range of soil types as this allows them to exploit a magnitude
of environments, including disturbed and degraded regions. The minor effect of pH levels on
successful weed seed germination has also been observed in Amaranthus retroflexus [
Galenia pubescens [
] and Nicotiana glauca [
]. The Gnarwarre population had higher
germination than the Ingliston populations at all pH levels, however the r-squared value of 36.6%
indicates that this is unlikely to be a result of the pH treatment. Overall, the Gnarwarre
population had higher seed viability than the Ingliston population. The variation observed in weight
could account for the difference in the total proportion of germination. The Gnarwarre seeds
were heavier and denser than the seeds collected from Ingliston, and greater seed density has
been observed to promote higher germination yields [
The results of this study highlight that N. trichotoma seeds are non-photoblastic, and
dormancy break can be triggered by favourable of alternating temperatures of approximately 25/
15ÊC and ample water availability. Radiant heat was also observed to have a positive effect on
total germination yields. Under osmotic stress and salinity, germination was significantly
reduced, and water appeared to be the most important limiting factor on germination. Seeds
are able to germinate when buried to a depth of at least 4cm, and seedling emergence can
occur at this depth, although the success of emergence appears to be linked to seed weight,
with the denser Gnarwarre seeds having higher emergence than the lighter Ingliston seeds at
this depth. Germination was not enhanced or inhibited by pH level, suggesting that soil pH is
not a limiting factor on this species recruitment.
These findings suggest that light reducing management techniques will be unsuccessful for
preventing germination. Tilling the seeds to a depth of at least 4cm may reduce the emergence of
seedlings, and because the seeds still germinate when buried, this may quickly reduce the
seedbank. The effect of seed burial on emergence should be further explored by investigating the
effect of greater seed burial depths under controlled and field conditions so that better
recommendations can be made for using tillage as a control method. Land managers should look for
N. trichotoma recruitment after good rainfall events and suitable temperature regimes,
particularly after fire treatments. By understanding the climatic conditions that significantly enhance
recruitment, management techniques can be applied accordingly to maximise their productivity.
This study observed variations in the seed ecology between the two populations of N.
trichotoma, and it is likely that greater variations would be observed between populations with
greater differences in selective pressures. It would be beneficial to observe the spatial variations
between populations across different states of Australia, or even internationally in order to
develop a more thorough understanding of this species seed ecology so that management
recommendations can be made confidently across wide geographical gradients.
22 / 25
Conceptualization: Singarayer K. Florentine.
Data curation: Talia Humphries.
Formal analysis: Singarayer K. Florentine.
Methodology: Talia Humphries, Singarayer K. Florentine.
Resources: Singarayer K. Florentine.
Supervision: Bhagirath S. Chauhan, Singarayer K. Florentine.
Writing ± original draft: Talia Humphries.
Writing ± review & editing: Bhagirath S. Chauhan, Singarayer K. Florentine.
23 / 25
24 / 25
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