Germination biology of Hibiscus tridactylites in Australia and the implications for weed management

Hibiscus tridactylites is a problematic broadleaf weed in many crops in Australia; however, very limited information is available on seed germination biology of Australian populations. Experiments were conducted to evaluate the effect of environmental factors on germination and emergence of H. tridactylites. Germination was stimulated by seed scarification, suggesting the inhibition of germination in this species is mainly due to the hard seed coat. Germination was not affected by light conditions, suggesting that seeds of this species are not photoblastic. Germination was higher at alternating day/night temperatures of 30/20 °C (74%) and 35/25 °C (69%) than at 25/15 °C (63%). Moderate salinity and water stress did not inhibit germination of H. tridactylites. Seedling emergence of H. tridactylites was highest (57%) for the seeds buried at a 2 cm depth in the soil; 18% of seedlings emerged from seeds buried at 8 cm but no seedlings emerged below this depth. Soil inversion by tillage to bury weed seeds below their maximum depth of emergence could serve an important tool for managing H. tridactylites.

Scientific RepoRts | 6:26006 | DOI: 10.1038/srep26006 be useful, the germination requirements of H. tridactylites in Australia may be different from those of H. trionum in Greece.
A study was conducted to determine the effects of scarification, temperature and light, salt and osmotic stress, and burial depth on seed germination and seedling emergence of H. tridactylites. It was hypothesized that: a) the germination percentage of H. tridactylites seeds will increase with scarification, b) the germination percentage of H. tridactylites seeds will decrease linearly with increasing salt and water stress, and c) the percent emergence of H. tridactylites seedlings will decrease linearly with increasing seed burial depth in soil.

Results and Discussion
Seed scarification. As hypothesized, chemical scarification with sulfuric acid (H 2 SO 4 ) released the seeds from dormancy and stimulated germination. Seed germination increased with the time of H 2 SO 4 scarification up to 20 minutes and decreased after that (Fig. 1). Only 13% seeds germinated without scarification. The fitted model suggested 20 minutes of scarification with H 2 SO 4 was required to achieve maximum germination (80%). Therefore, in the subsequent experiments, seeds were scarified with H 2 SO 4 for 20 minutes.
The previous study in Greece suggested that the optimum acid soaking time to soften the hard H. trionum seeds without reducing germination was 30 minutes; however, the authors did not show the data 3 . The seeds of H. tridactylites released from dormancy by chemical scarification, suggesting that this species has hard-seeded/ physical dormancy. Some other weeds that can break coat-imposed dormancy by scarification are Malva parviflora L., Mimosa invisa Mart. ex Colla, Corchorus olitorius L., and Melochia concatenata L 12,15,16 .
In a previous study in Queensland, Australia, 38% of H. trionum seeds remained viable at 0-2 cm depth after 2 years 17 . In another study in Turkey, seeds of H. trionum persisted in the soil longer with 23% seeds remaining viable after 7 years 7 . The results of this study suggest that germination of H. tridactylites seeds will remain very low in the field unless scarified. Under natural environments, extreme changes in temperature and moisture conditions, soil acids, fire (e.g., window burning practiced in Australia), damage by predators (insects, rodents, microorganisms, etc.), and passage through the digestive system of animals help in seed scarification 9,18 . No-till farming systems leave most of the weed seeds on the soil surface after seed rain and crop planting, where they may experience fluctuating temperature and moisture regimes. Viability is usually lost more rapidly for seeds present on the soil surface through germination and mortality than when buried in the soil 19 . In addition, weed seeds are most vulnerable to seed predators when present on the soil surface 9,20,21 . These observations suggest that H. tridactylites seeds can persist for a long time when buried in the soil.
Similar germination responses to light and darkness in H. tridactylites indicate that seeds of this species are not photoblastic, and therefore, these seeds may germinate even when buried in the soil or after canopy closure in a crop. These results also suggest that farming practices involving no-till or mulch systems will have no influence in terms of light exposure affecting germination of H. tridactylites 16 . Previously, light-independent germination has been reported in several species (e.g., C. olitorius and M. invisa) who also have a hard seed coat 15,16 .
Temperature affects seed germination and governs seasonality and range expansions 16 . Seeds of H. tridactylites germinated over the tested range of temperatures, suggesting that seeds of this species can germinate throughout the spring, summer, and autumn seasons in Queensland. These results are consistent with the suggestions provided in a previous study that this species has successive seedling flushes after rainfall or irrigation events throughout spring, summer, and autumn 1 . The previous study using the Greece population also reported highest Salt stress. In contrast to the proposed hypothesis, a sigmoid response (rather than a linear response) was observed in the germination of H. tridactylites seeds in response to salt concentrations. Maximum germination (77%) was obtained in no-salt conditions (Fig. 2). Germination was greater than 60% up to the concentration of 150 mM sodium chloride (NaCl), 15% germination occurred at 250 mM NaCl, although germination was completely inhibited at 300 mM NaCl. The concentration required for 50% inhibition of maximum germination was 193 mM NaCl.
These results suggest that H. tridactylites seeds can germinate in high saline conditions, which could be a key feature of this species enabling it to colonize saline areas. Salinity is an important abiotic stress to crop production worldwide, including Australia. Soils with more than 100 mM NaCl are considered to have high salt contents. In Queensland alone, the area of saline land was 107,000 ha in 2002 22 . Crop production may be affected by soil salinity as well as weed competition. Similar to H. tridactylites, seeds of M. invisa (another hard-seeded weed species) germinated at very high salt concentrations 15 . Water stress. A sigmoid response was observed in the germination of H. tridactylites seeds with decreased in osmotic potential from 0 to − 0.8 MPa (Fig. 3). However, it was hypothesized that a linear decline in percent germination would be observed in response to increasing water stress. Germination was greater than 74% at  The results of this experiment suggest that H. tridactylites seeds can germinate under moderate water stress conditions, which can occur temporarily between the rainfall events at the start of the summer seasons in the northern region of Australia. The ability to germinate under moderate water stress conditions could enable H. tridactylites to gain an advantage due to earlier seedling emergence. Similar results were reported in the previous study from Greece and the authors suggested that H. trionum germination and establishment would not be halted in poorly drained or mostly dry soil conditions 3 .

Seed burial depth.
Seedling emergence of H. tridactylites was greatly influenced by seed burial depth. The proposed hypothesis was that the percent emergence of H. tridactylites seedlings would decrease linearly with increasing seed burial depth in soil. However, this response was not observed. Only 28% of seeds produced seedlings when planted on the soil surface (Fig. 4). Seedling emergence increased with shallow burial at depths of 1 and 2 cm but declined progressively as depth increased. The fitted model predicted a maximum of 57% emergence at a burial depth of 2 cm. Up to 18% of seedlings emerged beyond that depth. Similar results were reported for the Greece population in the previous study 3 . In that study, seedling emergence of H. trionum was higher for seeds buried at 2-cm than those placed on the soil surface (54 vs 38%germination).
Seedling emergence on the soil surface was lower than the emergence from the seeds buried at 1 and 2 cm depths. As light is not inhibitory for seed germination in H. tridactylites, reduced seedling emergence on the soil surface was probably due to limited soil-seed contact and, consequently, poor seed imbibitions 23 . Low or no emergence from deeply buried seeds could be due to fatal or no germination. Previous studies also suggested limited energy reserves to support hypocotyl elongation from deeper depths 9,24 .
The results of this experiment suggest that deep tillage operations, that bury the weed seeds below 8 cm depth, would suppress emergence of H. tridactylites seedlings. Subsequent tillage operations, however, should be shallow to avoid the possibility of bringing back the seeds on the soil surface.

Conclusions
Scarification stimulated germination of H. tridactylites, indicating that the hard seed coat is the primary reason for the inhibition of germination. In natural conditions, scarification usually occurs more in seeds present on or near the soil surface than in deeply buried seeds. These observations indicate that the seeds of H. tridactylites can persist for a long time when buried deep in the soil. Light did not influence seed germination, suggesting that H. tridactylites seeds are not photoblastic. It also indicates that no-till systems will not make any difference in term of light exposure. Seed germination of H. tridactylites was moderately tolerant to salt and water stress. Seedling emergence was optimal at shallow burial depths. In case seeds are concentrated in the top soil layer, a deep tillage operation that buries seeds below 8 cm depth would be appropriate. Conclusions drawn from the results of this study should be limited to the weed population sampled because weed populations often vary in their germination requirements. Seed-germination behavior may also differ among seeds produced in different seasons and years. Therefore, conclusions are most relevant to the northern Australian weed population tested.  Queensland, Gatton, Queensland. Seeds from these plants were collected in May 2015 for use in the study. Seeds were cleaned, placed in plastic containers, and stored in a laboratory until used in the experiments. Experiments were conducted from May to October 2015.

Material and Methods
Germination test. Seed germination of H. tridactylites was evaluated by placing 25 seeds in a 9-cm-diameter Petri dish containing two layers of filter papers. These filter papers were moistened with 5 ml of distilled water or a treatment solution. Seeds used in the experiments were scarified with concentrated sulfuric acid (H 2 SO 4 ) for 20 minutes, unless otherwise specified. Petri dishes were placed in transparent plastic bags (to reduce moisture loss from the dishes) and incubated at fluctuating day/night temperatures of 30/20 °C in light/dark conditions. The photoperiod in the incubator was set at 12 h to coincide with the higher temperature interval. Germination was evaluated 14 days after the start of the experiment, with the criterion for germination being visible protrusion of the radical.
Scarification. An experiment was conducted to investigate whether the seeds of H. tridactylites could be released from dormancy by seed scarification. The experiment also aimed to test how long scarification time is needed so that the information can be used for the other experiments of the study. Seeds were scarified with concentrated (98%) H 2 SO 4 at different time intervals (2,5,10,20, and 30 minutes). The seeds were washed for 5 minutes in running tap water before placing them in dishes. There was also a control treatment in which seeds were not scarified (i.e., 0 minutes with H 2 SO 4 ).

Temperature and light.
To evaluate the effect of temperature and light on germination, the chemically scarified seeds of H. tridactylites were incubated at three alternating day /night temperatures (25/15, 30/20, and 35/25 °C) in two light regimes [complete darkness (24 h) and light /dark (12 h/12 h)]. These temperature regimes were chosen to reflect the temperature variation during the spring to summer seasons in Queensland, Australia. In the complete darkness treatment, the dishes were wrapped in three layers of aluminum foil (to ensure no light penetration).

Salt stress.
To determine the effect of salinity on germination, the chemically scarified seeds of H. tridactylites were incubated in sodium chloride (NaCl) solutions of 25,50,100,150,200,250, and 300 mM. There was also a control treatment (0 mM), where distilled water was added.

Water stress.
To determine the effect of water stress (or osmotic stress) on germination, scarified seeds of H. tridactylites were incubated with osmotic potential of − 0.2, − 0.4, − 0.6, − 0.8, and − 1.0 MPa. These solutions were prepared by dissolving polyethylene glycol 8000 in distilled water, as described earlier 25 . There was also a control treatment (0 MPa), in which distilled water was added.
Seed burial depth. The effect of seed burial depth on the seedling emergence of H. tridactylites was studied in a growth chamber (set at 30/20 °C) by placing 50 chemically scarified seeds on or in soil within 15-cm diameter plastic pots. The seeds were placed on the soil surface and covered with the same soil to achieve soil burial depths of 0, 1, 2, 4, 8, and 12 cm. The soil used in this experiment was collected from a nearby field and it was red clay with a pH of 5.5. There was no background seed bank of H. tridactylites in the soil. The experiment was initially irrigated with an overhead mist sprinkler and later subirrigated. Seedling emergence of H. tridactylites was counted when the cotyledon was easily visible. The experiment was carried out until 35 d after seed burial.

Statistical analyses.
All the experiments were conducted in a randomized complete-block design with three replications. All the experiments were repeated over time (and, therefore a total of six replications) and the second run was started within a month of the termination of the first run. The data variance was visually inspected by plotting the residuals to confirm homogeneity of variance before statistical analysis. ANOVA indicated no difference between the experiments conducted at the different times and therefore, the data from the two experimental runs were combined for analysis.
The data obtained from the temperature and light experiments were separated using the least significant difference (LSD) at 5% (Gen Stat 16 th Edition, VSN International Ltd, United Kingdom), while regression analysis was used for other experiments (SigmaPlot 10.0, Systat Software, Inc; Pint Richmond, CA).
A three-parameter sigmoid model was fitted to the germination values resulting from salt and water stress experiments. The model was where, G the total germination (%) at NaCl or osmotic potential x; G max is the maximum germination; x 0 is the NaCl or osmotic potential required for 50% inhibition of maximum germination; and b is the slope of the curve. A three-parameter Gaussian model was fitted to the germination values resulting from the scarification experiment and seedling emergence values resulting from the seed burial experiment. The model was: The graph of a Gaussian is a "bell curve" shape. In the model, a is the height of the curve's peak (i.e., maximum germination or emergence); b is the position of the center of the peak (i.e.; the duration of scarification required to achieve maximum germination or the depth of seed burial to achieve maximum seedling emergence); and c is the width of the "bell".