10.7251/AGRENG1607255

DO BUR CUCUMBER POPULATIONS EXHIBIT DIFFERENCES IN SEED DORMANCY?

Cumali OZASLAN1*, Sonnur TAD2, Huseyin ONEN2, Shahid FAROOQ2 1Department of Protection, Dicle University, Diyarbakir, Turkey 2Department of Plant Protection, Gaziosmanpasa University, Tokat, Turkey *Corresponding author: [email protected]

Abstract Bur cucumber (Sicyos angulatus L., SIYAN) is a troublesome invasive vine. Seeds of the plant possess a water-impermeable, hard seed coat exhibiting physical dormancy. Seed dormancy is employed by the invasive for persistence in soil seed bank, seedling recruitment and avoiding unfavorable environmental conditions. The seed dormancy differences among populations offer valuable insights for understanding biology and devising effective management practices against invasive plants through experimental studies. Therefore this study was carried out to evaluate the dormancy differences among 3 populations collected from Black Sea region of Turkey. Chemical scarification with H2SO4, wet and dry pre-chilling (for 2 weeks) and mechanical scarification (girdling with scalpel) were tested along with an untreated control for comparison. Seeds were dipped in concentrated (98%) H2SO4 solution for 1, 2, 3, 4 and 6 minutes for chemical scarification. Whereas, a small portion of the seed coat was removed with the help of scalpel in mechanical scarification. Experiments were incubated at 25 ºC with 12 hours light and dark period for 30 days and repeated. Freshly harvested seeds exhibited higher dormancy with slight differences among populations. Chemical scarification and pre-chilling treatments were ineffective in overcoming the dormancy. Mechanical scarification significantly promoted the final germination percentage with slight differences (82-90%) among populations. The results of the mechanical scarification were consistent in both experimental runs. It is concluded that slight scarification of SIYAN seeds imparts water-permeability in seeds leading to higher germination. The persistence of the seeds in soil seed banks probably leads to the deterioration of seed coat till next growing season resulting in effective seedling recruitment. Mechanical scarification can be successfully employed to break the dormancy in SIYAN for experimental studies.

Keywords: Sicyos angulatus, Populations, Physical dormancy, Mechanical scarification, Chemical scarification.

Introduction Biological invasions are considered as the most serious threat to native biodiversity throughout the world (Lee et al., 2015). Among invasive plant species, vines are more dangerous as they readily get established in introduced ranges, spread and replace native biodiversity by blocking sunlight and competing for resources. Sicyos angulatus L. (Bur cucumber, SIYAN) is an annual vine, native to North-eastern USA and currently invades several regions of the world (Kurukowa et al., 2009; Onen et al., 2015a, b; Lee et al., 2015;). It has been regarded as invasive plant in Europe as well and its range is continuously expanding (Hulina, 1996). SIYAN interferes with several crops, such as maize, soybean, hazelnut, and pumpkin etc. (Hulina, 1996; Shimizu, 1999; Esbenshade et al., 2001; Watanabe et al., 2002; Larché, 2004; Gibson et al., 2005; Onen et al., 2015a, b). SIYAN is also commonly observed along watercourses, forest sites and in other open spaces in the natural habitats where it suppresses native vegetation by completely covering the ground and

1682 blocking sunlight (Watanabe et al., 2002; Onen et al., 2015a, b). The presence of the plant alongside rivers and streams restricts water flow and creates several problems for stream managers (Lee et al., 2015). SIYAN is currently infesting considerable portion of Black Sea region in Turkey and exerted several negative effects on the native plant communities (Onen et al., 2015a, b). Different invasive plant species and/or even different populations of the same species successfully manipulate seed dormancy to persist and germinate under diverse range of environmental conditions. Dormancy influences the survival, colonization, adaptation, speciation and extinction both at population and species levels (Willis et al., 2014). However, different life forms of plants exhibit different extent and type of seed dormancy (Willis et al., 2014). The type and degree of dormancy strongly affects the germination especially in newly colonizing populations (Huang et al., 2010). Therefore, seed dormancy is considered as the major determinant of species distribution as seeds first need to get germinated for the evolution of adaptive traits (Donohue et al., 2010). Environmentally cued seed dormancy through evolving specialized structures is known as physical dormancy. Physical dormancy has been documented in nine orders and 17 families of angiosperms (Baskin and Baskin, 2003; Baskin et al., 2006). Cucurbitaceae is one of the families containing species which exhibit physical dormancy via developing water- impermeable seed coat. To date physical dormancy has been reported only in the two plants of Sicyos in the Cucurbitaceae family i.e., Sicyos deppei (Orozco-Segovia et al., 2000) and Sicyos angulatus (Lim et al., 1994). The existence of physical dormancy in SIYAN seeds and role of mechanical scarification in overcoming it is well documented (Qu et al., 2010). However, seed dormancy differences among different populations of SIYAN have been less explored although, higher genetic diversity has been reported for the plant (Kurokawa et al., 2009; Dlugosch et al., 2015). SIYAN can grow under different shade and water availability conditions indicating the presence of higher genetic diversity (Oh et al., 2015). Therefore seed dormancy differences among different populations may help to infer the mechanisms of introduction and further spread (Kurokawa et al., 2009; Dlugosch et al., 2015). As described earlier, seed dormancy is the most initial trait expressed in the life history of plants, hence it is critical determinant of survival and colonization success (Willis et al., 2014). Therefore a thorough understanding of seed dormancy differences among different populations might help to understand population dynamics and further invasion trends. Although invasion status of SIYAN is well studied in Black Sea region of Turkey however, no literature exists on the germination biology of different populations infesting the region. The current study was therefore planned to infer the seed dormancy differences among different SIYAN populations and potential of different dormancy breaking techniques to overcome dormancy. The results of the study will help to better understand the germination biology of different populations infesting the region.

Materials and Methods Seed Collection: Sicyos angulatus have extensively infested the eastern parts of Black Sea region in Turkey and started to interfere with different agricultural crops. Seeds were collected from three different sites (Dernekpazar, Dereli and Camlihemsin counties) highly infested with the plant. Physiologically mature fruits were collected and brought to the laboratory where they were dried under shade and seeds were separated from fruit coats. Seeds were stored in glass jars until the start of experiments.

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Experimental treatments Mechanical and chemical scarification techniques along with pre-chilling were tested to evaluate their role in overcoming the dormancy of SIYAN seeds. A small proportion of the seed coat was removed with the help of a scalpel (girdling) in mechanical scarification treatment. Seeds of the tested populations were dipped in concentrated (98%) H2SO4 solution for different time intervals (1, 2, 3, 4 and 6 minutes). Seeds were then thoroughly rinsed, surface dried and used. Seeds were also stratified (pre-chilling) either wet (between moistened layers of filter paper) or dry at 4 °C for two weeks. The untreated seeds (control treatment) was also included in the study for comparison purpose.

Experimental Layout Experiments were conducted in randomized complete block design with split plot arrangements. The SIYAN populations were taken as main plots whereas dormancy breaking techniques were randomized in sub plots.

Experimental procedure Twenty five seeds were placed on the double layer of Whatman no. 1 filter paper in 5 replicates and the papers were moistened with deionized water. The moisture was supplied to the germinating seeds throughout the experimental duration according to the needs. The petri dishes were kept at 25 °C for 30 days with 12 hours light and dark period. The cool, while fluorescent lamps were used for the provision of light in the incubators and relative humidity was adjusted to 60%. The place of the petri dishes was changed every day within the incubators. Germination was observed on daily basis and germinated seeds were counted up to 30 days after the initiation of experiments. A seed with 2mm radicle prolonged was considered as “germinated”. The germinated seeds were removed from the petri dishes. Final germination percentage of the seeds was computed at the end of experiments and used in the statistical analysis.

Statistical Analysis All of the tested seed dormancy breaking techniques except dry pre-chilling and chemical scarification for lesser time (i.e., 1, 2 and 3 minutes) improved final germination percentage compared to control. Therefore the treatments resulting higher germination than control were included in statistical analysis. A two way analysis of variance was used to test the significance for populations, treatments and their all possible interactions. Least significant differences test at 5% probability was used to separate means where ANOVA indicated significant differences. The SPSS computer program version 23 was used for the statistics analyses whereas graphical representation of the data was accomplished using Microsoft Excel version 2013.

Results and Discussion The individual and interactive effects of SIYAN populations and dormancy breaking techniques proved significant in the current study (Table 1). Freshly harvested seeds of different SIYAN populations exhibited extensive dormancy (92.8 to 94.8% dormant seeds). Similarly, different seed dormancy breaking techniques significantly affected the final germination percentage of tested SIYAN populations (Table 1). Mechanical scarification with scalpel proved as the most effective in breaking dormancy. Chemical scarification with H2SO4 for 6 minutes also slightly stimulated the germination whereas chemical scarification for lesser time proved ineffective in this regard (Figure 1). Among the remaining seed dormancy breaking techniques, wet pre-chilling slightly stimulated the germination whereas dry pre- chilling proved ineffective. Therefore these treatments cannot be considered as potential

1684 techniques for breaking dormancy of SIYAN seeds. Regarding interactions among populations and seed dormancy breaking techniques, highest final germination in all populations was observed with mechanical scarification while the untreated (control) seeds result in the lowest germination (Figure 2). The population collected from Dereli exhibited slightly higher final germination compared to the other SIAYN populations studied. Seed dormancy delays or prevents germination under unfavorable conditions thus ensuring germination over the next growth season (Willis et al., 2014). It is evident from the results that SIYAN seeds exhibit physical dormancy as all the seed dormancy breaking techniques except mechanical scarification failed to promote germination of different tested populations. Physical dormancy allows the plants to colonize in novel environments by adjusting timing of germination (Donohue et al., 2005). The results of the study reveal that all of the populations undergo similar adaptations for persistence in the currently invaded region of Turkey. The development of water impermeability in SIYAN seeds soon after physiological maturity is well reported (Qu et al., 2010). Different weed populations exhibit high genetic variations among different populations or even within the same population (Dlugosch et al., 2015). Large scale genetic variations have been observed in different populations of SIYAN collected from Japan (Kurokawa et al., 2009). Therefore the difference in seed dormancy of different Turkish SIYAN populations are probably the result of genetic variations among populations.

Table 1. Two-way ANOVA different seed dormancy breaking techniques, bur cucumber populations and their interactions on final germination percentage. Source df SS MS F value P value Populations (P) 2.00 187.55 93.77 10.33 0.00* Treatments (T) 4.00 57117.65 14279.41 1572.62 0.00* P × T 8.00 197.79 24.72 2.72 0.01* Error 60.00 544.80 9.08 Total 75.00 128336.00

90 a 80 70 60 50 40 30 b 20 c c

Final germination Finalgermination (%) 10 d 0 SCA SA6 SA4 STR CTR Treatments Figure 1. Effect of different seed dormancy breaking treatments on the final germination % of different populations (average of 3 populations). Here; SCA = mechanical scarification, SA4 and SA6 = chemical scarification with H2SO4 for 4 and 6 minutes, respectively, STR = wet pre-chilling at 4 °C for 15 days, and CTR = control treatment, (LSD 5% = 2.20).

Dormancy breaking requirements are highly specific for a given taxa which indicate that dormancy is developed by complex adaptations (Donohue et al., 2010). Physical dormancy due to the presence of water-impermeable seed coat have been widely cited in different studies (Lim et al., 1994; Qu et al., 2010). Seeds of SIYAN become water-impermeable after drying followed by physiological maturity (Qu et al., 2010). Since, the water imbibition is the

1685 core requirement of the seeds for enzymatic activities during seed germination. Therefore, it was hypothesized that chemical scarification will impart the water permeability to SIYAN seeds and possibly overcome the seed dormancy. However, the results indicated that chemical scarification, even for longer time (6 minutes) almost failed to stimulate germination and hence proved ineffective for breaking dormancy. As chemical scarification slightly improved the germination with increasing exposure time, it is predicted that chemical scarification for longer time may possibly overcome dormancy.

Figure 2. Effect of different seed dormancy breaking treatments and on the final germination % of different populations (effect on individual population). Here; SCA = mechanical scarification, SA4 and SA6 = chemical scarification with H2SO4 for 4 and 6 minutes, respectively, STR = wet pre-chilling at 4 °C for 15 days, and CTR = control treatment, (LSD 5% = 3.81).

Conclusion It is concluded that SIYAN populations undergo similar adaptations to develop physical seed dormancy for persistence and colonization in novel environments. The presence of physical dormancy will help the plant to explore and invade novel environments in the country. Slight differences in the populations are owed to inherent genetic variations among populations. Secondly, the seed coat must be ruptured prior to germination which naturally happens due to longer burial time and presence of ample soil moisture in the currently invaded range of the plant in Turkey. It is recommended that proper management measures must be taken to tackle the existing populations and to prevent the further spread.

Acknowledgements The current study was funded by the Scientific and Technological Council of Turkey (TUBITAK) with a Grant Number 113 O 790 as a part of the COST Action (FA 1209 - European Information System for Alien Species).

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