Impact of temperature and germination time on the success of a C4 weed in a C3 crop: Amaranthus retroflexus and spring barley

Elevation in temperatures due to climate change could promote the invasion by C4 weed species of arable fields in the boreal region, which are dominated by C3 crops. The success of Amaranthus retroflexus L. (a C4 weed) in spring barley (a C3 crop) was studied at current and elevated temperatures (3 °C difference) in a greenhouse experiment in southern Finland. The competition treatments included no competition and four levels of competition with barley, differing in terms of germination time. The success of A. retroflexus was measured as growth (height and biomass) and seed production (number and biomass). Elevation in temperature enhanced seed production of A. retroflexus, but the impact on growth was minor (only difference in plant height in one treatment). The growth and seed production of A. retroflexus in competition with barley was minimal although the growth of barley decreased with the rise in temperature. The results indicate that climate change could improve growth of a C4 weed such as A. retroflexus, but it is unlikely to succeed in spring barley.


Introduction
Climate change has been predicted to broaden the distribution areas of arable weed species (Patterson 1995).A successful range expansion of arable weeds requires not only adaptation to the climatic conditions of a new region but also colonisation of cultivated fields.Therefore, crop-weed interactions must be considered in connection with range expansion of arable weeds as a consequence of global warming.
The key issue to understanding crop-weed interactions with respect to climate change lies in the different responses of C 3 and C 4 species to elevation in temperature and CO 2 concentration (Patterson and Flint 1980, Ziska 2001, 2003).C 3 plants benefit from the elevated CO 2 levels, whereas C 4 plants benefit from the elevated temperature (Pearcy et al. 1981, Sage andKubien 2007).As a result climate change will affect crop-weed interactions disproportionately depending on the photosynthetic pathway of the crop and weed species (Ziska 2001(Ziska , 2003)).C 3 and C 4 species occupy a temporal niche in natural vegetation that is determined by temperature (see Sage and Pearcy 2000).This is also evident in arable weed communities since C 4 weeds usually occur in C 4 crops (e.g.Schroeder et al. 1993, Bunce andZiska 2000), which are alike with respect to temperature requirements for germination and growth.Since the majority of the "world's worst weeds" are C 4 plants (Holm et al. 1977) and most crops are C 3 plants (Patterson 1995), global warming can be assumed to strengthen the competition from these weeds in the future (Ziska 2003, Tungate et al. 2007).Regarding range expansion, the pressure on C 4 weeds to invade northern regions, where they are currently rare, can be assumed to increase (Holm et al. 1997).This will require, however, the colonisation of C 3 crops by C 4 weeds.
One of the most widely distributed C 4 weeds in the world is Amaranthus retroflexus L., which is found in 60 crops in 70 countries (Holm et al. 1997, Costea et al. 2004).The main distribution area of A. retroflexus in Europe is in southern and central Europe, the northern border of the distribution area being in the Nordic and Baltic countries.At the margins of the distribution area, including Finland and Estonia, A. retroflexus is regularly found as a casual alien (Hämet-Ahti et al. 1998, Kukk andKull 2005), i.e. a species that cannot establish permanent populations and is dependent on the regular supply of new seeds (Richardson et al. 2000).Based on its current distribution, A. retroflexus can be regarded as one of the most probable C 4 weed species to extend its distribution into the boreal region as a consequence of climate change.Previous studies showed that elevation in temperature increases growth of A. retroflexus (Guo and Al-Khatib 2003) but decreases seed produc-tion (Weaver 1984), indicating opposing responses regarding success as a consequence of climate change.Seed production is lowered in competition with a crop (McLachlan et al. 1995).The success of A. retroflexus as a competitor with C 3 cereals has not been established experimentally, but field observations suggest it is poorer than with C 4 crops (Costea et al. 2004).
The aim of the present study was to explore the growth response and the seed production of A. retroflexus at an elevated temperature with and without competition from a C 3 crop (spring barley).The study was conducted in Finland, where the dominant C 3 crop species is spring barley and which represents the northern distribution limit of A. retroflexus.Since the timing of germination is a critical factor affecting crop-weed competition (Knezevic et al. 1997) and the optimum germination temperature for A. retroflexus is high compared with that for barley (Ghorbani et al. 1999, Guo andAl-Khatib 2003), which indicates later germination and emergence times, several germination dates for A. retroflexus were included in the study.I expected the elevation in temperature to enhance growth but decrease seed production of A. retroflexus.

Plant material
The seeds of Amaranthus retroflexus were collected from southern Sweden (55°45´ N; 13°19´ E) in September 2004.In summer 2008, the seeds were sown in pots and grown both outdoors and in a greenhouse in Jokioinen, southern Finland (60°49' N; 23°29' E) in order to produce seeds for the experiment.The seeds were stored at +4 °C but kept at -18 °C for two days in October 2008.The seeds of spring barley, Hordeum vulgare L., cultivar Rolfi, were obtained from a local agricultural market.
The plants for each treatment (see below) were grown in 3.5 l pots (diameter 18 cm) in a commercial peat-sand mixture ('Kylvöseos' by Kekkilä Oyj, Mellilä, Finland; pH 6.0, N: 70 mg l -1 , P: 21 mg l -1 , K: 140 mg l -1 ) and fine sand (grain size 0.2-1 mm) at a ratio of 2:1.The pots were watered regularly and fertilised (a mixture of 2.5 kg fertilization [N-P-K content 14-5-21 %, respectively] with 25 litres water mixed with irrigation water in a proportion of 1:50) 7 times during the experiment (total amount of fertiliser-water mixture per pot was 2.7 l).
The sowing density of barley was 12 seeds per pot sown in two rows (row width 7 cm), the same sowing density as in field cropping, and one seed or seedling of A. retroflexus placed in the middle of the pot.The seeds of A. retroflexus were either sown in the pot (treatments 1 and 2) or planted as 1-2 cm long seedlings (treatments 3-5).The seedlings were germinated on Petri dishes in a growth chamber at a temperature regime of 35/10 °C (16/8 h) with full light.If a seedling of A. retroflexus died within two weeks after the sowing date it was replaced by a new seedling of the same age grown in the same greenhouse.Seventy-seven (32.1% of all plants in the experiment) such replacements were made during the experiment.At the end of the experiment, 12 A. retroflexus individuals were dead of which 9 were replacement seedlings.Two dead barley plants were also replaced with new seedlings after 10 days from the beginning of the experiment.

Experimental design
The greenhouse experiment was conducted in Jokioinen between 10 th February and 8 th May 2009.The experiment was conducted in four greenhouses of which two had a temperature regime of 20/10 °C (18/6 h) (hereafter, current) and the other two 23/13 °C (18/6 h) (hereafter, elevated).The temperature regimes were randomized among greenhouses.The temperature regime with lower values was in line with the long-term  average minimum and maximum temperatures for the summer months (June-August) recorded at Jokioinen meteorological station (Drebs et al. 2002).The 3 degree elevation in temperature conforms to the predicted median temperature value for summer months (June-August) for the time period 2040-2069 according to four climate scenarios (Jylhä et al. 2004).The light/dark regime was 19/5 h for both temperature regimes, equivalent to the day length for Jokioinen in June.
The experiment included the following treatments: 1) A. retroflexus seedling planted on the 1 st day of the experiment, no barley, 2) A. retroflexus seedling planted and barley sown on the 1 st day of the experiment, 3) A. retroflexus seedling planted on the 7 th and barley sown on the 1 st day of the experiment, 4) A. retroflexus seedling planted on the 14 th and barley sown on the 1 st day of the experiment, 5) A. retroflexus seedling planted on the 21 st and barley sown on the 1 st day of the experiment and 6) Barley sown on the 1 st day of the experiment, no A. retroflexus.Treatments 1-5 were placed on the same table as a row column design with three replicates (10 cm between pots).Four such tables were placed in each greenhouse (n = 12 for each treatment per greenhouse).The distance between the tables was about 1 metre.Barley control (treatment 6) consisted of two pots placed in close proximity to each table (n = 8 per greenhouse).

Measurements and data analyses
At the end of the experiment, plant height, above ground biomass (air-flow drying at 40 °C) and number and weight of seeds of A. retroflexus were recorded.The height of each barley plant and the above ground biomass of 12 barley plants per pot were also measured.Prior to analyses, the heights of barley plants were averaged and the data were pooled for treatments 1-5.The averages for barley controls were calculated for above ground biomass and height.
The data were analysed using linear mixed models.The treatment and greenhouse were fixed factors and the interaction of greenhouse and table a random factor in the models used for the analyses of all barley data sets and the height of A. retroflexus.However, for the data sets of seed number and weight, as well as for the biomass of A. retroflexus, comparisons within treatment 1 only were possible due to missing data (seeds) or highly skewed data distributions (biomass).In these analyses, the greenhouse was a fixed factor and the interaction of greenhouse and table a random factor.In both analyses, the paired comparisons between temperatures within treatment or between treatments within temperature were made using two-sided t-tests.All the A. retroflexus data were square root-transformed and the height and biomass data for barley (treatments 1-5) log (x+1) transformed prior to analyses.The statistical analyses were performed using the MIXED procedure in SAS (Littell et al. 2006).

Results
Seed production of A. retroflexus was affected both by competition with barley and temperature.The impact of competition was so strong that only 9 A. retroflexus plants in treatment 2, 2 plants in treatment 3 and a single plant in treatments 4 and 5 produced seeds.The majority of these cases were in the greenhouses at an elevated temperature: only one plant produced seeds in competition with barley at the current temperature.Due to the scattered data for seed production, the impact of competition and the interaction of competition and temperature could not been verified using statistical testing.In the comparison of treatment 1 (i.e.no competition with barley) at different temperatures, a greater number (1655 ± 896 vs. 5465 ± 2711 seeds per plant, t = 4.3, df = 12, p < 0 .001)and weight (0.7 ± 0.4 vs. 2.1 ± 1 g per plant, t = 4.2, df = 12, p < 0.01) of seeds were associated with an elevated rather than a current temperature (Fig. 1).

Discussion
Contrary to expectations, elevation in the temperature enhanced seed production and had a minor impact on the growth of the C 4 weed A. retroflexus.
The growth and seed production of A. retroflexus in competition with a C 3 crop, spring barley, was minimal although barley growth was decreased by the elevation in temperature.The results indicate that climate change could improve the success of A. retroflexus populations but success in spring barley is unlikely.
Previous studies showed C 4 weeds to respond positively to elevation in temperature (Guo and Al-Khatib 2003).However, the temperature optimum for photosynthesis of C 4 plants is typically high (Sage and Kubien 2007); for A. retroflexus over 35 °C (Pearcy et al. 1981).This suggests that the maximum temperature used in the present study (23 °C) was too low to give an advantage to A. retroflexus in terms of photosynthetic rate.Interestingly, the 3 degree elevation in temperature used in the present study was high enough to limit the growth of barley, in accordance with earlier findings (Peltonen-Sainio et al. 2010, 2011).This suggests a decline in the competitive ability of spring cereals against weeds, in a future climate.For C 4 weeds this would mean potential improvement in the success in the crop rotations typical of boreal regions.The results of the present study, however, suggest that even these changes will not promote the success of A. retroflexus in competition with spring cereals in the boreal region.
Previous competition experiments (e.g.Knezevic et al. 1997) showed considerable yield losses due to competition by A. retroflexus.In the present study, yield loss was not evident but A. retroflexus was unable to effect any decline in the biomass of barley either.Previous studies included crops other than spring cereals as study species.Under field conditions A. retroflexus is more common in such crops than in cereals (e.g.Costea et al. 2004), which suggests the growth patterns to be more alike with these species.One of the factors related to growth patterns is the germination temperature which determines the emergence patterns of seedlings.In the present study, the germination temperature (35 °C) was high compared with the day temperatures used during growth (20 or 23 °C).Under field conditions, A. retroflexus would have germinated later than barley.Therefore, the treatment used in the study benefited A. retroflexus.However, the seedlings of A. retroflexus were germinated on Petri dishes and the seedlings planted in the pots to ensure the same germination date for each individual within treatments.This procedure appeared not to be successful since one third of the seedlings had to be replaced with new seedlings during the experiment.This likely contributed to the poor success of A. retroflexus in competition with barley.
Even though A. retroflexus did not affect decrease in barley biomass, seed production showed a positive response to elevation in temperature.The finding was contradictory to previous findings by Weaver (1984), who reported an increase in seed production when the temperature regime was changed from 28/22 °C to 22/14 °C.The latter temperature regime is comparable with the elevated temperature regime used in this study.Therefore, the results are not entirely contradictory.The seed production level recorded in the present study was lower compared with those reported earlier (Weaver 1984, Costea et al. 2004).The increase in seed production at the elevated temperature suggests an improvement in the success of establishment of permanent populations.The finding suggests that some individual plants could produce seeds even in competition with barley.However, the building up of the populations, which would effect considerable yield losses, would require a crop rotation including a crop species less competitive against A. retroflexus (e.g.sugar beet).Even though the present study focused only on seed production, neglecting many components of the population demography (e.g.Morse and Bazzaz 1994), the suggestion is that A. retroflexus could survive under these conditions.Range expansion of C 4 weeds into the boreal region of Europe as a consequence of global warming is of concern.Several C 4 weed species comprise an important part of the weed community in central Europe, but C 4 weed species typically occur in C 4 crops (Schroeder et al. 1993).The results of the present study suggest that A. retroflexus is unlikely to take hold in spring cereals.This is confirmed by the field observations: none of the 188 weed species found in a Finnish weed survey (Salonen et al., 2001) was a C 4 species.In addition, none of the C 4 species belong to the most important weeds of C 3 crops in Europe (Schroeder et al. 1993).The results suggest that even though the 3 degree elevation in average temperature decreased the growth of a C 3 crop and improved the seed production of a C 4 weed, C 3 crop species could act as a barrier to invasion by the "world's worst weeds" into the boreal region.Furthermore, climate warming will advance the sowing times of C 3 crops (Kaukoranta and Hakala 2008), thus reinforcing the competitive benefit of C 3 crops in spring time.The invasion of C 4 weeds cannot be expected to take place before the introduction of C 4 crops (e.g.maize), which has been predicted to occur not before the end of this century in Finland (Peltonen-Sainio et al. 2009).

Fig. 1 .
Fig.1.The number and biomass of Amaranthus retroflexus seeds per plant (mean and SD) at the two temperature regimes.

Fig. 2 .
Fig.2.The height and biomass of Amaranthus retroflexus and barley individuals (mean and SD).In T1, no barley included.The treatments T2-T5 differed in terms of sowing date of A. retroflexus.