Materials and Methods
This study was conducted from April to July 2019 under greenhouse conditions (had a temperature of 25±5oC with 60% relative humidity) to simulate the natural condition. The greenhouse is located at Jiangsu University, Zhenjiang, China (32.20°N, 119.45°E).Wedelia trilobata and Wedelia chinensis were chosen in this study. Wedelia trilobata (WT) is an annual invasive plant species in China, while Wedelia chinensis(WC) is its congener native species, belonging to theAsteraceae family. WT is a clonal evergreen creeping herb that was also found in the southern region of China in the 1970s on a large scale (Qi et al., 2014). In China, initially, it was introduced as a groundcover species but later it spread rapidly from gardens to roadsides, and then to the agricultural fields, and nowadays, it is found near riverside as well (Song et al., 2010, Talukdar and Talukdar, 2013). WC is the native congener of WT, and is mostly used as a medicinal plant. The growth rate of WCis very slow as compared to WT(Dai et al., 2016a, Talukdar and Mukherjee, 2008). WT in China was mostly found in the arid and semi-arid regions. Some of the population of WT was also found near the Yangzi River in Jiangsu province of China, which probably indicates that water fluctuation and nutrient enrichment made WT successful in these environments. It prefers growing in nutrient and moisture-rich soils (Dai et al., 2016a). Ramets of WT and WC were collected from the same study site of Jiangsu University for the experiment. Ramets of WT and WC were growing in the seedling tray with sand as the growth medium. These trays were placed in a greenhouse. The ramets were irrigated with distilled water every day, while nutrition was provided weekly through Hoagland solution. When ramets had two fully expanded leaves, these were transferred to plastic pots (height = 10 cm, diameter = 13 cm) containing sand as a growing-medium. The ramets in the pots were placed in the greenhouse for one week to let them adapt to the greenhouse condition. The two plant species were left growing under two cultures, i.e. mono and mixed cultures. In monoculture, one plant of each species grew; in mixed culture one plant of WT and WC grew together. Subsequently, the treatments were implemented as three levels of nitrogen (control = 0.043 g, denoted as CK; additional = 0.130 g, denoted as N and double additional = 0.261 g, denoted as 2N) and two levels of water (normal water = 0.450 L/week and flooding = 0.9 L/week) (Fig. 1). Nitrogen treatments prepared according to (Wan et al., 2018), comprised of equal proportions of KNO3 and NH4Cl, and water treatment was made according to (Rahlao et al., 2010). Nitrogen treatments were renewed once a week. The required water amount for water treatments were given three times a week. Two environmental factors were subjected to all pots after transplanting based on factorial design: water (normal or flooding), nitrogen (control, additional, or double additional), and three cultures (two monocultures and one mixed culture), with five replicates. According to this experimental design, there were 90 pots in total (3 nitrogen levels x 2 water levels x 3 cultures x 5 replicates).
Growth and physiological traits measurement
Two months after the treatment, i.e. in the month of July, leaf chlorophyll content (CHI) and leaf nitrogen of both species were measured with portable chlorophyll meter, SPAD; Oakoch OK-Y104, China. Leaf area was measured with ImageJ software every plant with five replicates. Plant height of every plant with five replicates was measured with a measuring scale. The dry weight of above ground (leaf and stem) and below ground (root) biomass of each individual was measured separately, after oven drying to constant weight at 72oC for 48 hours (Parepa et al., 2019).