Littleseed canarygrass (Phalaris minor Retz.) is widely distributed in subtropical and temperate regions in Yunnan Province, Southwest China [14,17]. Since 2013, littleseed canarygrassseeds have been collected from wheat fields in Songming County of Yunnan Province and propagated in the glasshouse of the Agricultural Environment and Resource Research Institute, Yunnan Academy of Agricultural Sciences, China. The average weight of 1000 seeds was 1.49 ± 0.05 g and the germination rate was 91.8%, as tested before the experimentation.
Rapeseed (Brassica napus L.) is a dominant oil crop in subtropical and temperate regions of Yunnan Province . Our previous studies showed that rapeseed variety Yunyou No. 2 had strong competitive ability and could be used to control littleseed canarygrass in agroecosystems . In this experiment, the seeds of Yunyou No. 2 were obtained from the Food Research Institute, Yunnan Academy of Agricultural Sciences (YAAS)
Experiment design and data collection
We conducted a field experiment from September, 2018 to April, 2019 at the Agricultural Environment and Resource Research Institute, Yunnan Academy of Agricultural Sciences, Kunming China (25°12′–25°39′ N, 102°76′–102°89′ E) in the same field as Xu et al. . This area is characterized by a subtropical and temperate monsoon climate. Rainfall averages 1000–1300 mm per year and the annual mean temperature is 14.1°C . The soil type of test site is yellow-brown, with a total N content of 1.32g·kg–1, Olsen P content of 29.5 mg·kg–1, and available K content of 86.4 mg·kg–1, respectively.
The experiment was a split-plot design with basal N fertilization rates as the whole-plot factor (four replications for each N application rate treatment), topdressing N fertilization rates and plant ratios were split-plot factors, utilizing a de Wit replacement series method . The rapeseed and littleseed canarygrass were sown on 24 September 2018 in the greenhouse. On 27 October 2018, similar-sized seedlings of both species were transplanted into 9 m2 plots (3 m × 3 m) and subjected to different N fertilization treatments. Three basal N fertilization rates (30 (BN1), 90 (BN2) and 150 (BN3) kg·ha–1) were applied in the form of urea at pre-plant (26 October 2018), and three topdressing N fertilization rates (30 (TN1), 60 (TN2) and 90 (TN3) kg·ha–1) were also applied in the form of urea at bolting stage (26 January 2019). In each plot, the amount of other fertilizers used in the whole growth period was the same, P2O5 90 kg·ha–1, K2O 120 kg·ha–1, B 1.6 kg·ha–1, respectively.
We utilized a de Wit replacement series incorporating three ratios of rapeseed and littleseed canarygrass densities and nine different N regimes in replicated 9 m2 plots. A combination of three ratios (1:0 (180:0 plants), 1:1 (90:90 plants) and 0:1 (0:180 plants)) of rapeseed and P. minor were studied at nine N fertilization rates (BN1TN1, BN1TN2, BN1TN3, BN2TN1, BN2TN2, BN2TN3, BN3TN1, BN3TN2, BN3TN3). A total of 180 plants were grown at three ratios of rapeseed andP. minor while maintaining a constant planting density of 20 plants m−2 (0.25 m × 0.20 m space) in each plot. All plants were distributed uniformly within the plot. All plots were arranged in a complete randomized block design with four replicates per ratio and per nutrient level (total n = 4 replicates × 3 ratios × 9 nutrient levels = 108). A 1.0 m border was constructed between plots and each plot was fenced with 0. 5 m high glass panels to prevent the plants from climbing beyond the plots.
The net photosynthetic rate (Pn) measurements on leaves for rapeseed andlittleseed canarygrass conducted using a Portable Photosynthesis System (LI-COR Biosciences LI–6400XT, Lincoln, Nebraska, USA), between 10:00 am and 16:30 pm, with a 6400–02 LED source and 1000 μmol m–2 s–1 photosynthetically active radiation . During sampling, CO2 concentration in surrounding air, air temperature and relative humidity (RH) in the chamber were measured. In December 2018, two months after transplanting, 40 fully expanded leaves (flag leaf and the top second leaf) of each species were randomly sampled from each plot and immediately scanned using an LI–6400XT . Then, the leaves were cleaned for chlorophyll content determination. We cut 0.1g pieces from fresh leaves avoiding the main vein, and soaked the leaf fragments in 25 ml 95% ethanol at room temperature for 48 h. The detector was set at 665 nm in order to calculate the total chlorophyll content (Chl) . In February 2019, four months after transplanting, twenty plants of each species were selected randomly and forty fully expanded sun leaves (flag leaf and the top second leaf) of each species were sampled for net photosynthetic rate and chlorophyll content, following thesame method as above. Plants were manually uprooted and then cut at ground level for determination of aboveground biomass. Fresh plants were heated for 30 min at 105 °C to halt metabolic processes, and then dried at 80 °C in a forced-draft oven until reaching a constant weight before weighing. Rapeseed yield and littleseed canarygrass seed number were determined in a 2m2 area in each plot; rapeseed yield was adjusted to a moisture content of 10.0%.
To evaluate the effect of littleseed canarygrass on rapeseed yield under various N regimes, the yield losses of rapeseed were determined from rapeseed yield comparing monoculture versus mixed culture according to the formula: the yield reduction rate of rapeseed(%) = (1 yield in mixed culture / yield in monoculture)×100%. To evaluate the ability of rapeseed to control littleseed canarygrass under various N regimes, control efficacy of littleseed canarygrass was determined from seed number comparing monoculture versus mixed culture according to the formula: the control efficacy of littleseed canarygrass (%) = (1 seed number in mixed culture / seed number in monoculture)×100%.
Relative yield (RY) per plant  and competitive balance index (CB)  were calculated from the final aboveground biomass obtained for each species in each plot. Relative yield per plant of species a or b in a mixed culture with species b or a was calculated as RYa = Yab/Ya or RYb = Yba/Yb. Competitive balance index was calculated as CBa = In (RYa/RYb), where Yab is the yield for species a growing with species b (g/individual), Yba is the yield for species b growing with species a, Ya is the yield for species a growing in pure culture (g/individual), and Yb is the yield for species b growing in pure culture. Values of RYab measure the average performance of individuals in mixed cultures compared to that of individuals in pure cultures. An RYab of 1.00 indicates species a and b are both equal in terms of intraspecific competition and interspecific competition. An RYab greater than 1.00 means intraspecific competition for species a and b is higher than interspecific competition, and an RYab of less than 1.00 implies intraspecific competition of species a and b is less than interspecific competition . Values of CBa greater than 0 indicate that species a is more competitive than species b .
The rapeseed yield and its yield reduction rate, seed number of littleseed canarygrass and its control efficacy, aboveground biomass, physiological (Pn) and chlorophyll content of rapeseed and this weed were analyzed by analysis of variance (one-way ANOVA) using IBM SPSS 23.0 software (Armonk, New York, USA). The F and partial eta squared statistics were calculated considering density ratio and N level with their interaction as factors at a 5% level of significance. Relative yield from each mixed culture were compared to the value of 1.00 using t-tests (P = 0.05), and values of CB for deviation from 0 using a paired t-test.