Experimental site
All experiments were conducted at National Experimental Station for Precision Agriculture in Xiaotangshan Town (40°10′ N, 116°26′ E, hereinafter referred to as Xiaotangshan Station), in Beijing suburb, Northern China. Xiaotangshan Station is characterized by a continental, semi-humid, and monsoonal climate. The mean annual rainfall is 640 mm, of which 80% occurs between June and August. The mean annual temperature is 11.5 ℃, and the lowest and highest monthly mean temperatures are − 4.1 ℃ in January and 25.8 ℃ in July, respectively. The annual sunshine hours are 2641.4 h. The active accumulated temperature (≥ 10 ℃) is 4188.3 ℃. The frost-free period is 198.0 days, approximately from mid-April to mid-October.
The topsoil, to a depth of 0–10 cm in the field, is clay loam, composed of 41% sand, 24% silt, and 35% clay, with bulk density of 1.37 g·cm− 3, pH of 7.6, organic matter content of 1.4%, total nitrogen of 2.46 g·kg− 1, and total phosphorus of 0.63 g·kg− 1.
Bioassay experiment
During the growing season in 2017, a bioassay experiment was carried out to evaluate the allelopathic effects of redroot pigweed and crabgrass root aqueous extracts on switchgrass germination in Petri dishes. Plump seeds of switchgrass and roots of these two weed species were collected from the field at Xiaotangshan Station during growing seasons in 2016 and 2017, respectively.
After being washed with distilled water three times, redroot pigweed and crabgrass roots were lyophilized for at least 48 h at − 80 ℃ in a freeze dryer, and then ground into powder to pass a 1-mm screen mesh. 10 g powder was homogenized in 100 mL distilled water for 30 min. The homogenate was filtered, and the filtrate was used as the root aqueous extract at 0.1 g·mL− 1 concentration, which was diluted to prepare a series of root aqueous extracts at concentrations of 0.005, 0.01, 0.02, and 0.05 g·mL− 1.
Each Petri dish, with 90 mm inner diameter, was lined with two layers of filter paper and moistened with addition of 5 mL root aqueous extract at different concentrations. Control Petri dishes only received 5 mL distilled water. Plump switchgrass seeds were surface sterilized with 75% alcohol for 5 min, and then with 3% sodium hypochlorite for 10 min, before being rinsed with distilled water three times. Fifty seeds were placed on the filter paper in each Petri dish. All Petri dishes were covered, sealed with parafilm and placed in a controlled environmental chamber with a 16:8 h light:dark photoperiod, 30:25 ℃ light:dark temperatures, and 70% relative humidity. A randomized complete block design with three replicates was used and the positions of all Petri dishes were randomized once a day.
After 12 days, germinated switchgrass seeds with radicle length (≥ 1 mm) were counted for germination rate calculation. Ten germinated switchgrass seeds were selected randomly from each Petri dish, for plumule and radicle lengths measurement.
Glass jar experiments
Mixture determination for weed-switchgrass system
During the growing season in 2016, a replacement experiment was conducted in glass jars, 100 mm inner diameter and 90 mm height, containing 500 g soil, in a greenhouse with 32:21 ℃ day:night temperatures and 65–90% relative humidity at Xiaotangshan Station. The objective of this experiment was to identify the optimal mixture proportion for the subsequent root segregation experiment.
Plump seeds of redroot pigweed, crabgrass, and switchgrass were collected from the field during the growing season in 2015, and seedlings were cultured in the greenhouse in mid-February 2016. A total of ten weed and switchgrass seedlings at three-leaf stage were planted in each glass jar. The proportions of weed to switchgrass were 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, respectively. A monoculture of ten switchgrass seedlings served as the control (Fig. 1).
Root segregation of weed and switchgrass
During the growing season in 2016, a root segregation experiment of weed and switchgrass was conducted in glass jars, in the same greenhouse as described in the experiment on mixture determination. The objective of this experiment was to evaluate the role of different components involved in the soil root system on switchgrass seedling growth at 5:5 proportion with weeds.
The experiment was carried out in a series of glass jars, with the same dimensions as described above. A total of sixteen glass jars were divided into four groups: root segregation with plastic film (hereinafter referred to as complete segregation, as the control), root segregation with 0.45 µm Millipore filtration (preventing both mycorrhizal hyphae linkages and root penetration but allowing chemical and bacterial interaction, hereinafter referred to as partial contact 0.45), root segregation with 30 µm Millipore filtration (preventing root penetration but allowing microbial penetration and chemical interaction, hereinafter referred to as partial contact 30), and without root segregation (hereinafter referred to as full contact) (Fig. 2). Each glass jar contained a central cylinder, 70 mm inner diameter and 90 mm height, made of plastic mesh frame, where plastic film or Millipore filtration could be inserted.
Each glass jar was filled with 500 g soil, 250 g inside and 250 g outside the central cylinder. Seedlings of redroot pigweed, crabgrass, and switchgrass were cultured in the greenhouse in mid-May 2016. Five switchgrass seedlings and five weed seedlings, at three-leaf stage, were planted inside and outside the central cylinder, respectively, in each glass jar.
In the two glass jar experiments described above, the soil used was naturally air-dried topsoil sampled in the field at Xiaotangshan Station. Four replicates were used for each treatment or control in a randomized complete block design, and the positions of all glass jars were randomized once a day.Glass jarswere irrigated daily with distilled water. Any seedlings, except those of tested species, were manually removed as soon as they were detected. After 30 days, switchgrass seedlings were harvested, washed three times, and theirshoots and roots weredried for at least 48 h at 80 ℃in an oven to measure their biomass.
Field experiment
During growing seasons from 2014 to 2016, a field experiment was conducted to evaluate the inhibitory effects of local weeds on transplanted switchgrass seedlings’ growth at Xiaotangshan Station over a three-year period.
During the growing season in 2013, plump switchgrass seeds were collected from the field and were sown in the same greenhouse as described in the above experiment on mixture determination in mid-February 2014. In mid-May 2014, six field plots of 4.0 × 4.0 m2 were prepared and sixteen switchgrass seedlings at six-leaf stage were transplanted into 2.4 × 2.4 m2 central area of each field plot with inter-plant and inter-row spacing of 0.8 m. In three field plots, all weeds were allowed to grow naturally with switchgrass, while, in the other three field plots, all weeds were manually removed as soon as they were detected, as the control. All field plots, with weeds and without weeds, were arranged in a randomized complete block design with three replicates.
The field plots were irrigated immediately after transplantation to ensure switchgrass establishment, but not thereafter during the experimental period. The equivalent of 150 kg·ha− 1 N:P:K fertilizer (15:15:15) was applied as basal fertilizer, followed by 150 kg·ha− 1 urea application in mid-June during the first growing season.
During growing seasons from 2014 to 2016, a sampling quadrat of 1.6 × 1.6 m2 was set in the central area of each field plot. In each sampling quadrat, there were four switchgrass plants, of which three were labeled randomly. After anthesis, tiller number was counted, and twenty tillers were selected randomly from these three plants for plant height measurement in situ. At the end of November in each year, when switchgrass stems had fully senesced, shoots of these three plants were harvested at stubble height of 5 cm. Twenty tillers with relatively uniform size were selected from these three plants for phytomer number calculation in situ. These twenty tillers and other shoots were washed three times, and then dried for at least 96 h at 80 ℃ in an oven, for shoot and phytomer biomass measurement, respectively.
Data analysis
Plant biomass was calculated by shoot biomass + root biomass in glass jar experiments. Weed effects, as percentages, were calculated by (control – treatment)/control × 100%, based on germination characteristics in the Petri dish experiment, and growth characteristics in glass jar and field experiments, respectively.
Analysis of variance, multiple comparisons, and independent t-test were performed in Microsoft Excel 2007 for Windows. Data were presented as means ± standard error in each experiment.