Collection of study species
Seeds of P. americana were collected in August 2019 on the campus of Yunnan University (24°83′N, 102°85′E), Kunming City, Yunnan Province, and seeds of P. acinosa were collected at the same time in Fuyuan County (25°27′N, 104°19′E), Qujing City, Yunnan Province. After harvesting the ripe berries, we washed off the pulp and washed the seeds, air-drying and storing them at 25°C.
Experimental Design
We conducted the experiments in a greenhouse at Yunnan University with suitable temperature and relative humidity (average temperature of 25°C and average relative humidity of 60%) for the growth of P. americana and P. acinosa.
In August 2020, we planted the seeds of P. americana and P. acinosa in cavity trays in the greenhouse and watered them every 2–3 days. The seedlings were transferred to the pots (d = 9 cm) and watered them every 2–3 days when the seeds in the cavity trays had germinated and the fourth cotyledon had grown. The healthy seedlings up to 10 cm were selected and transferred to 35 cm diameter pots for one of the following three culture types: (1) two P. americana monoculture, (2) two P. acinosa monoculture, and (3) a mixed culture comprising one P. americana and one P. acinosa in a pot. At the same time, we sprayed the plants to assess their resistance with the following treatments: (1) spraying insecticide; (2) spraying fungicide; (3) spraying insecticide + fungicide; and (4) spraying water (Fig. 1). Specifically, the insecticide used was Esfenvalerate, which can be used against a broad spectrum of insects but does not negatively affect the plants themselves. The fungicide used was Benomyl. According to the application instructions, the dose of insecticide is 3 ml per plant and the dose of fungicide is 7 ml per plant. Both insecticide and fungicide were sprayed on the leaves and the control treatment was sprayed with 10 ml of water, with 12 replicates of each treatment. Spraying was done once every two weeks.
During the experiment, watering was done once every 2–3 days, and the position of the pots was changed randomly once a month. We harvested all the plant materials on January 17 and 18, 2021, and measured the plant height, basal diameter, and the number of leaves of each plant, determined the chlorophyll content of plant leaves with a portable chlorophyll meter and recorded the number of racemes and the fruits of P. acinosa and P. americana.
Competitive Ability Analyses
In order to investigate the competitive ability of the invasive plant P. americana and the native plant P. acinosa, we used the Relative Yield (RY, (Danieli-Silva et al. 2010)), the Relative Yield Total (RYT, (Fowler 1982)) and Competitive Balance Index (CBI, (Wilson 1988))to determine species competitive ability.
The equations were calculated as:
$${\text{RY}}_{\text{α}}\text{ = }{\text{Y}}_{\text{αβ}}/{\text{Y}}_{\text{α}}$$
(Eq. 1)
$$\text{RYT = }{\text{RY}}_{\text{α}}\text{+}{\text{RY}}_{\text{β}}$$
(Eq. 2)
$$\text{CBI = }\text{ln}\left({\text{RY}}_{\text{α}}/{\text{RY}}_{\text{β}}\right)$$
(Eq. 3)
Where Yα denotes the mean value of plant α biomass per plant in the same planting treatment and Yαβ denotes the mean value of plant α biomass per plant in the mixed planting treatment between the two species. When RYα > 1, it means that the interspecific competition between two species is less than the intraspecific competition of species α; when RYα = l, it means that the intraspecific competition of species α is equal to the interspecific competition between two species; when RYα < l, it means that the interspecific competition between two species is greater than the intraspecific competition of species α. When RYT > 1, it means that the invasive species and the native species are in a reciprocal symbiotic relationship; when RYT < 1, it means that the invasive species and the native species are in a competitive antagonistic relationship; when RYT = 1, it means that the two species need the same nutritional resources for survival, belonging to the contest competition. When CBI > 0, it indicates that the competitiveness of species α is greater than that of species β, moreover, and the larger the CBI, the stronger the competitiveness of species α is; and vice versa; when CBI = 0, it indicates that the competitiveness of species α and species β is equal.
Determination Of Chemical Substances
After the flowering period of the plants, the leaves of P. acinosa and P. americana were collected from the same parts under different treatment conditions, Further, we dried and ground at 60°C, and stored at -20°C for determing nutrients (starch and soluble sugars) and resistant substances (total phenols, total flavonoids, tannins, and lignin). The kits used for measuring the above six substances were: starch content kit (anthrone colorimetric method), soluble sugar content (SS) kit, total phenol (Tp) kit, total flavonoid (TF) kit, tannin (Tannin) content kit, lignin content (Lignin) kit (acetylation method), and all the kits were purchased from Suzhou Grace Biotechnology Co., Ltd., China.
Statistical analysis
The individual and interactive effects of the two competition types (inter-specific and intra-specific competition) as well as the three spray treatments (insecticide, fungicide, and insecticide + fungicide) on 16 studied traits of P. americana and P. acinosa were assessed using general linear models with SPSS software (IBM SPSS Statistics 25). The interaction effect had a significant effect on root-crown ratio, the number of racemes, soluble sugars, total phenols, tannins, and lignin content, whereas we detected no significant effect on the other traits (P > 0.05). In addition, we performed multiple comparison analysis (LSD test) for the above six traits. Some data were transformed appropriately to meet the assumptions of chi-square and normal distribution before analysis (Table S1). Plots were performed using Origin Pro 2021.