Polygonum cuspidatum Sieb. Et Zucc is a medicinal plant, whose active ingredients contain stilbene compounds (e.g., resveratrol) and anthraquinones (e.g., emodin) (Kong et al., 2020). In East Asia, P. cuspidatum is widely used in the therapy of hepatitis, cough, jaundice, and other diseases (Zhang et al., 2013). Resveratrol, emodin, physcion, and chrysophanol of P. cuspidatum have been used as monomeric ingredients in pharmaceutical, chemical, and food applications, and thus P. cuspidatum becomes the raw material for these monomeric ingredients (Sun, 2007). Among them, resveratrol can not only improve the disease resistance of plants, but also have antioxidant and anti-tumor effects, along with great demand market (Wu et al., 2019); emodin has the functions of lowering blood pressure, protecting liver and anti-tumor, and immunomodulatory effects (Wu et al., 2022). Of all reported plant species, P. cuspidatum has much greater resveratrol concentrations than other plants (Wu et al., 2019).

Arbuscular mycorrhizal (AM) fungi are beneficial fungi in soil that colonize the roots of approximately 72% of terrestrial plants, thus establishing a reciprocal symbiosis (Genre et al., 2020). AM fungi affect primary metabolic processes of plants and also change secondary metabolites in various medicinal plants (Zeng et al., 2014; Sun et al., 2021). In Glycyrrhiza uralensis plants, AM fungi distinctly raised concentrations of glycyrrhizic acid, and the increase became greater with the prolongation of inoculated time (Liu et al., 2007). In Angelica dahurica plants, AM fungi also dramatically induced the increase in total coumarin and imperatorin concentrations (Zhao and He, 2011). However, in Ocimum basilicum plants, Glomus intraradices triggered an increase in anthocyanin levels, but it did not affect concentrations of polyphenolic substances (Lee and Scagel, 2009). These results imply the potential of AM fungi in increasing specific medicinal ingredients in certain medicinal plants.

Substrate phosphorus (P) levels have an impact on the colonization response of AM fungi (Campos et al., 2018; Cao et al., 2021). High levels of substrate P inhibit AM fungal colonization (Breuillin et al., 2010), and low levels of P and AM fungi have improved effects on the accumulation of secondary metabolites in plants (Zeng et al., 2014). Inoculation with G. mosseae promoted artemisinin content in the medicinal plant Artemisia annua under low P conditions (40 mg/kg), while high P (120 mg/kg) significantly decreased artemisinin content by 42.5% (Tan et al., 2013). P fertilizer supply could achieve similar results to AM fungal inoculation in promoting the content of active ingredients in medicinal plants, such as castanospermine levels in Castanospermum austral plants and essential oils in Foeniculum vulgare plants (Kapoor et al., 2004; Bastami and Majidian, 2016). In Anadenanthera colubrina seedlings, AM fungi had an increased effect on the content of total phenols, total flavonoids, and total tannins in leaves, even under high P supply (30 and 50 mg/dm³) (Pedone-Bonfim et al., 2013). Interestingly, G. mosseae inoculation, but not P fertilization application, accelerated the production of essential oils in oregano plants (Khaosaad et al., 2006), suggesting different potential mechanisms for P fertilization and AM fungi to affect secondary metabolism.

Polygonum cuspidatum is mostly planted in mountainous areas, where the soil is poor, especially with low levels of P (Yu, 2016). An AM fungus, Funneliformis mosseae, could establish mycorrhizal symbionts in roots of P. cuspidatum and improve plant growth and root development, coupled with the increase in root medicinal components (Sun et al., 2022a). However, it is not clear whether the AM fungus promotes these medicinal components of P. cuspidatum under different P levels and what the underlying molecular mechanisms are involved in the promotion of medicinal components. The purpose of the present study was to assess the effects of F. mosseae on plant growth, P acquisition, soil phosphatase activity, concentrations of medicinal ingredients, and relative expressions of related genes in potted P. cuspidatum under low and appropriate P conditions.

AM fungal colonization in roots is often influenced by substrate P levels and root architecture (Wang et al., 2020). Low P levels favor root AM fungal colonization, but high P levels inhibit AM fungal colonization (Mora et al., 2008; Breuillin et al., 2010). The present study revealed that root mycorrhizal colonization rate was significantly lower at P₀ than at P_0.2, which is consistent with the findings of Shao et al. (2021), who reported that under P deficit conditions, P uptake by tea plants relied more on root hairs than on AM fungi, resulting in low AM fungal colonization under low P levels. Therefore, future work is needed to evaluate the response of root hairs and AM fungal colonization of P. cuspidatum to different P levels.

P supply and AM fungal inoculation collectively improved growth performance of P. cuspidatum, and the improvement was better at P_0.2 than at P₀. This results indicated that P is a key mineral element for vigor growth of P. cuspidatum, and P supply can promote their growth (Feng et al., 2019). On the other hand, AM symbiosis promoted the growth performance of P. cuspidatum, independent of substrate P levels, which is consistent with our earlier findings of inoculating P. cuspidatum with symbiotic fungi, including F. mosseae (Sun et al., 2022a). AM fungi also improved biomass production of Salvia miltiorrhiza and Artemisia annua plants (Huang et al., 2011; Jia et al., 2020). AM fungi can improve rhizospheric microenvironment and enhance nutrient uptake and utilization as well as water uptake by the plant after establishing a symbiosis, thus promoting plant growth (Xie et al., 2020).

Root morphology is plastic and can be influenced by various external factors (Wang et al., 2020). This study indicated that P supply distinctly promoted the establishment of root architecture, especially volume. Generally, P supply accelerates the formation and development of lateral roots to allow the root to penetrate into a larger soil volume to facilitate P acquisition (Williamson et al., 2001). Inoculation with AM fungi also dramatically improved root average diameter, total length, and projected area independent of P levels, being superior at P_0.2 than at P₀. Similar results were also observed in tea (Shao et al., 2021), walnut, and trifoliate orange exposed to P stress (Wu et al., 2015b; Huang et al., 2020). The improvement of root morphology triggered by AM fungi may be linked to the increase of root auxins and polyamine levels by AM fungi (Zhang et al., 2019; Zou et al., 2021).

AM fungi play an essential role in nutrient acquisition by plants grown in nutrient-poor soil (Ren et al., 2022). In this study, AM fungi promoted the acquisition of root P in P. cuspidatum, accompanied by an increase in soil acid and neutral phosphatase activities, independent of substrate P levels. Phosphatases, which are P-solubilizing enzymes, are responsible for the mineralization of organic P to provide available inorganic P (Redel et al., 2019). AM soil possessed higher activities of acid and neutral phosphatase in P. cuspidatum, which can hydrolyze more organic Pi to enhance P acquisition (Hofmann et al., 2016). AM fungi secrete phosphatases and organic acids into mycorrhizosphere, and also possess phosphorus transport proteins that contribute to the uptake and transport of P, thereby enhancing P acquisition of plants (Bagyaraj et al., 2015; Yang et al., 2021).

In this study, HPLC was used to determine the medicinal components of P. cuspidatum, in which the concentrations of polydatin, resveratrol, and emodin were 1.89–8.64, 2.69–7.19 and 0.24–1.65 mg/g DW, respectively, which were much lower than those detected in polydatin (5.12–47.51 mg/g DW), resveratrol (3.73–15.16 mg/g DW), and emodin (2.23–17.33 mg/g DW) concentrations of P. cuspidatum by She et al. (2021) in Hubei, China. The difference could be attributed to different years of P. cuspidatum tested and different extraction methods. Future work should be made around extraction methods and gas chromatography–mass spectrometry assays. AM symbionts influence the production of secondary metabolites in various medicinal plants, and medicinal plants also regulate AM symbiosis through their secondary metabolites (Sun et al., 2021). Earlier studies also showed that Ocimum basilicum plants inoculated with Glomus caledonium had higher leaf rosmarinic and caffeic acid levels at three P levels (0.1, 0.2, and 0.3 g/kg of CaHPO₄), as compared with uninoculated controls (Toussaint et al., 2007). G. mosseae at P levels of 40–80 mg/kg significantly promoted the accumulation of artemisinin in Artemisia annua plants, while high P (120 mg/kg) supply significantly reduced artemisinin content (Tan et al., 2013). In our study, P supply promoted the concentrations of aloe-emodin, physcion, and resveratrol in non-mycorrhizal plants as well as the concentrations of chrysophanol, physcion, polydatin, and resveratrol in mycorrhizal plants, implying that P is an important regulator of the synthesis of medicinal components. At P₀ and P_0.2 levels, AM fungi promoted chrysophanol, emodin, physcion, polydatin, and resveratrol accumulations, and the promote effect was better under P-deficient conditions than under adequate P conditions.

AM fungi, after coexisting with host plants, are able to affect expressions of synthetic enzyme genes related to plant secondary metabolites (Zeng et al., 2013; Merlin et al., 2020; Oliveira et al., 2022; Sun et al., 2022a). The synthesis of resveratrol in P. cuspidatum is regulated by key enzymes such as resveratrol synthase (RS), stilbene synthase (STS), supposed stilbene synthase (CRS), and resveratrol-forming stilbene synthase (Zheng et al., 2021). In the present study, both AM fungi and P supply up-regulated PcCRS1, PcRS11, and PcSTS expression, except for down-regulation of PcRS expression, suggesting that both P supply and AM fungi were able to accelerate the expression of resveratrol-associated enzymes in P. cuspidatum, which, in turn, promoted the accumulation of resveratrol levels in roots. Although chalcone synthase (CS) is not directly involved in resveratrol biosynthesis, the enzyme can compete with RS for the same substrate (Flores-Sanchez and Verpoorte, 2009), which thus triggered down-regulated expression of PcRS and elevated expression of PcCHS1 and PcCHS2 by AM fungi and P supply. In Paris polyphylla var. yunnanensis plants, AM fungi also up-regulated PpSE expressions to accelerate polyphyllin accumulation (Li et al., 2021). Therefore, AM fungi as a biostimulator has the potential ability to regulate the expression of secondary metabolite-associated synthases, thereby promoting the level of secondary metabolites. Nevertheless, the underlying mechanisms of how AM fungi and P supply regulate the expression of these genes have yet to be studied.

In short, both P supply and AM fungal inoculation were able to dramatically enhance the concentration of root medicinal components (chrysophanol, physcion, polydatin, and resveratrol) of P. cuspidatum, together with up-regulated expression of associated synthase genes. Therefore, AM fungi as a biostimulator can be introduced or appropriate P fertilizer can be provided in P. cuspidatum cultivation to promote the production of medicinal components.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

R-TS and Q-SW designed the experiment. R-TS, Z-ZZ, and H-DF prepared the materials for the experiment. R-TS, M-YL, NZ, and X-CF conducted the experiment. R-TS, M-YL, and X-CF analyzed the data. R-TS wrote the manuscript. AH, EA, WH, and Q-SW revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the 2021 Undergraduate Innovation and Entrepreneurship Training Program of Yangtze University (Yz2021329). The authors extend their sincere appreciation to the Researchers Supporting Project Number (RSP-2021/134), King Saud University, Riyadh, Saudi Arabia.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.