4.1 Response of S. miltiorrhiza productivity to AMF inoculants
Plant-related microorganisms play an important role in many aspects of life, including nutrition, growth, flowering, etc., and affect plant productivity through various means. AMF colonization of roots was one of the oldest and most common interactions in ecology [33] and facilitated the absorption of macronutrients by plants, especially N and P [34–36], through arbuscular (a specialized structure in the host root that was the site of nutrient exchange between the plant and the fungi). In return, plants allocate up to 20% of photosynthetic products (carbon sources) to AMF, ensuring their growth. Root colonization was the premise of plant-microbe interaction. In our study, Root colonization occurs as a prerequisite for plant-microbe interactions. In our study, all thirty-one treatments formed a good symbiosis with AMF, but there was not necessarily a correlation between the degree of root colonization and plant promotion benefits (Fig. 7). For example, when C was colonized in the root alone, the colonization rate was the lowest (54.83%), while the FW and DW increased. On the contrary, some groups with higher colonization rates (such as communities AC and CE) had lower root biomass. This has been demonstrated in many studies [37, 38].
Due to the strong host specificity of AMF, it is necessary to explore the interaction characteristics between host plants and AMF(s). It has been widely reported that AMF formed symbiotic relationships with host plants and increased plant productivity [16, 39, 40], and the beneficial effect is often attributed to AMF improving the absorption of mineral nutrients [41]. While some AMF communities in this study still had adverse effects on herb productivity, such as the inoculation of E, AC, and DE. It seemed that the symbiotic benefits did not always exceed the cost of symbiosis between AMF and plants, meaning that not all AMF symbiosis provided benefits to plants but instead adversely affected plant productivity [42]. Nevertheless, most treatments still effectively promoted the formation of S. miltiorrhiza productivity, in which the DW of communities BD, ABE, ACE, ABCE, and BCDE increased significantly by more than 80% (p < 0.05), which had great potential in the application of AMF(s) to the improvement of root yield. Interestingly, the combination of more closely related AMFs, such as any combination of inoculation with A, B, and C with the same family, resulted in lower biomass than the mono-inoculation treatment. This is in agreement with Maherali et al. [43], indicating that when multiple AMFs of the same family are contained in the same inoculant, the growth-promoting effect on the plant is diminished due to competition between ecological niches triggered by their functional redundancy [44]. We also found that overdispersal affinities in complex communities (e.g., community ACDE) were also detrimental to root biomass accumulation, suggesting that plant growth is influenced by AMF affinities in the community and that species compositions that are too close or too distant are not conducive to productivity. Plant productivity also appears to be influenced by species richness [25], which increases with AMF species richness and has been confirmed for Plantago lanceolata [43]. This suggests that the productivity of S. miltiorrhiza is influenced by both species’ richness and AMF affinities in the communities, and that community members who are too closely or too distantly related are not conducive to plant productivity.
MD was often used to reflect the degree of compatibility between plants and AMF and the effectiveness of mycorrhizal agents. In this study, the MD of most groups was greater than 100%, which promoted the formation of root biomass (Table 2), and both communities ABE and ACE of MDs reached more than 200%, with significant plant proliferation (p < 0.05). This was consistent with many previous studies, indicating that S. miltiorrhiza was a mycorrhizal-dependent medicinal plant [22], which could form a good symbiosis with various AMFs. Because of these impacts on plant health and adaptation, AMF inoculants could be an important strategy for sustainable agriculture [45].
4.2 Bioactive constituent’s content and accumulation response diversity
Secondary metabolites were the critical components of the interaction between plants and the environment to adapt to biological and abiotic stresses and the vital basis for the medicinal efficacy of herbal medicines. Due to people's significant interest in the secondary metabolites of S. miltiorrhiza root as pharmaceutical raw materials [46], an increasing number of studies tended to enhance the production of active metabolites, including the use of biotic and abiotic exciters to stimulate metabolite accumulation in S. miltiorrhiza, cellular, or hairy root cultures [46–50]. Among the new plant production strategies, AMFs were considered the most reliable soil microbiome for promoting secondary metabolite production [51]. AMF was intimately involved in plant physiological and biochemical metabolic processes promoting the production and accumulation of important active components of medicinal plants, such as terpenoids, phenols, and alkaloids [18]. And these secondary metabolites possess a variety of pharmacological properties (anti-diabetes, anti-cancer, anti-hypertension, anti-cardiovascular, etc.) [52], which were widely used in some agricultural, industrial and medical applications [53], making it of great interest in the potential of AMF to regulate the production of secondary metabolites.
It is generally accepted that the chemical compositions of plants are directly or indirectly related to endophytic microbes and their interactions with host plants [54]. There is evidence that AMF colonization had qualitative or quantitative effects on the secondary metabolites of medicinal or aromatic plants [55], either altering their content or changing their composition [56], which has been widely confirmed on the aromatic plants Salvia officinalis L. [57] and Angelica archagelica L. [58]. It has been shown that the inoculation of a single AMF effectively improved the growth and accumulation of secondary metabolites, increasing the content and accumulation of tanshinones and SB in roots [20, 21, 59]. This study showed that the content of TTS and TP in roots was influenced by AMF species richness, with an increase in species richness facilitating an increase in TTS content and a less favorable increase in TP content (Figs. 3e and 3f). Overall, simultaneous inoculation with three or four species was more advantageous for TP and TTS formation. In addition, the contents of active ingredients were influenced by the affinities of AMFs in the community, and the combinations of species with more distant affinities (such as communities ABD and BCD) seemed to be more favorable for the simultaneous enhancement of TP and TTS contents. Notably, both the more dispersed community ACDE and the more closely related AMF community ABC increased both TP and TTS content, and both combinations showed inhibitory effects on root biomass, suggesting that the symbiotic relationship between microbial communities and plants influences the plants to continuously balance growth and defense functions during growth and development, ultimately affecting the yield and quality of medicinal plant. Secondary metabolites are an important means of phytochemical defense produced during the competition between plants and microorganisms [60]. Phenolic compounds have various plant protective functions due to their high antioxidant potentials [61], such as resistance to invasion by pathogens, osmotic regulation and hormone production [62, 63]. Terpenoids were also often considered as a defense product of plants against fungal colonization [64]; AMF symbiosis was shown to upregulate the transcription of genes encoding enzymes involved in isoprene-like biosynthesis and was strongly correlated with terpenoid concentrations [65]. However, the effect of AMF colonization on plant active ingredients varies depending on the plant as well as the fungal species [17, 59, 66], thus the present study systematically investigated the inoculation effect of single to multiple AMFs on the active ingredients of S. miltiorrhiza and found that inoculation with A was most favorable for the TP content, while community ACDE was most favorable for the TTS content. The inoculation effect of AMF communities was influenced by the species richness and the members' affinity, and the simultaneous inoculation of three or four AMFs of different families with distant affinities was more favorable for the formation of TP and TTS contents. Our results further indicate that selecting and applying the appropriate AMF could effectively improve the quality of the herbs obtained while improving plant productivity [16].
4.3 Difference in response to plant growth and secondary metabolism between a single AMF and AMF communities
Several recent studies suggested that microbial communities sustain additional ecosystem functions and services, including plant-promoting and plant-protective effects. For example, when functionally complementary AMF species colonized plant roots, the colonization benefits of mixed inoculants were higher than those of a mono-inoculant [23, 67, 68]. Conversely, it has been shown that certain single species were more favorable for plant growth [69] and that increasing inoculants diversity did not lead to greater benefits [70, 71]. The focus of research on the role of microbes in promoting plant growth has gradually shifted from mono-inoculants to microbial communities in recent years [72–74]. Despite the widely established beneficial effects of AMF on plant growth and metabolism, the effects of single or combined applications were inconsistent and highly variable on plants. Therefore, this study systematically explored the responses of mono-inoculants to mixed inoculants and found that all thirty-one inoculation treatments formed a good symbiosis with S. miltiorrhiza (up to 89.97% colonization), and the mixed inoculation treatments possessed higher mycorrhizal colonization rates compared with mono-inoculation treatments (Fig. 3c) [75]. Colonization rate differences among communities may be related to multiple group interactions [76].
Consistent with most studies, our results also indicate that mixed inoculants are more beneficial to plant growth than single inoculants (Figs. 3a and 3b) [75, 77], but most current studies are limited to two or three AMFs [78, 79], ignoring the fact that AMFs are complex and diverse in the field environment. In this study, communities BD, BE, ABE, ACE, ABCE, BCDE, and ABCDE had a greater enhancement benefit for both DW and FW than a single inoculant. This beneficial effect may result from functional complementarity among the species, allowing the resources to be used more efficiently through their differential ecological niches [24]. Whereas the remaining AMF inoculant mixes did not show, synergistic effects on plant growth may be attributed to the fact that one of the species may have become the dominant species [71] or may be related to complex interactions between the species or competition for similar ecological niches [80].
In addition to differences in regulating plant growth, secondary metabolites also varied depending on the inoculum. For example, the combined inoculation of Cynara cardunculus L. var. scolymus with G. intraradices and G. mosseae under field conditions was more beneficial in terms of increased total phenolic acid content in leaves and flowers compared to mono-inoculation [66]. Combined inoculation ach community in this study did not appear to be more effective than mono-inoculants in increasing the TP content (Figs. 3e and 5a), while the AMF communities seemed to have more potentials in increasing TTS content (Figs. 3f and 5b). Only B increased both TP and TTS content, while communities ABC, ABD, BCD, BCE, ABDE, and ACDE all increased TP and TTS content. Communities ABD, BCE, and ABDE increased plant root biomass, while some communities did not show simultaneous promotion effects, which might be attributed to the strong antagonistic effect between species, suggesting that the effectiveness of mixed inoculants might be related to the compatibility between different microorganisms [68]. These variations further indicate that the mycorrhizal benefits largely depend on plant-fungi combinations [81]. Although only a few communities were able to increase the yield of medicinal parts of S. miltiorrhiza and the content of secondary metabolites more effectively than e than a single AMF, we still believed that higher AMF richness might have more potential in promoting S. miltiorrhiza growth and yield, especially when three to four distantly related AMF species are co-inoculated. This might be attributed to the fact that biodiversity is essential for maintaining ecosystem function, and communities with higher richness might contain more ecological functions with specific buffering properties, which could maintain ecosystem stability and function in more complex environments [82, 83].
4.4 The key to biofertilization
Applying beneficial microbes to herbal cultivation is highly promising, and biofertilizers could improve soil fertility and achieve higher yields and quality without negatively affecting the agricultural environment [49, 84]. Despite the widely recognized efficacy of microbial fertilizers, it was a fact that biofertilizers sometimes provided little benefit in growth promotion [42, 85]. Several pieces of evidence show that the introduction of foreign AMFs does not bring more benefits than native AMFs [86, 87] because they have to compete with native AMFs [88]. This could be why most commercial mycorrhizal fungi have little effect on plant growth promotion. More interestingly, some researchers have found large differences in the effects of using the same inoculum in different locations [89, 90], making AMFs much less effective as biofertilizers. Therefore, we designed experiments in this study using native AMFs to maximize biofertilizers' effectiveness. In this study, B, communities BD, ACD, ACE, and BDE effectively promoted biomass formation (more than 60%) and increased the ATP and ATTS as well as the accumulation of active ingredients, which can be considered as potential communities for AMF application to improve the economic yield of S. miltiorrhiza, with communities ACD and ACE having more potential.
Biodiversity affects many ecosystem functions, and plant productivity often increases with increasing species diversity [91], and this positive effect could often be explained by functional complementarity, whereby different species occupy different ecological niches and thus perform different functions [92]. The results of this study showed an increasing trend of FW, DW and ATTS with increasing species richness (Figs. 3a, 3b, and 3h), while ATP showed fluctuations, with greater benefits when a single or four AMF species were co-inoculated (Fig. 3g). All these results strongly emphasize that the AMF species richness influenced the inoculation benefits of AMFs in the inoculant and that mixed AMF inoculants showed greater potential for application than mono-inoculants. In addition, the plant growth promotion effect as well as lower ATP and ATTS of any inoculants of A, B, and C (with the same family) was lower than that of mono-inoculants (Figs. 4 and 6), suggesting that species with closer relatives might have similar ecosystem functions and competing effects and that combinations in groups might have reduced the beneficial effects of single species. And there were positive or negative effects of the different family combination communities on root growth, depending on the species composition, which may be related to the complex interactions between different species [80]. However, in general, the more distantly related combinations of two-family AMFs (e.g., communities ACD and ACE) better promoted S. miltiorrhiza root growth and facilitated TP and TTS accumulation (Figs. 4 and 6), showing a better potential for application.
Overall, the application of AMF did not always promote the growth and active ingredient production of S. miltiorrhiza, while the inoculant benefit of mixed inoculants appeared to be higher than that of single inoculants. The inoculum benefits of mixed inoculants were limited by the AMF species richness and member affinities in the community. Therefore, we believe the key to biofertilization is to select candidates from indigenous AMFs and choose three to four distantly related AMFs in combination according to their phylogenetic relationships as microbial inoculants to ensure the yield of S. miltiorrhiza.