The inoculation of beneficial microorganisms to improve plant growth and soil properties is a promising strategy in the soil amendment. However, the effects of co-inoculation with phosphate-solubilizing bacteria (PSB) and N2-fixing bacteria (NFB) on the soil properties of typical C-deficient soil remain unclear. Based on a controlled experiment and a pot experiment, we examined the effects of PSB (M: Bacillus megaterium and F: Pseudomonas fluorescens), NFB (C: Azotobacter chroococcum and B: Azospirillum brasilence), and combined PSB and NFB treatments on C, N, P availability, and enzyme activities in sterilized soil, as well as the growth of Cyclocarya Paliurus seedlings grow in unsterilized soil. During a 60-day culture, prominent increases in soil inorganic N and available P contents were detected after bacteria additions. Three patterns were observed for different additions according to the dynamic bacterial growth. Synergistic effects between NFB and PSB were obvious, co-inoculations with NFB enhanced the accumulation of available P. However, decreases in soil available P and N were observed on the 60th day, which was induced by the decreases in bacterial quantities under C deficiency. Besides, co-inoculations with PSB and NFB resulted in greater performance in plant growth promotion. Aimed at amending soil with a C supply shortage, combined PSB and NFB treatments are more appropriate for practical fertilization at intervals of 30–45 days. The results demonstrate that co-inoculations could have synergistic interactions during culture and application, which may help with understanding the possible mechanism of soil amendment driven by microorganisms under C deficiency, thereby providing an alternative option for amending such soil.

Research Highlights: We firstly interpreted nutritional mechanisms involved in growth regulation and phytochemical accumulation in Cyclocarya paliurus (Batal.) Iljinskaja under three inoculant types, and selected bacterial inoculations for multiple purposes of C. paliurus plantation. Co-inoculation with phosphate-solubilizing bacteria (PSB) and N2-fixing bacteria (NFB) performed better in growth promotion and nutrient uptake than single bacterial inoculation. Background and Objectives: C. paliurus is a well-known medicinal plant as it accumulates bioactive compounds (BC) such as flavonoids, triterpenoids, and polysaccharides, in its leaves. However, the effects of plant growth-promoting rhizobacteria (PGPR) on the growth and BC yields in C. paliurus are not known. To fill this gap, the effects of different inoculants should be examined. Materials and Methods: A pot experiment was conducted and two-year-old C. paliurus seedlings were inoculated with three inoculant types (PSB, NFB, PSB + NFB). After four rounds of inoculation, the growth characteristics and concentrations of flavonoids, triterpenoids, and polysaccharides, as well as the nutrients in soil and leaves, were measured. Results: The inoculations resulted in the elevation of soil available nutrients, with improvements in plant growth, BC yield, and N and P uptake in leaves. However, the changes in BC yields were mainly a result of elevated leaf biomass rather than BC concentrations, and leaf biomass was regulated by C:N:P stoichiometry. Co-inoculation with PSB and NFB was applicable for leaf production, while inocula related to NFB resulted in higher BC yields than PSB and control. Conclusions: Our results implied that bacterial inoculants improved plant growth and BC yield by altering the nutrients in soil and leaves, while three inoculant types showed a different pattern in which co-inoculation with four strains presented a greater performance than single bacterial addition.

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To maintain the beneficial effects of microbial inoculants on plant and soil, repeated inoculation represents a promising option. Until now, the impacts of one-off inoculation on the native microbiome have been explored, but it remains unclear how long and to what extent the periodic inoculations would affect the succession of the resident microbiome in bulk soil. Here we examined the dynamic responses of plant growth, soil functions and resident bacterial community in the bulk soil to periodic inoculations of phosphate-solubilizing and N2-fixing bacteria alone or in combination. Compared to single-strain inoculation, co-inoculation better stimulated plant growth and soil nutrients. However, the benefits from inoculants did not increase with repeated inoculations and were not maintained after transplanting to a different site. In response to microbial inoculants, three patterns of shifts in bacterial composition were observed – fold increased, fold decreased, and resilience. The periodic inoculations impacted the succession course of resident bacterial communities in bulk soil, mainly driven by changes in soil pH and nitrate, resulting in the development of three main cluster types throughout the investigation. The single and mixed inoculants transiently modulated the variation in the resident community in association with soil pH and C/N, but finally the community established and showed resilience to following inoculations. Consequently, the necessity of repeated inoculations should be reconsidered, and while the different microbial inoculants showed distinct impacts on resident microbiome succession, communities ultimately exhibited resilience. IMPORTANCE Introducing beneficial microbes to the plant-soil system is an environmentally friendly approach to improve crop yield and soil environment. Numerous studies have attempted to reveal the impacts of inoculation on rhizosphere microbiome. However, little is known about the effectiveness of periodic inoculations on soil functioning. In addition, the impact persistence of repeated inoculations on the native community remains unclear. Here, we track the succession traits of resident microbiome in the bulk soil across a growing season and identify the taxa clusters that diversely respond to periodic inoculation. Crucially, we compare the development of resident community composition with and without inoculation, thus providing new insight into understanding the interactions between resident microbes and intruders. Finally, we conclude that initial inoculation plays a more important role in influencing the whole system, and the native microbial community exhibits traits of resilience, but no resistance, to the subsequent inoculations.

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