Succession of the Resident Soil Microbial Community in Response to Periodic Inoculations

Abstract

To maintain the beneficial effects of microbial inoculants on plants 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 the 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, coinoculation better stimulated plant growth and soil nutrients. However, the benefits from inoculants did not increase with repeated inoculations and were not maintained after transplantation to a different site. In response to microbial inoculants, three patterns of shifts in the bacterial composition were observed: fold increase, fold decrease, 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 the C/N ratio, but finally, the community established and showed resilience to subsequent inoculations. Consequently, the necessity of repeated inoculations should be reconsidered, and while the different microbial inoculants showed distinct impacts on resident microbiome succession, the communities ultimately exhibited resilience.IMPORTANCE Introducing beneficial microbes to the plant-soil system is an environmentally friendly approach to improve the crop yield and soil environment. Numerous studies have attempted to reveal the impacts of inoculation on the rhizosphere microbiome. However, little is known about the effectiveness of periodic inoculations on soil functioning. In addition, the long-term impact of repeated inoculations on the native community remains unclear. Here, we track the succession traits of the resident microbiome in the bulk soil across a growing season and identify the taxon clusters that respond differently to periodic inoculation. Crucially, we compare the development of the resident community composition with and without inoculation, thus providing new insight into 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.

Keywords: beneficial microorganisms; inoculant type; microbial community succession; periodic inoculation; resident microbiome; soil remediation.

FIG 1

Dynamic growth of C. paliurus height (a) and ground diameter (d) in Baima (inoculation period), final height (b) and ground diameter (e) of C. paliurus in Taizhou (transplantation period), and relative growth rates of height (c) and ground diameter (f) in Baima and Taizhou. The sampling days were I-, II-, III-, and IV-45 (45 days after the first, second, third, and fourth inoculations, respectively). The treatments were M or C (single application of Bacillus megaterium or Azotobacter chroococcum, respectively), MF (dual application of B. megaterium and Pseudomonas fluorescens), CB (dual application of A. chroococcum and Azospirillum brasilense), MFCB (application of all four strains), and CK (noninoculation).

FIG 2

Heat map of the bacterial community at the family level (top 50) under periodic inoculations over time. Black boxes indicate the statistical significance of differences between treatments at each time point. a, b, and c show different changing patterns of bacterial taxa across all sampling time points clustered based on abundance similarities between taxa. The sampling days were 0d (the day before microbial inoculation); I-10, I-30, and I-45 (10 days, 30 days, and 45 days after the first inoculation, respectively); and II-, III-, and IV-45 (45 days after the second, third, and fourth inoculations, respectively). The treatments were M or C (single application of Bacillus megaterium or Azotobacter chroococcum, respectively), MF (dual application of B. megaterium and Pseudomonas fluorescens), CB (dual application of A. chroococcum and Azospirillum brasilense), MFCB (application of all four strains), and CK (noninoculation).

FIG 3

Temporal variation of bacterial community structure under different soil managements. (a) Succession of the resident soil bacterial community as revealed by principal coordinates of Bray-Curtis similarities. (b) Bacterial community clusters (PCoA plot) and their dominations (bar plot) in the succession of noninoculated soils across all time points. NonIno_0-10d and NonIno_30-180d indicate two main clusters for noninoculated samples as derived from community type analysis. (c) Bacterial community clusters and their dominations in the succession of inoculated soils across all time points. Ino_0-10d, Ino_30-45d, and Ino_90-180d indicate three main clusters for inoculated samples as derived from community type analysis. (d) Differences in phylum abundances among the three clusters found in the inoculated soils according to linear discriminant analysis (LDA) scores. The sampling days were 0d (the day before inoculation); I-10, I-30, and I-45 (10 days, 30 days, and 45 days after the first inoculation, respectively); and II-, III-, and IV-45 (45 days after the second, third, and fourth inoculations, respectively). The treatments were M or C (single application of Bacillus megaterium or Azotobacter chroococcum, respectively), MF (dual application of B. megaterium and Pseudomonas fluorescens), CB (dual application of A. chroococcum and Azospirillum brasilense), MFCB (application of all four strains), and CK (noninoculation).

FIG 4

Redundancy analysis illustrating the effects of environmental factors on the succession of the bacterial community and top 10 families across all treatments. The sampling days were 0d (the day before inoculation); I-10, I-30, and I-45 (10 days, 30 days, and 45 days after the first inoculation, respectively); and II-, III-, and IV-45 (45 days after the second, third, and fourth inoculations, respectively).

FIG 5

Typing analysis of the temporal variation of bacterial community structure under different inoculations at I-10, I-30, I-45, and IV-45. Different routes from type 1 to type 4 were identified: type 1-2-4 (for treatments with MF, CB, and MFCB) or type 1-3-4 (for treatments with M and C). The column diagram indicates a single-complex-single pattern of change in the presence of the four cluster types at the five sampling time points. The sampling days were 0d (the day before inoculation); I-10, I-30, and I-45 (10 days, 30 days, and 45 days after the first inoculation, respectively); and IV-45 (45 days after the fourth inoculation). The treatments were M or C (single application of Bacillus megaterium or Azotobacter chroococcum, respectively), MF (dual application of B. megaterium and Pseudomonas fluorescens), CB (dual application of A. chroococcum and Azospirillum brasilense), and MFCB (application of all four strains).

FIG 6

Timeline for microbial inoculation, soil sampling, and plant growth measurement. The major part of the experiment was conducted in 2018 in Nanjing (inoculation period). After that, seedlings were transplanted to Taizhou in 2019 (transplantation period).