Interaction between arbuscular mycorrhizal fungi and native soil microbiome on early stage restoration of a coal-mine soil

Abstract

The recovery of the soil ecosystem after severe disturbances, such as coal-mining activities, depends on both abiotic and biotic improvements. This study assessed the influence of arbuscular mycorrhizal (AM) fungal consortia on microbial community dynamics across two stages of soil recovery - 2 years (2Y) and 15 years (15Y) post-disturbance - using a secondary succession forest (SSR) as a reference. We analyzed bacterial community composition via 16 S rRNA gene amplicon sequencing and evaluated key soil quality indicators. While inoculation with AM fungal consortia had minimal effects on most soil parameters, significant differences were observed between recovery stages. The 15Y recovery site exhibited improved soil structure, microbial activity, and aggregate stability compared to the 2Y site, highlighting the importance of long-term restoration. However, potential overlap in ecological roles among native microorganisms likely mitigates the impact of AMF inoculation. These findings suggest that AM fungal consortia alone may not drive immediate improvements in soil quality but can contribute to microbial interactions and recovery processes over time. This study highlights the complexity of soil restoration and emphasizes the need for strategies that integrate plant cover with microbial community development to enhance long-term ecosystem stability. Further research should explore the specific roles of AM fungi and native soil microbes in promoting soil structure and accelerating recovery.

Keywords: AM fungal consortia; Combined inoculation; Glomeromycota; Microbiome; Simplified community.

Fig. 1

Field sampling locations in Siderópolis, Brazil, highlighting sites with distinct restoration durations: 2 years (2Y) and 15 years (15Y) post-reforestation, alongside a secondary succession forest (SSF) serving as a reference site. Scheme generated in BioRender.com

Fig. 2

Soil parameters post-greenhouse experiment. Soil attributes measured include (A) percentage of soil macroaggregates, (B) total glomalin-related soil protein (T-GRSP), (C) acid phosphatase activity measured as p-nitrophenyl phosphate (PNP), (D) metabolic quotient (qCO₂), and (E) microbial biomass carbon (C-MB). Comparisons were made between control samples and various treatments: AM consortium1 (Acaulosporaceae), AM consortium2 (Gigasporaceae), AM consortium3 (Acaulosporaceae and Gigasporaceae), AM consortium4 (AM fungal richness, n = 4), AM consortium5 (AM fungal richness, n = 8), and AM consortium6 (AM fungal richness, n = 16). Analyses were conducted within each site: 2 years post-reforestation (2Y), 15 years post-reforestation (15Y), and a secondary succession forest (SSF) was used as a reference. Statistical significance between treatments and sites was assessed using ANOVA, with significance levels denoted by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001). Vertical bars represent contrasts between sites (Two-way ANOVA- Contrast analysis), and standard errors are depicted as black lines

Fig. 3

Total AM spore count and AM fungal colonization of Urochloa brizantha across mining sites with short-term recovery (2Y), long-term recovery (15Y), and a secondary succession forest (SSF) observed post-greenhouse experiment. (A) Total number of AM spores per 50 g of soil (mean ± standard deviation); (B) Percentage of AM fungal colonization in U. brizantha roots (mean ± standard deviation); (C) Total AM spore count and species recovered from each microcosm. Comparisons between control and treatment samples [AM consortium1 (family Acaulosporaceae); AM consortium2 (family Gigasporaceae); AM consortium3 (families Acaulosporaceae and Gigasporaceae); AM consortium4 (AM richness, n = 4); AM consortium5 (AM richness, n = 8); AM consortium6 (AM richness, n = 16)] at each site (2Y, 15Y, and SSF) were performed using Two-way ANOVA with contrast analysis. Vertical bars represent species’ presence across sites. Statistical significance is denoted by asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001). Standard errors are shown as black lines. Data is presented in triplicate

Fig. 4

Principal Component Analysis (PCA) and Multivariate Analysis of Variance (MANOVA) were used to analyze how AM inoculation treatments relate to soil quality indicators in soils from mining sites with short-term recovery (2Y), long-term recovery (15Y), and secondary succession forest (SSF). Comparisons are made between control and treatment samples, which include AM consortium1, family Acaulosporaceae; AM consortium2, family Gigasporaceae; AM consortium3, families Acaulosporaceae and Gigasporaceae; AM consortium4, AM richness (n = 4); AM consortium5, AM richness (n = 8); AM consortium6, AM richness (n = 16), within each site, (A) short-term recovery (2Y); (B) long-term recovery (15Y), (C) secondary succession forest (SSF)

Fig. 5

Analysis of microbial diversity and composition based on 16 S rRNA gene amplicon sequencing in different treatments, AM consortium1 (Acaulosporaceae), AM consortium2 (Gigasporaceae), AM consortium3 (Acaulosporaceae and Gigasporaceae), AM consortium4 (AM fungal richness, n = 4), AM consortium5 (AM fungal richness, n = 8), and AM consortium6 (AM fungal richness, n = 16), within each site, short-term recovery (2Y), long-term recovery (15Y), and a secondary succession forest (SSF). (A) Alpha diversity is represented by Shannon indices, comparing diversity across different treatments to control samples. Statistical significance was assessed using the Mann-Whitney U test with p-values adjusted by the Bonferroni method. Standard errors are shown as vertical black lines. (B) Beta diversity displays differences in microbial community composition among treatments using multidimensional scaling (MDS). (C) Relative abundance (%) of the most prevalent phyla in the soil is shown. Taxa with fewer than 350 sequences in less than three samples are grouped as “Other”

Fig. 6

Relative abundance of Myxococcota across different sites (2Y, 15Y, and SSF) and treatments, AM consortium1 (Acaulosporaceae), AM consortium2 (Gigasporaceae), AM consortium3 (Acaulosporaceae and Gigasporaceae), AM consortium4 (AM fungal richness, n = 4), AM consortium5 (AM fungal richness, n = 8), and AM consortium6 (AM fungal richness, n = 16)