Effect of arbuscular mycorrhizal fungi (AMF) and plant growth promoting rhizobacteria (PGPR) on microbial community structure of phenanthrene and pyrene contaminated soils using Illumina HiSeq sequencing

In order to determine the influence of arbuscular mycorrhizal fungi (AMF, Glomus versiforme) and plant growth promoting rhizobacteria (PGPR, Pseudomonas fluorescens, PS2-6) on degradation of phenanthrene (PHE) and pyrene (PYR) and the change of microbial community structure in soils planted with tall fescue (Festuca elata), four treatments were set up in phenanthrene (PHE) and pyrene (PYR) contamined soil: i.e., tall fescue (CK), AMF + tall fescue (GV), PGPR + tall fescue (PS) and AMF + PGPR + tall fescue (GVPS), PHE and PYR dissipation in the soil and accumulated in the tall fescue were investigated. Our results showed that highest removal percentage of PHE and PYR in contaminated soil as well as biomass of tall fescue were observed in GVPS. PHE and PYR accumulation by tall fescue roots were higher than shoots, the mycorrhizal status was best manifested in the roots of tall fescue inoculated with GVPS, and GVPS significantly increased the number of PGPR colonization in tall fescue rhizosphere soil. And paired-end Illumina HiSeq analysis of 16S rRNA and Internal Transcribed Spacer (ITS) gene amplicons were also employed to study change of bacterial and fungal communities structure in four treatments. GVPS positively affected the speices and abundance of bacteria and fungi in PHE and PYR contaminated soil, an average of 71,144 high quality bacterial 16S rDNA tags and 102,455 ITS tags were obtained in GVPS, and all of them were assigned to 6,327 and 825 operational taxonomic units (OTUs) at a 97% similarity, respectively. Sequence analysis revealed that Proteobacteria was the dominant bacterial phylum, Ascomycota was the dominant fungal phylum in all treatments, whereas Proteobacteria and Glomeromycota were the most prevalent bacterial and fungal phyla in GVPS, respectively. And in the generic level, Planctomyces is the richest bacterial genus, and Meyerozyma is the richest fungal genus in all treatments, whereas Sphingomona was the dominant bacterial genus, while the dominant fungi was Fusarium in GVPS. Overall, our findings revealed that application of AMF and PGPR had an effective role in improving the growth characteristics, root colonization of F. elata and soil microbial community structure in PHE and PYR contaminated soils, but no obvious in degradation efficiencies of PAHs as compared to the control.

which were cultured in beef extract peptone medium and inorganic salt medium for standby.

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Other samples of roots and shoots were then freeze-dried and ground, in preparation for PAHs analysis. The entire 113 soil in each pot was thoroughly homogenized, ground sufficiently to pass through a 100-mesh sieve, and divided into two 114 sets. One was stored at −20°C for DNA extraction, and the other was stored at 4 °C for PAHs analysis.   (Table 3).

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The total number of OTUs detected at 97% shared sequence similarity was very high in PHE and PYR contaminated  (Table 3).

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Venn diagrams were performed in R, based on the shared OTU tables from 4 different soil groups ( Fig. 2A). The

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At phylum level, the CK, GV, PS and GVPS samples shared common phyla, Proteobacteria was the most prevalent 233 bacteria phylum, while different proportions of valid reads from 33.80% to 41.73% were observed for all treatments.

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More Proteobacteria taxa (41.73%) were detected in GVPS than in GV, PS and CK (Fig. 3A). Fungal classification 235 results showed that the dominant phylum was Ascomycota, accounting for 33.13-52.04% of all valid reads, with an 236 average relative abundance of 43.56%. The next most dominant fungal phyla were Chytridiomycota (average abundance 237 12.13%) and Basidiomycota (average abundance 6.60%), and the abundance of Glomeromycota (0.27%) in GVPS was 238 significantly higher than that in GV, PS and CK treatment (Fig. 3B).

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Planctomycetaceae (average abundance 6.25%), and Sphingomonadaceae (average abundance 3.59%). The abundance 241 of Xanthomonadaceae (12.58%), Phytophthoraceae(6.76%) and Sphingomycidae(4.43%) in GVPS was significantly 242 higher than others (Fig. 3C) bacterial genera had high abundances in the CK, GV, and PS, but the Sphingomonas, Planctomyces, and Arenimonas 260 genera had a relatively high abundance in the GVPS (Fig. 4A). For fungi, heatmap clustering analysis showed that 261 Meyerozyma and Spizellomyces fungus genera had relatively high abundances among all the treatments, while Fusarium 262 had a high abundances in GVPS (Fig. 4B). These finding were consistent with previous results (Fig. 3). and fungi calculated at 97% levels showed that the order of OTUs numbers from high to low among samples both were 268 GVPS > PS > GV > CK. And the OTU densities of GVPS was higher than the other three treatments (Fig. A.1). The 269 bacteria and fungi richness based on rarefaction curves were strongly supported by statistical diversity estimates, based and ACE of GVPS treatment were higher, which indicated that the richness of microbial community under GVPS

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In addition, the GV samples had a relatively higher PC1 value, followed by PS and GVPS treatment, whereas the CK 284 samples had a higher PC2 value at OTU level (Fig. 5A). In fungi, the GVPS groups had a relatively higher PC1 value, 285 followed by PS and CK, while the samples from GV were closer than the other groups. Meanwhile, No significant 286 gathering were observed among four groups (Fig. 5B).

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UPGMA clustering obtained a phylogenetic tree by using unweighted group averaging method (Fig. A.2.). Result 288 indicates that same type of samples showed high similarity of bacterial communities (Fig. A.2-A), while similarity of 289 fungal communities from the same treatment is relatively weaker (Fig. A.2-B).