Overview of the soil microbiome
For each sample, six repetitions were collected, for a total number of 24 samples in four gropes. A total of 170,115 and 192,199 CCS sequences were obtained from the 16S rRNA gene and ITS sequencing of the four groups of samples by the PacBio Sequel, respectively. Of them, 143646 and 183025 effective CCS were obtained after identified PacBio barcode sequences, quality-filtering, and removing chimaera, respectively. Each sample sequence numbers of bacteria ranged from 3,790 to 7,058 with a median of 6,684, and the average read length was 1,457 bp (median of 1,457 bp). Each fungal sample sequences ranged from 7,057 to 7,831 with a median of 7,669, and the average read length was 631 bp (median 620 bp).
These CCS sequences were divided into 1,624 bacterial OTUs and 1,080 fungal OTUs. Each sample contained 424 to 885 bacterial OUTs and 214 to 450 fungal OUTs. Of the 1,624 bacterial OTUs, 644 were shared among the four samples, 153 were specific to pitaya groups (Hp) and 217 were specific to grape soils (Vv) (Fig. 1a). There were 194 common fungal OTUs were found in all the four soils, meanwhile 193 in the soil planted pitaya and 82 in grape soils (Fig. 1b).
Microbiota diversity is conditioned by site, cultivation and titanium ions
The Shannon and Chao1 indexes of bacteria ranged from 7.71 to 8.89 and 635.58 to 1152.52, while those of fungi ranged from 5.40 to 7.34 and 217 to 476, respectively (Fig. 2). In the pitaya soil, there were no significant differences (p>0.05) in the Shannon and Chao1 indexes of bacterial communities between the HpCon and HpTi groups (Fig. 2a). However, the Shannon indexes of fungal communities of HpCon (Fig. 2b) were higher than that of HpTi (p<0.05). Moreover, in the grape soil, the Shannon indexes of bacterial communities and Chao1 indexes of fungal communities were significantly decreased after using titanium (p<0.05).
The PCoA (Fig. 3) showed that the soil community were distinct. The four groups were separated, and the repetitions of each group clustered together. The difference between communities were significant (p=0.001) in both bacterial and fungal as PERMANOVA showed. In addition, the two agroecosystems of pitaya and grape were separated and the distance within a same site was relatively close. For subsequent analyses, the data for using titanium ions in pitaya and grape were discussed separately.
The microbiome structure is altered by location, cultivation and titanium ions
The change of bacterial community structure
The structure and richness of bacteria were intensely altered by titanium. In the pitaya soil, the bacterial communities were composed of 25 (HpCon) and 27 (HpTi) phyla, respectively. The seven most abundant bacterial phyla (Fig. 4a) were Proteobacteria (mean relative abundance: 39.2% in HpCon and 46.0% in HpTi), Acidobacteria (16.3% and 14.4%), Bacteroidetes (10.1% and 10.3%), Planctomycetes (12.5% and 8.2%), Actinobacteria (7.0% and 4.5%), Verrucomicrobia (5.7% adn 5.8%) and Gemmatimonadetes (4.7% and 4.1%). These phyla represented 95.6% and 93.2% of sequences from HpCon and HpTi, respectively. The relative abundance of Proteobacteria and Rokubacteria (0.3% in HpCon and 2.3% in HpTi) were significantly higher and Planctomycetes were significantly lower in the soil using titanium ions (p<0.05).
Further classified in the family level, there were 24 families with average relative abundance >1% were identified in the pitaya soils, accounted for 70.8% and 63.6% in HpCon and HpTi respectively. Chitinophagaceae (7.6% in HpCon and 8.5% in HpTi, belong to Bacteroidetes), uncultured_bacterium_c_Subgroup_6 (8.0% and 6.6%, Acidobacteria), Rhodanobacteraceae (0.6% and 9.4%, Proteobacteria), Nitrosomonadaceae (4.9% and 3.8%, Proteobacteria) and Gemmatimonadaceae (4.4% and 3.9%, Gemmatimonadetes) were the most dominant bacterial families (Fig. 5a). The relative abundance of Rhodanobacteraceae were dramatically (p<0.001) higher in the applied-titanium soil.
For the soil samples planted grape, at the phyla level, all the sequences were classified into 27 phyla. Distribution of the 10 most abundant phyla were showed in Fig. 4a. Proteobacteria (45.6% in VvCon and 45.6% in VvTi) was the most abundant phyla, followed by Acidobacteria (14.2% and 10.8%), Planctomycetes (11.1% and 10.3%), Gemmatimonadetes (8.1% and 12.7%) and Bacteroidetes (8.1% and 6.6%). The abundances of Acidobacteria were significantly lower in VvTi than that in VvCon, conversely Gemmatimonadetes showed higher abundances after using titanium (p<0.05).
As for the family level in grape soils, there were 232 families were identified, although 94 of which were uncultured. Bacterial communities were dominated by uncultured_bacterium_c_Subgroup_6 (9.9% in VvCon and 7.9% in VvTi), Nitrosomonadaceae (9.0% and 13.5%) and Gemmatimonadaceae (5.2% and 9.8%). Fellow 20 dominated family with mean relative abundance >1% accounted for 64.6% and 66.2% of the sequences in VvCon and VvTi respectively. The abundances of Nitrosomonadaceae and Gemmatimonadaceae were remarkably higher in the titanium-applied soil than that control group (p<0.01). In contrast, the abundance of Chitinophagaceae (3.7% and 1.8%, Bacteroidetes), Comamonadaceae (1.7% and 0.6%, Proteobacteria), Xanthomonadaceae (3.3% and 1.3%, Proteobacteria) and TRA3-20 (3.7% and 2.5%, Proteobacteria) were substantially lower in the titanium-applied soil (p<0.05).
The change of fungal community structure
In the pitaya-planted soil, a total of 13 fungal phyla were identified. The most important phyla accounted for 98.3% of the sequences in each group, including Ascomycota (51.9% in HpCon and 56.7% in HpTi), Basidiomycota (23.5% and 12.3%), Mortierellomycota (11.0% and 14.7%), Chytridiomycota (2.7% and 9.0%), Glomeromycota (8.6% and 2.9%), and Rozellomycota (0.6% and 2.8%)., The relative abundance of Chytridiomycota increased significantly (p<0.05) and the Basidiomycota and Glomeromycota decreased significantly (p<0.05) in the titanium-applied soil.
At the genus level, a total of 237 genera were identified, with 39 genera having a relative abundance greater than 1% in at least one sample, while 22.3% of sequences could not be classified to unknown genera. Furthermore, the dominant genera of fungal communities were Mortierella (10.8% in HpCon and 14.5% in HpTi, belong to Mortierellomycota), Fusarium (12.8% and 7.3%, Ascomycota), Penicillium (2.6% and 11%, Ascomycota) and Thanatephorus (5.7% and 3.4%, Basidiomycota). The relative abundance of Penicillium and Cladosporium (1.0% and 3.1%, Ascomycota) dramatically increased response to titanium ions, in contrast, the relative abundance of Fusarium and Purpureocillium (3.5% and 0.99%, Ascomycota) significantly decreased (p<0.05).
In the grape-planted soil, the fungal community were composed of 11 phyla and other unclassified phyla (3.1%~4.1%) (Fig. 5). The five most abundant phyla were Ascomycota (51.4% in VvCon and 34.2% in VvTi), Basidiomycota (21.2% and 19.1%), Mortierellomycota (16.6% and 25.0%), Chytridiomycota (2.8% and 14.4%), and Glomeromycota (3.5% and 3.1%). The relative abundance of Chytridiomycota and Rozellomycota were significantly higher in the soil using titanium ions (p<0.05) and the abundance of Ascomycota and Basidiomycota were significantly lower (p<0.05).
As for the genus level of fungi, about 69.8% to 82.0% of sequences were classified into 222 genera, and the 10 most abundant genera are shown in Fig. 4. The communities were dominated by Mortierella (16.0% in VvCon and 25.0% in VvTi), Fusarium (7.0% and 7.9%), Botryotrichum (7.0% and 1.3%, Ascomycota), Leucoagaricus (0.22% and 6.4%), Colletotrichum (4.4% and 1.7%), Wallemia (5.2% and 0) and Cladosporium (2.2% and 1.5%). The relative abundance of Leucoagaricus and Penicillium (0.7% and 1.6%) were elevated significantly in the titanium-applied soil and the Dactylonectria (3.3% and 0.14%) were reduced (p<0.001).
Titanium ions enhanced the cooccurrence relationships among the bacteria and fungi
The co-occurrence and co-exclusion network of OTUs in bacteria and fungi in soil community were analyzed (Fig. 6). We found that the co-occurrence was dominant in the interaction network. Fungi with high abundance were important nodes in the interaction network, including Ascomycota, Glomeromycota, Chytridimycota and Basidiomycota, etc. Co-occurrence relationship exists not only in the interior of high abundance phylum, but also between high abundance and other low abundance phylum, as well as between bacteria and fungi. In pitaya soil interaction network, the co-occurrence relationship of HpCon accounted for 67%, while that of HpTi accounted for 81%. As for grape soils, the proportion of lines representing co-occurrence increased from 67% to 70% after titanium application.