3.2.3 Changes in the Structure of Bacterial and Fungal Communities
ASV-level analyses showed that the community compositions under different treatments were significantly different. Non-metric multidimensional scaling (NMDS) analysis (Figure S2) showed extremely significant differences (P < 0.001) in the bacterial and fungal communities under different treatments. For bacteria, the community compositions of the two groups (CB2, V2) that underwent the same base fertilizer treatment were more similar, as were the community compositions of the three groups (CB3, V3, CV3) that underwent the 50-day planting experiment. The community structure of the dried vermiculite group (V1), which was classified as mineral, had significant differences from all other groups. For fungi, there were significant differences in community composition among all groups.
Trends in community changes at the phylum level. To reveal the changes in the microbial community in the same substrate and the differences in these changes in different substrates, we first referred to the corresponding database and analyzed the total bacterial and fungal phylum and genera in the samples, obtaining 38 bacterial phylum and 15 fungal phylum. Figure 2 shows the top ten phylum in terms of relative abundance. The core bacterial phylum were Proteobacteria (19.8% ~ 87.8%), Bacteroidota (1.1% ~ 23.6%), Actinobacteriota (2.0% ~ 19.7%), Firmicutes (0.3% ~ 15.9%), Acidobacteriota (0.1% ~ 20.5%), Patescibacteria (0.5% ~ 8.8%), Verrucomicrobiota (0.1% ~ 8.9%), Bdellovibrionota (0.1% ~ 1.7%), Myxococcota (0.0% ~ 2.9%), Gemmatimonadota (0.0% ~ 2.8%), accounting for more than 93% (93.3% ~ 99.7%) of all treatments.
The composition of the bacterial communities in each group at the phylum level was different (Fig. 2a). In the three treatments of the coconut bran substrate, compared to the dry coconut bran, the relative abundance of Bacteroidota, Patescibacteria, and Bdellovibrionota all increased after the addition of base fertilizer for 3 days, while the relative abundance of Actinobacteriota, Verrucomicrobiota, and Firmicutes significantly decreased. Compared to the addition of base fertilizer for 3 days, after 50 days of planting, there was no significant change in the content of Bacteroidota, but the relative abundance of Proteobacteria and Verrucomicrobiota increased, while the relative abundance of Patescibacteria and Bdellovibrionota decreased to the level of dry coconut bran. The content of Acidobacteriota and Firmicutes continued to decrease in all three groups. In the three treatments of the vermiculite substrate, compared to the dry vermiculite, the relative abundance of Proteobacteria and Actinobacteriota significantly increased after the addition of base fertilizer for 3 days, while the relative abundance of Bacteroidota, Firmicutes, Acidobacteriota, Patescibacteria, Verrucomicrobiota, Myxococcota, Gemmatimonadota significantly decreased. After 50 days of potato planting, the relative abundance of Bacteroidota, Actinobacteriota, Fifty days after planting potatoes, the relative abundance of Bacteroidota, Actinobacteriota and Patescibacteria significantly increased, while that of Proteobacteria and Firmicutes decreased. Overall, Proteobacteria, Bacteroidota, and Actinobacteriota dominated all groups. Unexpectedly, dry vermiculite exhibited greater species diversity than dry coconut bran, but this advantage gradually disappeared after fertilization and planting.
Fungal communities also showed different compositions at the phylum level (Fig. 2b). In the three treatments of coconut bran substrates, the most important fungal phylum were Ascomycota, Basidiomycota, Chytridiomycota, and Rozellomycota. The relative abundance of Ascomycota significantly decreased with the addition of base fertilizer and fermentation, while Basidiomycota significantly increased. After 50 days of potato cultivation, Ascomycota rebounded to the level of dry coconut bran, and Basidiomycota significantly decreased. Meanwhile, the presence of Chytridiomycota and Rozellomycota indicated their advantage in this sample.
For the three treatments of vermiculite substrates, the main fungal phylum were Ascomycota, Basidiomycota, Mortierellomycota, Chytridiomycota, Rozellomycota, and Glomeromycota. Compared to dry vermiculite, the addition of base fertilizer and potato planting caused the relative abundance of Ascomycota, Mortierellomycota, and Chytridiomycota to continuously decrease, while Basidiomycota continuously increased. Overall, Ascomycota and Basidiomycota dominated in the three treatments after the planting experiment, but the dominant phylum differed among samples.
Community changes at the genus level. In total, 1360 bacterial genera and 690 fungal genera were identified, including 71 core bacterial genera (5.2%) and 133 core fungal genera (19.3%). There were significant differences in species composition and dominant genera among groups (Figure S3). For example, the 15 most abundant bacterial genera showed varying degrees of environmental adaptation (Fig. 3a, Table S2a). For instance, most of Massilia was distributed in CB2 and V2, Pseudomonas was mostly found in Group V2, Allorhizobium_Neorhizobium_Pararhizobium_Rhizobium were mostly in V3, CV3, and CB1, Mucilaginibacter mostly in CB2, Flavobacterium mainly in V3, CB3, CV3, Sphingomonas adapted to environments with coconut bran (CB2, CV3, CB3, CB1), Burkholderia_Caballeronia_Paraburkholderia adapted to CV3, Stenotrophomonas was almost entirely in V3, Asticcaulis mainly in CB3, and Noviherbaspirillum mostly in V2. Dyella and Rhodanobacter were almost entirely distributed in CV3 and V3, which included diatomite. On the other hand, there are significant differences in dominant bacterial genera among the different samples (Fig. 3a, Table S2b). In groups A, B, and E, the dominant bacterial genera, ranked from highest to lowest, include Nocardioides, Allorhizobium_Neorhizobium_Pararhizobium_Rhizobium, Sphingomonas (CB1); Mucilaginibacter, Massilia, Sphingobacteriaceae (CB2); Massili, Pseudomonas, Noviherbaspirillum (V2). The distribution uniformity of dominant genera in the rhizosphere samples(CB3, V3, CV3) was significantly higher than that of other groups.
The main fungal genera (Fig. 3b) include Coniochaeta, Rhodotorula, Aspergillus, Papiliotrema, Cladosporium, Alternaria, Plectosphaeralla, Hannaella, Fusarium, and Sampaiozyma. Their distributions and dominant genera varied significantly among groups (Tables S2c, S2d). Coniochaeta almost only existed in groups containing coconut bran, especially in CB3; Rhodotorula was almost only present in groups 3 days after base fertilizer fermentation, particularly in CB2; Aspergillus was found almost exclusively in dry coconut bran (CB1); Papiliotrema was primarily found in groups with diatomite rhizosphere samples (V3, CV3), especially in V3. Sampaiozyma and Plectosphaeralla also adapted to this environment. Furthermore, Hannaella was almost only present in CV3, where it showed a certain advantage.
Lefse analysis revealed taxa that made a significant contribution to the community's uniqueness in different groups. The threshold for bacteria was set at LDA > 4.2, with 39 taxon groups obtained in total (Fig. 4, Table S3a). At the phylum level, in the two groups with a single type of dry substrate, Actinobacteriota was enriched in CB1, while Acidobacteriota and Firmicutes were enriched in V1. At the genus level, Nocardioides and Saccharopolyspora were enriched in CB1, while unclassified Acidobacteriales, Prevotella, and Rikenellaceae RC9 gut group were enriched in V1. In the two groups treated with large amounts of nutrients, Bacteroidota and Patescibacteria were enriched in CB2 at the phylum level, while Proteobacteria were enriched in V2. At the genus level, Mucilaginibacter and Sphingomonas were enriched in CB2, while Blastomonas, Acinetobacter, Noviherbaspirillum, Pseudomonas, and Massilia were enriched in V2. A large number of Proteobacteria (phylum) taxa were enriched in all three rhizosphere soil groups, including Asticcacaulis (from Caulobacterales (class) to genus), unclassified Comamonadaceae (genus), Methylophilaceae (family) in CB3; Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium (genus), Enterobacteriaceae (family) under Enterobacterales (order), Pectobacterium (from Pectobacteriaceae (family) to genus), and Stenotrophomonas (from Xanthomonadales (order) to genus) in V3; and Rhizobiaceae and Xanthobacteraceae (from Rhizobiales (order) to family), Sphingomonadaceae (from Sphingomonadales (order) to family), as well as Burkholderia-Caballeronia-Paraburkholderia (from Burkholderiaceae (family) to genus), Dyella and Rhodanobacter (from Rhodanobacteraceae (family) to genus), Thermomonas (genus) in CV3. In addition, CB3 was also enriched with Opitutaceae (from Verrucomicrobiae (class) to family) under Verrucomicrobiota (phylum), and V3 was enriched with Microbacteriaceae (from Micrococcales (order) to family) and Flavobacterium (from Flavobacteriales (order) to genus).
For fungi, the LDA threshold was set greater than 4.0, and a total of 22 taxon groups were located (Figure S4, Table S3b). Among the two groups with dry substrates, at the phylum level, Ascomycota was enriched in CB1, and Glomeromycota and Mortierellomycota were enriched in V1. At the genus level, Aspergillus was enriched in CB1, while Mortierella, Fusarium, and Cladosporium were enriched in V1. Among the two groups treated with a large amount of nutrients, at the phylum level, Basidiomycota was enriched in CB2. At the genus level, unclassified_Sporidiobolaceae and Rhodotorula were enriched in CB2, while unclassified_Ascomycota, Fusicolla, and Filobasidium were enriched in V2. Among the three rhizosphere soil groups, Coniochaeta (from Sordariomycetes (class) to genus) was its biomarker in CB3; in V3, Eotryotinia (from Helotiales (order) to genus), Hannaella and Papiliotrema under Tremellomycetes (class) were its biomarkers; in CV3, Alternaria (from Pleosporales (order) to genus), Plectosphaerella (from Glomerellales (order) to genus), and unclassified_Basidiomycota (from order to genus) were its biomarkers.