1 Introduction
2 Materials and methods
2.1 Field experiment, soil sampling and soil properties analysis
2.2 Soil DNA extraction and quantitative PCR
2.3 MiSeq sequencing and bioinformatics analyses
2.4 Microbial network construction
2.5 Statistical analyses
2.6 Calculation of resistance
3 Results
3.1 Fungal abundances and communities under long-term chemical fertilizations
![](https://academic.hep.com.cn//article\2022\2662-2289/2662-2289-4-4-348/thumbnail/sel-00120-ghw-fig1.jpg)
Fig.1 Fungal abundance for NoF, for P, K fertilizations and for N fertilizations assessed by qPCR (A). Shannon diversity index for NoF, for P, K fertilizations and for N fertilizations (B). Dissimilarity of fungal community structure between and within fertilization regimes based on their Bray-Curtis distance (C). Index values of fungal community resistance after different fertilizers addition (D). P, K fertilizations include P, K and PK fertilization treatments, and N fertilizations include N, NK, NP and NPK fertilization treatments. Bars with different letters indicate significant differences (P<0.05, ANOVA). |
3.2 Fungal responding taxa to long-term chemical fertilizations
![](https://academic.hep.com.cn//article\2022\2662-2289/2662-2289-4-4-348/thumbnail/sel-00120-ghw-fig2.jpg)
Fig.2 Volcano plots showing the significantly enriched and depleted fungal OTUs after addition of chemical fertilizers (A). Percentage proportions of the significantly varied OTUs. Variation patterns of responding fungal OTUs after P, K fertilizers and N fertilizers addition (B). The x-axis indicates the number of fungal OTUs, and the y-axis indicates the variation of the relative abundance (VRA). P, K fertilizations include P, K and PK fertilization treatments, and N fertilizations include N, NK, NP and NPK fertilization treatments. |
Tab.1 Variation of fungal communities based on responding taxa after P, K fertilizations and N fertilizations addition relative to NoF. |
P, K fertilizations vs NoF | N fertilizations vs NoF | |||
---|---|---|---|---|
CRTa | Phylum | 0 | 0 | |
Genus | 15 | 44 | ||
OTU | 88 | 330 | ||
VRAb | Phylum | 0 | 0 | |
Genus | 0.58% | 10.9% | ||
OTU | 0.81% | 17.1% | ||
VRA/CRTOTU | 0.01% | 0.05% |
a CRT, count of responding taxa. b VRA, variation of the relative abundance. P, K fertilizations include P, K and PK fertilization treatments, and N fertilizations include N, NK, NP and NPK fertilization treatments. |
3.3 Prediction of fungal functional profiles
![](https://academic.hep.com.cn//article\2022\2662-2289/2662-2289-4-4-348/thumbnail/sel-00120-ghw-fig3.jpg)
Fig.3 Relative abundance of fungal functional modes and guilds assigned using FUNGuild database for NoF, for P, K fertilizations and N fertilizations (A). Relative abundance of responding functional guilds under P, K fertilizations and N fertilizations (B). P, K fertilizations include P, K and PK fertilization treatments, and N fertilizations include N, NK, NP and NPK fertilization treatments. |
3.4 Fungal networks under long-term chemical fertilizations
![](https://academic.hep.com.cn//article\2022\2662-2289/2662-2289-4-4-348/thumbnail/sel-00120-ghw-fig4.jpg)
Fig.4 Subnetwork of responding plant pathogen under P, K fertilizations (A) and N fertilizations (B), and relevant subnetwork parameters shown in table (C). P, K fertilizations include P, K and PK fertilization treatments, and N fertilizations include N, NK, NP and NPK fertilization treatments. Blue and red lines indicate positive and negative interactions between two individual nodes, respectively. The responding nodes assigned to potential plant pathogen are colored in red and identified at the genus level or higher. The size of each node is proportional to its degree (i.e., the number of edges of a node to other nodes). |
3.5 Soil properties and fungal communities associated with soybean yields
Tab.2 Effects of soil properties on fungal Shannon diversity, community structure, community resistance and network connectivity. |
Variables | Shannon diversity | Community structure | Community resistance | Network connectivity | |||||||
---|---|---|---|---|---|---|---|---|---|---|---|
ra | P | ra | P | rb | P | rb | P | ||||
pH | 0.625** | 0.001 | 0.938** | <0.001 | 0.918** | <0.001 | 0.368** | 0.001 | |||
TC | –0.404* | 0.048 | 0.704** | <0.001 | 0.547** | 0.009 | 0.097* | 0.010 | |||
TN | –0.469* | 0.021 | 0.845** | <0.001 | –0.742** | <0.001 | 0.294** | 0.001 | |||
TP | 0.130 | 0.543 | –0.242 | 0.254 | 0.439* | 0.046 | –0.176 | 1.000 | |||
TK | –0.183 | 0.393 | 0.365 | 0.080 | 0.040 | 0.864 | –0.043 | 0.867 | |||
NH4+-N | –0.238 | 0.262 | 0.319 | 0.129 | –0.349 | 0.121 | –0.095 | 1.000 | |||
NO3–-N | –0.514** | 0.009 | 0.8107** | <0.001 | –0.737** | <0.001 | 0.258** | 0.001 | |||
AP | 0.090 | 0.677 | –0.189 | 0.376 | 0.421 | 0.057 | –0.183 | 1.000 | |||
AK | –0.183 | 0.392 | 0.100 | 0.642 | 0.025 | 0.913 | –0.128 | 1.000 |
a Correlations between fungal Shannon diversity, community resistance and soil properties examined by Spearman correlation coefficient. b Correlations between fungal community structure, network connectivity and soil properties examined by partial Mantel test. **, P<0.01; *, P<0.05. |
![](https://academic.hep.com.cn//article\2022\2662-2289/2662-2289-4-4-348/thumbnail/sel-00120-ghw-fig5.jpg)
Fig.5 Structural equation modeling (SEM) relating responding taxa, fungal community indices and network structure affect soybean yields. Solid and dashed lines indicate significant and insignificant relationships, respectively. The widths of arrows represented the effect strength, with blue and red indicating positive and negative relationships, respectively. RT, responding taxa; RP, responding plant pathogen; RPN, responding plant pathogen detected in network. Alpha diversity represented by Shannon diversity, beta diversity represented by PCoA1 index, network connectivity as an indicator of network structure which is equal to average node degree. R2 values indicate proportion of the variance explained for the endogenous variable. , Chi-square value; df, degree of freedom; GFI, goodness of fit index; RMSEA, root mean square error of approximation. ***, p<0.001; **, p<0.01. |