The application of fertilizers can significantly change the nutrient availability of plants and the diversity and function of microorganisms [1]. The excessive use of chemical fertilizer has caused a series of environmental problems (such as the decrease in nutrient utilization efficiency, soil quality deterioration, etc.) [2]. Soil microorganisms not only have extremely rich genetic and functional diversity, but also play important roles in soil nutrient conversion, maintaining soil productivity, and promoting the sustainable development of ecosystems [3]. Soil microbial-community diversity and composition are also important indicators that reflect the evolution of soil quality. Studies have shown that long-term application of chemical fertilizers significantly decreased the bacterial richness of soil and its diversity, and disturbed the ecological balance of soil microbial communities [4,5]. Organic amendments have been shown to be more effective than inorganic fertilizer in improving soil biological characteristics [6], especially in terms of increasing the microorganisms beneficial to plant growth, and decreasing plant pathogenic microorganisms [7]. Thus, an effective way to solve the problems caused by the excessive application of chemical fertilizers is to apply organic amendments alone or in combination with inorganic fertilizers [8,9,10].

Compost is an important form of organic amendment, which plays an important role in improving soil properties and crop growth [11,12]. Some researchers found that compost addition could improve soil microbial activity and diversity [9,13]. However, some other studies have demonstrated that compost has neutral or negative effects on the soil microbial community, which may be due to differences in compost type, dosage and duration time [14,15,16]. Therefore, it is necessary to use long-term experiments to study the effects of compost on soil microbial diversity and community structure. Bio-compost is a type of compost that is compounded by microorganisms with specific functions using organic waste (such as animal manure, straw and sewage sludge) [12]. Bio-compost is considered to be more effective than normal compost because it is rich in beneficial microbial flora and physiologically active substances (such as indoleacetic acid, gibberellin, vitamins and amino acids).

Network analysis is used to explore the microbial interactions between different microbial taxa under different fertilization treatments [17]. In addition, we can also find key functional microorganisms that have an important influence on the microbial community structure and potential functions by network analysis, which helps to deeply understand the diversity and function of the microbial community [18].

To sum up, it is of great significance to carry out research on the effects of bio-compost on the composition, structure and function of soil bacterial and fungal communities so as to elucidate the internal mechanism of soil microbial changes after the application of bio-compost, and the promotion of soil ecological health. Therefore, based on a 25-year long-term field experiment, we systematically studied the effect of long-term application of bio-compost on soil microbial composition, co-occurrence and function using high-throughput sequencing of soil bacteria 16S rRNA gene and fungal ITS gene data, network analysis, and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) and FUNGuild. The objectives of this study were to (i) study the differences in soil microbial diversity, composition and community structure under long-term different fertilization treatments; (ii) analyze the correlation between soil properties and microbial communities under different fertilization treatments; (iii) analyze bacterial and fungal co-occurrence patterns and predict the function of bacteria and fungi; (iv) preliminarily reveal the response mechanism of soil microorganisms to long-term different fertilization. We hypothesized that the application of bio-compost would significantly improve soil microbial diversity, increase beneficial microorganisms, decrease disease-causing microorganisms, and alter soil microbial co-occurrence patterns and metabolic functions.

The richness and diversity of the microbial community play a critical role in the functions of soil, and they can be affected by fertilization. The Chao1 and Shannon indices describe the richness and diversity of microbial community, respectively, with the larger values indicating greater richness and diversity [26]. Our results have demonstrated that a high dosage of bio-compost had a greater impact on bacterial and fungal richness than CF and EMII treatments, which implied that bacterial and fungal richness were affected by the dosage of compost and the type of amendments [27,28]. The bacterial-richness index increased with increasing dosages of bio-compost, which could be attributed to the added nutrients with the compost application. Interestingly, fungal richness showed no dose-dependent effect by bio-compost. It was generally believed that microbial diversity increased with manure application [28,29], while our results showed that a high dosage of bio-compost increased fungal richness, while a conventional dosage of bio-compost reduced fungal richness. There were two reasons for this, one was that bio-compost stimulated the proliferation of certain specific microorganisms and inhibited the growth of other microorganisms, and the other was that most soil nutrients were negatively correlated with the fungal chao1 index. Thus, high dosages of bio-compost had a greater impact on soil microbial alpha-diversity. There is a high dosage of bio-compost had a greater impact on bacterial and fungal richness than CF and EMII treatments, which implied that bacterial and fungal richness were affected by the dosage of compost and the type of amendments [27,28]. The bacterial-richness index increased with increasing dosages of bio-compost, which could be attributed to the added nutrients with the compost application. The high dosage of bio-compost increased fungal richness, while a conventional dosage of bio-compost reduced fungal richness. There were two reasons for this, one was that bio-compost stimulated the proliferation of certain specific microorganisms and inhibited the growth of other microorganisms, and the other was that most soil nutrients were negatively correlated with the fungal chao1 index. Thus, high dosages of bio-compost had a greater impact on soil microbial alpha-diversity.

Soil bacterial richness and diversity increased with the increase in WC, and it was proposed that the main reason for this was that bacterial growth can promote a more positive response to the increase in soil moisture by bio-compost [33]. Soil bacterial richness and diversity increased with the increase in NH₄⁺-N, which may be because the increase in NH₄⁺-N caused by bio-compost provided a source of nitrogen for microorganism [34]. In addition, the fungi had a wide pH range (5–9) [32], which was conducive to the optimal growth of fungi, explaining that pH may increase fungal diversity.

2.2. Significant Effect of Bio-Compost on Soil Microbial Community Composition

Soil microorganisms play a key role in N cycle and organic matter dynamics. Changes in the structure and composition of the soil microbial community will lead to changes in soil quality [35]. It is well known that fertilization can significantly change the soil microbial-community structure and composition [13,33]. The bio-compost application altered the community structure of soil bacteria and fungi, because the specific functional microorganisms in the bio-compost entered the soil and interacted with the indigenous microorganisms of the soil, further altering soil microbial community structure [36].

2.3. Bio-Compost Changed the Dominant Community Representatives and Biomarkers of Bacteria and Fungi

The bacterial and fungal-dominant community representatives played a key role in soil microbial metabolism, nutrient transformation and crop quality. The shifts in soil microbial communities induced by bio-compost were reflected in changes in dominant community representatives at phylum and genus levels of soil bacteria and fungi, especially the enrichment of beneficial microorganisms in the soil and the reduction of harmful microorganisms.

For bacteria, bio-composts increased the relative abundance of Proteobacteria and some of its genera, which is consistent with Longa et al. [38]. The presence of Proteobacteria is characteristic of nutrient-rich and high-carbon substrates [39], and bio-compost significantly increased the content of soil organic matter and provided a carbon source for Proteobacteria. Bio-composts increased the functional bacteria (such as Skermanella and SC_I_84 order within Proteobacteria) participating in the soil N cycle [40]. The significant correlation between Proteobacteria and most metabolic pathways related to N cycle implied that Proteobacteria promoted soil nitrogen transformation. The conventional dosage of bio-compost increased the relative abundance of Sphingomonas, which was related to the high content of Sphingolipid metabolism in the conventional dosage of bio-compost. Sphingomonas was a functional microbe with strong metabolic ability and can degrade organic pollutants in the soil. Bio-composts increased the relative abundance of Actinobacteria. The reason may be that Actinobacteria participated in the degradation of organic matter, while bio-compost increased organic matter and provided carbon source for Actinobacteria. In addition, bio-composts increased the relative abundance of Nocardioides, Pseudarthrobacter and Streptomyces within Actinobacteria, which belong to beneficial bacteria and can secrete actinomycin and antagonize the growth of soil pathogens [40,41].

Bio-composts increased the relative abundance of Bacteroidetes, which can synthesize glycosyl hydrolases to break down cellulose and hemicellulose, help in the soil N cycle and nutrient turnover and be positively related to soil nutrients [42]. Firmicutes was enriched in EMI treatment, and its positive correlation with soil nutrients explained this. Firmicutes was a symbiotic bacteria, which contributed to the C cycle, degraded plant-derived polysaccharides, and had a significant positive correlation with most carbon metabolic pathways [43].

Soil fungi plays an important and complex role in maintaining the normal operation of biological communities, such as establishing symbiotic or pathogenic relationships with plants and animals, participating in C and N cycles, and promoting the decomposition of organic matter. The most abundant phylum in all treatments was Ascomycota, which contained many beneficial and pathogenic microorganisms. Bio-composts increased the relative abundance of Ascomycota, which may be due to the fact that the bio-compost can provide nutrients for the growth of Ascomycota. Bio-compost increased the relative abundance of beneficial fungi, such as Chrysosporium, Chaetomium, Acremonium and Trichoderma within Ascomycota, which were commonly found biocontrol fungi and had inhibitory effects on plant pathogenic microorganisms [12,44]. High dosages of bio-compost increased the relative abundance of Metarhizium that can prevent fusarium wilt [45]. Bio-compost decreased the relative abundance of Stachybotrys, which is a plant pathogenic fungus and indoor pollution fungus that can easily cause human diseases [46]. High dosage of the bio-compost decreased the relative abundance of Aspergillus that can cause ear rot in plants [47].

2.4. Bio-Compost Altered Bacterial and Fungal Co-Occurrence Patterns

The application of bio-compost had a significant impact on both the individual microbial groups and the overall microbial community patterns. The average cluster coefficient and closeness centrality in the bacterial and fungal network of bio-compost treatments were higher than that of the non-bio-compost treatments. This implied that the application of bio-composts might not only remit the competition within bacteria, but also reduce competition within fungi. The higher average betweenness centrality of the bacterial and fungal network under bio-compost treatments than non-bio-compost treatments suggested that bio-compost may increase the bacterial and fungal niches. However, bacteria occupied a larger core niche than fungi. The fact that the average betweenness centrality of the bacterial network under bio-compost treatment is higher than that of the fungal network confirmed this. The average closeness centrality of bacterial network under all treatments was greater than that of fungal network, indicating that information transmission between bacteria was greater than between fungi. Positive links dominated both the bacterial and fungal networks, implying that mutual cooperation rather than competitive exclusion played a more important role in microbial assembly. Mutual cooperation occurred more frequently in bacterial communities than in the fungal network because positive links in the bacterial network were much higher. The application of bio-compost provided a higher supply of nutrients for microorganisms, which reduced competition for limited resources and promoted cooperation between species. Bio-compost changed key nodes in the network structure, because the functional microorganisms of bio-compost entered the soil and interacted with the indigenous microorganisms of the soil.

2.5. The Application of Bio-Compost Changed the Overall Function of Soil Bacterial and Fungal Communities

The application of bio-compost changed the overall function of soil bacterial and fungal communities. In addition, the application of bio-compost increased the ratio of Metabolism among the six categories of biological metabolic pathways. The proportion of certain metabolic pathways related to C, N and P cycles under bio-compost treatments was larger than that of CK and CF treatments, indicating that the bio-compost shifted the metabolic function of microorganisms. The key genes related to C cycle were mainly involved in C degradation, C fixation, and Methane cycling. The key genes related to N cycle were mainly involved in fixation, ammonification, nitrification, denitrification, dissimilatory N reduction, and assimilatory N reduction. Additionally, the key genes related to P cycle were mainly involved in P oxidation, Phytic acid hydrolysis, polyphosphate degradation, and polyphosphate synthesis.