Vermicompost derived from mushroom residues improves soil C/P cycling, bacterial community, and fungal abundance

The utilization of agricultural waste organic materials through composting technology has gained significant traction in agricultural production as an effective means of crop nutrient management. However, the differences in the impact of organic amendments prepared by traditional composting and vermicomposting on soil properties still deserve further research. Based on field experiments conducted in greenhouse, compared to chemical fertilizer treatments as control, we utilized traditional compost (OF) and vermicompost (VcF) derived from agricultural organic waste edible mushroom bran and cow manure (2:8). Variations in soil physiochemical properties, activities of soil enzymes related C and P cycling, abundances and diversities of bacterial 16S rRNA and fungal ITS gene at total DNA level were analyzed. Both compost treatments enhanced soil organic carbon, soil total phosphorus, and soil available P content significantly and also increased the activities of soil α‐glucosidase, β‐glucosidase, acid phosphomonoesterase, and alkaline phosphomonoesterase significantly. The above results suggested that soil C and P transformations were stimulated effectively by both organic amendments. OF and VcF increased the fungal ITS absolute abundances significantly while diversity indices of soil bacterial community increased significantly under both treatments. Correlation analysis indicated that bacterial community composition was strongly correlated with several soil property indexes while fungal community composition was only significantly correlated with soil total phosphorous content. In conclusion, similar to traditional compost, vermicompost significantly improved soil nutrient cycling (especially C and P aspects). In terms of soil microbes, bacteria and fungi showed different responding mechanism to vermicompost: bacteria adjust microbial structure, while fungi tend to proliferated. In consideration of the advantages of vermicompost in technology and economic cost, it could be applied in the subsequent agricultural production more frequently.


| INTRODUCTION
The great improvement effect on soil fertility and health with combined application of organic amendments and chemical fertilizers has been confirmed (Darakeh et al., 2021;Ning et al., 2017), indicating that the important role of organic amendments in agricultural production has become increasingly prominent (An et al., 2022;Chen et al., 2022).There are around 15 million tons of waste edible mushroom bran produced each year in China, which is an important source for organic amendments.While the utilization rate of waste edible mushroom bran in China is lower than 40% (Chen et al., 2022).Abandoning edible mushroom bran not only led to resource waste but also caused potential environmental pollution (Hu et al., 2022).
Edible mushroom bran contains high content of organic matter, nitrogen, phosphorus, potassium, medium, other trace elements, and bioactive substances.Composting edible mushroom bran from organic amendment is an effective technology for recycling waste organic material in agricultural crop production (Lou et al., 2017).However, temperature and speed are easily influenced by environmental factors during traditional composting process, which resulted in unsatisfying compost quality (Lou et al., 2017).A large proportion of nutrients are lost as CO 2 and NH 4 , therefore composted organic amendment from edible fungi often showed unsatisfying resource utilization rate.Aiming to overcome various problems during the traditional composting process (such as slow fermentation, low resource utilization rate, and low compost quality), researchers have introduced earthworms as an improvement measure in the composting process.The vermicomposting technology has been introduced into the transform process of agricultural organic waste into edible fungi.Compared to traditional composting process, the vermicomposting technology takes less time on crushing and decomposing edible fungi bran, and degrades organic materials with less nutrient loss (Albanell et al., 1988).Vermicomposting technology gets products as vermicompost, which obtains more economic benefits than traditional composting (Wang et al., 2018).As bio-compost, vermicompost also has several advantages such as slighter environmental pollution and lower energy cost, meaning it could be widely used and meet the sustainable development demand (Wang et al., 2021).
Many researches paid attention on response of crop to vermicompost application (Carnimeo et al., 2022;López et al., 2021;Yu et al., 2022).The promoting effect of vermicompost application on crops is partly due to keeping humus organic matter and mineral nutrients, as well as containing amino acids, indoleacetic acid, digestive enzymes, numerous and diverse microorganisms, disease-resistant strains, and other beneficial substances (Wang et al., 2018).But vermicompost also has obvious improvement effect on soil physiochemical properties (Akinnuoye-Adelabu et al., 2019;Hebeková et al., 2020;Li, Nie, et al., 2019;Li, Wan, et al., 2019;Shi et al., 2020;Wang et al., 2018).Previous results indicated vermicompost could better promote nutrient mineralization in soil and has greater potential to improve soil nutrition status (Hebeková et al., 2020;Shi et al., 2020) and sustain agricultural development (Han et al., 2021;Zhang et al., 2021).Exogenous nutrients such as nitrogen, phosphorus, and other elements are utilized by crops after enzymatic hydrolysis and microbial transformation (Zuo et al., 2018), as the enzymes' substrate concentration increases with organic material incorporation, variations in soil properties typically induce changes in soil enzyme activities (Wu et al., 2021).On the other side, vermicompost contains abundant beneficial substances and microorganisms, which could significantly promote soil microorganism growth and improve soil microbial activity (Akinnuoye-Adelabu et al., 2019;Wang et al., 2018).But to our knowledge, there have been few mentions on the exact differences of relationships between soil enzyme catalytic intensity and soil microbial (bacteria and fungi) communities under traditional compost and vermicompost compost (Chatterjee et al., 2021;Ravindran et al., 2019;Song et al., 2015).Therefore, it is necessary and meaningful to ascertain their relationships and detect the key factors influencing soil nutrient supply after vermicompost compost addition.
The objectives of the present study were (1) to investigate the effect of vermicompost addition on soil properties and soil enzyme activities, and clarify the different effects of traditional compost and vermicompost compost on above soil quality indexes, (2) to explore quantity and structure of soil microbial groups (bacteria and fungi) by targeting bacterial 16S rRNA and fungal ITS (using realtime PCR and high-throughput sequencing), and (3) to explore the relationship among soil properties, soil enzyme activities, and soil microbial groups.The present study aims to provide theoretical basis for rational vermicompost application in future agricultural production.

| Experimental design
The experiment was conducted at the Shenyang Ecological Experimental Station, Chinese Academy of Sciences.The study region is characterized by the temperate subhumid continental climate, where the annual average temperature is 7.5°C, and the annual average rainfall is 700 mm.The soil type of the study site is brown soil (FAO/ ISRIC/ISSS 1998), the conventional cultivation was applied in this area for a long time.
The present experiment was carried out in a greenhouse with a length of 100 m and a width of 5 m.The central area of the greenhouse was selected for testing, dividing into nine districts, and each district area was 2 × 0.5 m.The spacing of each district is 0.5 m, in order to reduce water migration among adjacent districts.The tomato variety in this experiment was chardonnay tomato.Three treatments were set up, each treatment was evenly divided into three blocks (three replicates for each treatment), and three replicates were arranged randomly in the group.Three treatments were set as follows: CK: chemical fertilizer as control; OF: traditional compost; and VcF: vermicompost.The iso-nitrogen amount of three treatments was 750 kg N hm −2 , and base fertilizer and topdressing fertilizer combinations were used.Considering that the proportion of available nitrogen in organic amendment is lower than that in chemical fertilizer, more organic amendment is applied as base fertilizer.Fully decomposed mushroom bran compost cow manure was 40 t hm −1 (1.5% N, based on dry content) and mushroom bran compost cow manure/wormcast organic amendment was 60 t hm −1 (1% N, based on dry content).Chemical base fertilizers account for 60% of the total amount, and topdressing fertilizers account for 40%, while organic base fertilizer is 80% of the total amount, and using compound fertilizer as topdressing fertilizer accounted for 20%.Organic amendments used in this study were obtained by self-composting.The raw materials were mushroom bran and cow manure, and the ratio of mushroom bran to cow manure was 2: 8, and conventional compost method was used for production.

| Soil sampling and soil property/soil enzyme activity analysis
Five soil cores were randomly collected (0-20 cm) in each replicated block by soil auger (20 cm in diameter), then mixing them into one soil sample, after transporting back to laboratory, each soil sample was divided into three parts, one was stored at 4°C for soil enzyme activity determination, one was air-dried for soil properties analysis, and other part was stored at −80°C for DNA extraction.
Soil pH was determined in a 1: 2.5 soil/water suspension by a digital pH meter (pH 700 Bench Meter, Eutech Instruments).Soil organic C (SOC) was determined by chemical oxidation using a K 2 Cr 2 O 7 solution (Nelson & Sommers, 1982).Soil total C (TC) and total N (TN) were determined via combustion of ground subsamples (passed through a 0.16 mm mesh) using an automatic elemental analyzer (Analyzer vario MICRO cube, Elementar).Soil total P (TP) was determined by digestion with perchloric acid (HClO 4 ; Kuo, 1996).Available P (AP) was extracted with 0.5 M NaHCO 3 (Wu et al., 2021).The available potassium (AK) was determined by flame photometer using method described by Zhao et al. (2004).

| DNA extraction, quantifications of gene abundance, and high-throughput sequencing
Following manufacturer's protocol, we used the Power Soil® DNA Isolation Kit (MoBio) to extract total genomic DNA from 0.5 g frozen soil.The quality of the total DNA was determined using the Nano Drop ND-2000 spectrophotometer (Thermo Fisher Scientific).The abundance of 16S rRNA and ITS gene was all estimated by qPCR in an ABI 7500 Real-Time PCR System (Applied Biosystems™) following the manufacturer's protocol.For 16S rRNA gene, a universal primer was used to quantify the total bacterial abundance, which is 338F: 5′-ACTCC TAC GGG AGG CAGCAG-3′ and 518R: 5′-ATTAC CGC GGC TGC TGG-3′ (Chen et al., 2019), and for ITS, the primer was ITS1: 5′-CTTGG TCA TTT AGA GGA AGTAA-3′ and ITS2: (5′-TGCGT TCT TCA TCG ATGC-3′; Jiang et al., 2023).Cycling conditions were as follows: 30 s at 95°C followed by 40 cycles and annealing at 60°C for 40 s.Briefly, the PCR mixture contained 16.5 μL of Power SYBR® Green PCR Maste Mix (Applied Biosystems™, Thermo Fisher Scientific Inc.), 0.8 μL of each primer, and 2 μL of extracted DNA template.The amplification specificity of the reaction was confirmed by generating a melting curve.Standard curves were prepared by tenfold serial dilution of the cloned plasmid, and the copy numbers of various genes were determined per gram dry soil based on standard curves.For high-throughput sequencing, the amplification protocol was as follows: a hot start of 5 min at 95°C, 28 cycles of 95°C for 45 s, 55°C for 50 s, and 72°C for 45 s, and followed by a final extension step of 10 min at 72°C.The PCR products were purified using a Agencourt AMPure XP Kit.The sequencing was conducted using an MiSeq platform in Allwegene Company.Sequence data were submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive under the PRJNA808813.

| Statistical analyses
One-way ANOVA with Duncan's test (p < 0.05) was used to identify the variations in various indices by SPSS 19.0 (SPSS).Prior to using ANOVA analysis, all of these data were tested for normality via the Shapiro-Wilk normality test using SPSS.The αdiversity of bacteria and fungi was calculated using the "picante" package in R (version 4.1.2,http://www.r-project.org).Pearson's correlations between soil properties, the relative abundance of dominant phylum, genus, and OTUs of bacteria and fungi were calculated using the "Hmisc" package.The principal coordinate analysis (PCoA) based on the Bray-Curtis distance was performed to assess the βdiversity, using the "vegan" package in R.Meanwhile, the βdiversity of gene was compared using an analysis of similarities (ANOSIM, permutations = 999)."ggplot2" package in R was used to data visualization, and the heatmaps of phylum/genus and Spearman's correlations were visualized using the "pheatmap" and "corrplot" packages.

| Soil physiochemical properties
Compared to control, both traditional compost (OF) and vermicompost (VcF) significantly affected pH, soil organic matter content (SOM), total phosphorus (TP) content, and available potassium (AK) content in soil (Table 1).What deserves our attention was that both compost treatments significantly increased soil pH, but there existed no obvious differences between two composts (Table 1).SOM content also surged from 27.6 g kg −1 of chemical fertilizer treatment to 41.3 g kg −1 under traditional compost, to 44.6 under vermicompost treatment (Table 1).Both compost treatments also significantly increased the soil TP content, while traditional compost showed more obvious effect than vermicompost treatment (Table 1).Both compost treatments significantly decreased available potassium (AK) content.Similar to variation of TP, AK of traditional compost changed more obvious than that under vermicompost (Table 1).Compared to control, two compost treatments had no obvious effect on relative soil N property indexes, so we did not show N content results here (data not shown).

| Soil enzyme activities
In addition to N-cycling enzyme β-N-acetylglucosaminidase (NAG), OF and VcF significantly affected the activities of soil dehydrogenase (DHA), α-D-glucosidase (AG), β-Dglucosidase (BG), acid phosphomonoesterase (AcP), and alkaline phosphomonoesterase (AIP; Figure 1).Both treatments significantly increased DHA activity compared to control while vermicompost showed slighter advantage (Figure 1).Soil AG and BG activity surged extremely obvious under both treatments compared to chemical fertilizer treatment.VcF showed stronger promoting effect on soil AG activity, while OF showed stronger promoting effect on soil BG activity (Figure 1).Compared to control, both composts significantly increased AcP and AIP activities in soil.Vermicompost showed a more obvious effect on soil AcP activity, while traditional compost stimulated AIP extremely (Figure 1).Overall, similar to soil properties, traditional compost and VcF significantly affected C-cycling enzymes and P-cycling enzymes but had slight effect on Ncycling enzymes.

| Absolute abundance and community composition of soil bacteria and fungi
Compared to control, both traditional compost and vermicompost (VcF) significantly increased fungal ITS gene copy number (Figure 2).Absolute abundance of fungal ITS were average 71.4% and 59.1% higher under traditional compost and VcF compared to control, seperately (Figure 2).While both treatments showed a slight effect on the absolute abundance of soil bacteria 16S rRNA (Figure 2).In terms of soil bacteria αdiversity, Chao1 index (species richness) and Shannon index (species richness and evenness in individual allocation among species) were significantly increased under traditional compost and VcF compared to control (Figure 6A,B).However, both αdiversity indices, that is, Chao1 and Shannon of soil fungi were constant across the treatments (Figure 6C,D).All bacteria sequences were classified into 39 phyla and 490 genera.Proteobacteria (32.57%-38.41%),Acidobacteria (13.08%-22.58%),and Actinobacteria (13.99%-18.63%)were the top three phyla, and collectively accounted for >70.03% of all sequences in all samples (Figure 3a).All sequences of soil fungi were classified into 15 phyla and 323 genera.The dominant phyla were Ascomycota (52.83%-59.9%),Mortierellomycota (15.69%-31.77%),and Basidiomycota (1.02%-2.59%),and 13.2%-27.15%were unclassified, collectively accounted for >99.13% of all sequences in all samples (Figure 3b).
In terms of genera level, the dominant genera of soil bacteria were Rhodanobacter, RB41, Sphingomonas, Haliangium, Gemmatimonas, and Mizugakiibacter.Compared to control, the relative abundance of Rhodanobacter and Mizugakiibacter was significantly decreased in traditional compost and VcF.The relative abundance of Gemmatimonas in VcF treatment was significantly lower than that in traditional compost fertilizer and chemical fertilizer plots (Figure 4A).The  top three of soil fungi were Mortierella (15.94%-Geosmithia (3.91%-10.15%),and Fusarium (3.79%-7.06%).The relative abundance of Fusarium and Penicillium in VcF was significantly higher than OF and CF plots, while the relative abundance of Mortierella showed a reverse result (Figure 4B).

| Soil physiochemical properties
Both organic amendment treatments significantly increased soil pH, meaning that traditional compost and vermicompost all could effectively reduce soil acidification and improve soil quality, which is consistent with several previous studies (Bhardwaj et al., 2014;Mahanty et al., 2017;Yao et al., 2009).Also, both treatments significantly increased soil organic matter content compared to control, meaning both treatments improved soil fertility significantly.As one of most important soil nutrition indexes, several researches pointed out that organic amendments could effectively improve soil organic matter content (Fageria & Baligar, 2005;Williams & Haynes, 1993), so the results in our study are reasonable.Phosphorus, as an important limiting element for crop growth, always playing an important role in soil ecosystem (Vitousek et al., 2010), compared to control, two composts also significantly increased soil total phosphorus content, which means better nutrient status (Redel et al., 2008).In summary, both treatments could effectively improve soil properties and there nearly existed no significant difference between them, which means they all are great measures that could be applied in practical agricultural production.Considering the lower environmental pollution caused by vermicompost during the production process, it should be a better choice.

| Soil enzyme activities
As we know, extracellular enzymes are responsible for nutrient cycling in soil (Sinsabaugh et al., 1991;Tabatabai, 1994), so they are often be considered as important soil health indicators (Chen et al., 2018;Elifantz et al., 2011;Guo et al., 2017;Jia et al., 2020;Jian et al., 2016;Nannipieri et al., 2018), so it is necessary to evaluate enzyme activities under two compost ways.Both traditional compost and vermicompost significantly increased the activities of C-cycling enzymes (AG and BG) and P-cycling enzymes (AcP and AIP), this may be attributed to higher substrate and energy source.Several previous studies pointed out that exogenous nutrients generally increase the activities of hydrolytic enzymes (Carreiro et al., 2000;Chen et al., 2018;Keeler et al., 2009), because nutrient addition promotes the growth and nutrient demand of fast-growing soil microbes (Jia et al., 2020;Li, Nie, et al., 2019;Li, Wan, et al., 2019).Pearson correlation analysis results also showed that, compared to N-cycling enzyme (NAG), C-cycling enzymes and P-cycling enzymes were more closely related to soil properties (Figure 5).AG and BG are significantly positively correlated with total N and available P content (Figure 5).What deserves our attention are P-cycling enzyme results, AcP and AIP, which are significantly positively correlated with total C, total N, total P content, and available P content (Figure 5).Phosphorous is a necessary nutrient for soil microbial reproduction and growth (Craine et al., 2007), so an increased content of these two in our study would stimulate soil microorganisms, which means the increased nutrient contents caused by compost additions stimulated microbial enzyme production.On the other side, soil organic matter stands for soil carbon content, and carbon is an important energy source for soil microorganisms (Stone et al., 2012), so abundant energy source accompanied by compost additions means more active microbial enzyme production in soil (Keeler et al., 2009).Moreover, as substrate, the increase in organic matter also further stimulated the increase in Ccycling and P-cycling enzyme activity (Xiao et al., 2018), correlation results also indicated this point (Figure 5).So, the result about enzyme activities increased in present study is reasonable.The increase in C-cycling and P-cycling enzyme activities means a higher activation capacity of carbon and phosphorus in soil (Dong et al., 2019); therefore, we conclude that both traditional compost and vermicompost could significantly accelerate the enzymatic turnover of soil carbon and phosphorus, thereby improving soil nutrient status.

| Absolute abundance and community composition of soil bacteria and fungi
Bacterial 16S rRNA gene copy number changed subtly while fungal ITS gene copy number increased significantly under both compost treatments compared to control (Figure 2).In contrast, the responses of bacterial and fungal community composition were opposite to their gene abundance.Compared to fungal community composition, bacterial community composition varied obviously (Figure 6), which proves the different response mechanisms of both soil microbes to compost additions.In detail, soil fungi tended to selfproliferate, while soil bacteria tended to adjust their internal composition.Pearson correlation analysis showed that fungal ITS gene copy number was significantly positively correlated with soil pH, TC, TP, and AP (Figure 7), while the alpha-diversity indices of bacteria were significantly correlated with pH, SOM, TC, TN, and TP, on the contrary, fungi diversity indices showed no significant correlation with soil properties (Figure 7).Furthermore, we correlated the geographical distance-corrected dissimilarities of community composition with those of environmental variables using the partial Mantel test (Figure 8), bacterial community composition was strongly correlated with pH, SOM, TP, AP, and AK.However, the fungal community composition was only significantly correlated with TP (Figure 8).All these above results indicated that two soil microbial groups had different response mechanism to the nutrient change caused by compost additions.Previous research pointed out that long-term organic amendment addition could improve soil bacterial community richness and diversity indexes, and bacteria are also more sensitive to organic amendment compared to fungi (Ramirez et al., 2012).Consistent with that, in our study there were no significant changes in soil fungal community richness but abundance of soil fungi changed significantly under two compost additions compared to control.It was pointed out that the bacteria in soil were more sensitive to carbon substrate availability than the fungal community due to shorter turnover time of bacteria, so soil bacteria were also more sensitive to external environmental factors, thus soil bacteria diversity changed more significantly under amendments than fungi is rea- (Ai et al., 2018;Ren et al., 2021).results indicated that bacteria and fungi truly responded differently to compost fertilizers, but the internal correlation among the microbial groups containing functional genes and soil properties/enzymes deserves more attention, which we should discuss further in future research.

| CONCLUSION
Compared with the chemical fertilizer treatment, traditional compost and vermicompost significantly increased the soil organic matter content, soil total P and available P content, soil enzyme activities related C-cycling and Pcycling were also enhanced obviously under both compost treatments.Based on comprehensive analysis about variations in both soil physiochemical properties and enzyme activities, we infer that vermicompost could stimulate soil C and P transformation effectively as traditional compost.At the same time, the result about soil microbial groups is within our expectation, vermicompost and traditional compost insert different impact on soil microbial group characteristics.Both compost treatments significantly increased fungal ITS absolute abundances, while bacterial diversity indices increased but fungi diversity indices changed subtly under both compost treatments.Reforms of soil microbial groups indicated different response mechanisms between bacteria and fungi to compost additions.Results in this study pointed out that vermicompost truly has positive effects on soil properties and soil microbial community quantity and structure.In consideration of the advantages of vermicomposting in technology and environmental protection aspects, vermicomposting could be applied in the subsequent practical agricultural production on a larger scale.

ORCID
Yulan Zhang https://orcid.org/0000-0002-4041-9645Nan Jiang https://orcid.org/0000-0003-4913-4395F I G U R E 8 Spearman's correlation analysis of soil properties, bacteria, and fungi composition.Orange and green lines denote correlations as shown in figure.Blue and red squares denote negative and positive correlations, respectively.The thickness of links as well as the squares represents the values of Spearman's correlation coefficients.aK, available potassium; aP, available phosphorus; SOC, soil organic carbon; TC, total carbon; TN, total nitrogen; TP, total phosphorus; (for interpretation of the references to color in this figure legend, the reader is referred to the note in this figure).

F
The gene copy numbers of bacteria (16S rRNA) and fungi (ITS) in soil under different treatments.The values are means with standard errors as shown in figure.CK: conventional fertilizer; OF: traditional compost plus NPK fertilizer; VcF: earthworm compost plus NPK fertilizer.The lowercase letters in figure indicate significant differences among different treatments (p < 0.05, Duncan's test).

F
Bacterial taxonomy variations (A) and fungal taxonomy variations (B) at the genus levels under different treatments (relative abundance, most abundant genera >1%; p < 0.05).CK: conventional fertilizer; OF: traditional compost plus NPK fertilizer; VcF: earthworm compost plus NPK fertilizer.F I R E 5 The matrix plot of Pearson correlation coefficients for various soil properties and soil enzymes, only the significant correlations were shown (p < 0.05).Larger circles indicate larger correlation coefficients, the color of the circle represent the Pearson correlation coefficient (red-blue: −1 to 1).AcP, acid phosphatase; AG, α-Glucosidase; AIP, alkaline phosphatase; AK, available potassium; AP, available phosphorus; BG, β-Glucosidase activity; DHA, dehydrogenase; NAG, N -acetyl-β-D-glucosaminidase; TC, total carbon; TK, total potassium; TN, total nitrogen; TP, total phosphorus.F I G U R E 6 The diversity indexes of bacteria and fungi in soil under different treatments, respectively.Chao1 and Shannon are diversity indexes shown in figure.The values are means with standard errors, as shown in figure.CK: conventional fertilizer; OF: traditional compost plus NPK fertilizer; VcF: earthworm compost plus NPK fertilizer.The lowercase letters in figure indicate significant differences among different treatments (p < 0.05, Duncan's test).

F
The heatmap of Pearson correlation coefficients for bacterial and fungal gene abundances, microbial diversity indexes, and soil properties.***p < 0.001; **<0.01;*<0.05, the color of the square represents the Pearson correlation coefficient (blue-red: −1 to 1).(For interpretation of the references to color in this figure legend, the reader is referred to the note in this figure).

T A B L E 1
The values of soil properties under different treatments.The values are presented as the means with standard errors are shown in table.
Note: Different lowercase letters in table indicate significant differences among treatments detected using Duncan's test (p < 0.05).Treatments as follow: CK: conventional fertilizer; OF: traditional compost plus NPK fertilizer; VcF: earthworm compost plus NPK fertilizer.Abbreviations: AK, available potassium; AP, available phosphorus; SOM, soil organic matter; TK, total potassium; TP, total phosphorus.