1 Introduction

Oudemansiella raphanipes (Berk.) Pegler & T.W.K. Young, called “Heipijizong” or “Black Termite Mushroom,” is one of the high-grade health care fungi for both food and medicine, widely cultivated in India, South Korea, Japan, China, etc. (Hao et al. 2016). Fruiting bodies of O. raphanipes are rich in protein, amino acids, vitamins, and other nutrients. Fruiting bodies or their fermentation broth is reported to have antihypertensive and antitumor effects (Gao et al. 2018). In recent years, with the improvement of people’s living conditions and health care awareness, O. raphanipes has shown a broad development prospect.

Casing soil is a key step to obtain better commercial yield in the cultivation process of some edible fungi, which provides necessary environment for the edible fungi from the vegetative stage to the reproductive stage and induces the formation and supports the growth of fruiting body, as well as maintains the moisture of culture medium and reasonable air humidity through evaporation. The physicochemical properties of the casing soil and the associated microbial community play important roles in the mycelial growth, fruiting body differentiation, and development of edible fungi, which also affect their yield, quality, and fruiting uniformity (Cai et al. 2009). A large number of microorganisms including bacteria inhabit with special functions in the casing soil of edible fungi (Nazir et al. 2013; McGee 2018). They participate in the cycle of energy and nutrients, as well as the transformation of various types of organic matter; thus, the microorganisms are widely considered an importantly comprehensive evaluation index of soil quality. The dynamic changes of microbial community significantly affect the growth and development of edible fungi (Kertesz and Thai 2018). Oudemansiella raphanipes is a kind of edible fungus depending on casing soil, and the long fruiting period and strong dependence on the casing soils limit its large-scale popularization and cultivation. To better understand the microecological mechanism of casing soil and promote the large-scale production of O. raphanipes, it is necessary to understand the structure and function of microbial community in its casing soil.

Studies about microorganisms associated with edible fungi mainly focused on the common edible fungi like Agaricus bisporus and Pleurotus eryngii by using culture-dependent methods (Lim et al. 2008; Siyoum et al. 2010; Vieira and Pecchia 2021). However, the culture-dependent methods often underestimate the biodiversity and cannot objectively reflect the composition of microbial community. In recent years, the development of high-throughput sequencing technology accelerated understanding of the microbial community associated with edible fungi. Yang et al. (2019) revealed that bacterial diversity of Phlebopus portentosus increased sharply from the hyphal to the primordium stage and then decreased during harvesting stage. Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria were found as the dominant groups of A. bisporus. During the growth of A. bisporus, the relative abundance of casing bacteria in the primordial stage was significantly higher than that in other growth stages (Carrasco et al. 2019). Pseudomonas putida was reported to induce the fruiting body primordium formation of A. bisporus (Hayes et al. 2010). Gram-negative bacteria were dominant in the mycelial growth and fruiting body development of A. bisporus, indicating that they may play important roles (Cai et al. 2009). It is reported that A. bisporus or Ganoderma lucidum cropping could significantly affect the bacterial community structures in the casing soil during the production (Zhang et al. 2018; Ke et al. 2019); thus, we propose a hypothesis that the bacterial community in casing soil will shift before and after the cultivation of O. raphanipes. However, studies about O. raphanipes mainly focused on the screening of high-quality casing materials, evaluation of efficient strains, and development of potential bioactive components (Gao et al. 2018; Lian et al. 2019), and bacterial community diversity and functions in casing soil of O. raphanipes are still unknown.

In this study, bacterial community shifts in casing soil before and after the cultivation of O. raphanipes (CSBACO) were studied by using 16S rRNA high-throughput sequencing technology combined with biological information and statistics methods. The objectives of this study were (1) to explore the composition and diversity of bacterial community in CSBACO; (2) to determine the correlations between the soil physicochemical properties and bacterial community in CSBACO; and (3) to discern the potential metabolic function of the bacterial community in CSBACO. The findings will deepen the understanding of bacterial community in casing soil of O. raphanipes, and lay a foundation for promoting the large-scale production of O. raphanipes.

2 Materials and Methods

2.1 Cultivation of O. raphanipes and Sample Collection

The cultivation experiment was carried out in the greenhouse in Da Xing district of Beijing, China (39.62°N, 116.43°E) in 2020. The greenhouse was previously ventilated, cleaned, and simply disinfected with lime. The O. raphanipes cultivar and the artificial bed-logs were bought from a factory in Heze, Shandong Province, with the dry weight 550 g of each artificial bed-log. The casing material was sandy loam from the local greenhouse. The artificial bed-logs were arranged vertically and cultivated in three parallel borders, with 280 artificial bed-logs in each border. Thickness of the casing soil was 4 ~ 5 cm, and the interval of the artificial bed-logs was 2 ~ 3 cm. The temperature of the greenhouse was kept at 25 ~ 26℃. For the details of cultivation and management techniques of O. raphanipes, refer to Zhang et al. (2020).

All the casing soil samples were uniformly collected via five-point sampling method with sterile glasswares and brought back to the laboratory on ice within 24 h. The casing soil before the cultivation of O. raphanipes (CSBC) and the casing soil in fruiting stage (CSAC, 84 days after casing soil) were sampled respectively with three replicates for each treatment. The topsoil with the depth 1–2 cm was removed in order to avoid the interference of surface soil by external factors and ensure the consistency of samples, and then, the casing soil with the depth of 3–5 cm was collected. Five sampling points were randomly set, and soils taken from each sampling point were mixed as a casing soil sample, and uniformly treated using a 2-mm sieve to remove the tissues. Each sample was divided into two parts; one part was air-dried to measure the physicochemical properties, and the other part was stored at − 80℃ for DNA extraction.

2.2 Measurement and Analysis of Casing Soil Physicochemical Properties

To reflect the corresponding environmental conditions, physicochemical properties of the six soil samples were tested following the methods described by Bao (2000). The total nitrogen (TN) concentration in casing soil was determined by Kjeldahl method with a CHN elemental analyzer (PerkinElmer; Boston, MA, USA). Ammonium nitrogen (NH4+-N) and nitrate nitrogen (NO3−-N) in casing soil were extracted with 2 M potassium chloride solution and determined by an automated flow injection analyzer (FS3100; O.I. Corporation/Xylem Inc., USA). The available phosphorus (AP) in casing soil was extracted with 0.5 mol·L−1 sodium bicarbonate solution and determined by molybdenum blue method. The content of available potassium (AK) was determined by flame emission technique extracted by 1 mol·L−1 ammonium acetate solution. The total salt content is measured by mass method. The content of casing soil organic matter was determined by potassium dichromate volumetric method in an automated TOC analyzer (TOC-VCPH; Shimadzu, Shimaneken, Japan) following the manufacturer’s instructions. Casing soil pH was determined by a pH meter (ST2100; OHRUS, Co. Ltd., Jiangsu, China) with soil-to-water ratio of 1/2.5.

2.3 DNA Extraction and Illumina Sequencing

Microbial genomic DNA was extracted by using E.Z.N.A.® Soil DNA kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s instructions. DNA concentration and purity were subsequently determined by an ND-2000 spectrophotometer (Thermo Scientific, Wilmington, USA). The 16S rRNA genes were amplified with primer pairs 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Zhang et al. 2019) by an ABI GeneAmp® 9700 PCR thermocycler (ABI, CA, USA). The PCR mixtures contain 4 μL of 5 × TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of forward primer (5 μM), 0.8 μL of reverse primer (5 μM), 0.4 μL of TransStart FastPfu DNA Polymerase, 10 ng of template DNA, and finally ddH2O up to 20 μL. The PCR amplification system was as follows: initial denaturation at 95 ℃ for 3 min, followed by 27 cycles of denaturing at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, and extension at 72 ℃ for 45 s, and single extension at 72 ℃ for 10 min, and end at 4 ℃. PCR reactions were performed in triplicate. The PCR products were purified and subsequently quantified via AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and Quantus™ Fluorometer (Promega, USA). Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina MiSeq PE300 platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The raw reads were deposited into the NCBI Sequence Read Archive with the accession numbers SRR17687160–SRR17687165.

2.4 Processing of Sequencing Data

Quality control of the original sequence was conducted via Fastp software, and forward and reverse reads were merged to full-length sequences by FLASH (Magoc and Salzberg 2018). The operational taxonomic units (OTUs) were classified using UPARSE version 7.1 at a 97% similarity threshold (Edgar 2013). Representative sequences were compared with SILVA (SSU132) database via RDP classifier (https://sourceforge.net/projects/rdp-classifier/) to obtain annotations of taxa classification. Detailed experimental steps and data pre-processing process were referred to Caporaso et al. (2012). To perform a relatively fair comparison among all samples, a sequencing depth of 30,524 was used.

Alpha diversity indices such as richness (Sobs), Shannon–Wiener index, Chao1, inverse Simpson (Invsimpson) index, and evenness were calculated to evaluate microbial taxonomic diversity by R version 3.4.2. The bacterial community composition was analyzed by Circos (Krzywinski et al. 2009) and principal coordinates analysis (PCoA). Linear discriminant analysis effect size (LEfSe) was used to recognize the differential microbes (biomarkers), and linear discriminant analysis (LDA) score was then used to estimate the effect sizes of different biomarkers (Segata et al. 2011). T test was performed to investigate the statistical significance of the soil physicochemical properties and the relative abundance of bacteria using the SPSS 17.0 software (SPSS Inc., Chicago, USA). Redundancy analysis (RDA) was used to discern the important physicochemical factors influencing bacterial community. Predictive functional analyses were performed by using FAPROTAX (Functional Annotation of Prokaryotic Taxa) (Louca et al. 2016), PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) (Douglas et al. 2019), and Bugbase (Ward et al. 2017) via Galaxy platform (http://mem.rcees.ac.cn:8080/root/) with the default parameters (Feng et al. 2017) and the cloud platform of Majorbio Bio-pharm Technology Co., Ltd., Shanghai, China (https://cloud.majorbio.com/).

3 Results

3.1 Physicochemical Properties of the Casing Soil

Physicochemical properties of the casing soil changed after the cultivation of the O. raphanipes (Table 1). The concentrations of total nitrogen (TN), available phosphorus (AP), ammonium nitrogen (NH4+-N), and nitrate nitrogen (NO3−-N) significantly decreased, while pH and available potassium (AK) content significantly increased. Notably, the concentration of NH4+-N and NO3−-N decreased from 33.10 and 27.87 to 7.89 mg/kg and 8.69 mg/kg, respectively. The concentration of AK increased and reached 433.33 g/kg after the cultivation of the O. raphanipes.

Table 1 Physicochemical properties of the casing soil before and after the cultivation of O. raphanipes
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3.2 Diversity Shifts of the Bacterial Community in Casing Soil

After quality filtering, denoising, and chimera removal, a total of 183,144 effective sequences were obtained and assigned into 3871 OTUs from six soil samples. Bacterial community before and after the cultivation of O. raphanipes shared 2899 OTUs, while 491 and 481 unique OTUs were exclusively detected in the CSBACO, respectively (Fig. 1a).

Fig. 1
figure 1

Diversity of bacterial community in the casing soil before and after O. raphanipes cultivation. a Venn diagram showing the common and exclusive OTUs in the bacterial community in casing soils before and after O. raphanipes cultivation. b Alpha diversity of bacterial community in the casing soil before and after O. raphanipes cultivation. c Principal coordinates analysis for bacteria community in the casing soil before and after O. raphanipes cultivation. *P < 0.05, **P < 0.01

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Alpha diversity indices of bacterial community including the Sobs, Shannon–Wiener, Invsimpson, and evenness were calculated, and results showed alpha diversity in casing soil after the cultivation of O. raphanipes decreased tremendously (Fig. 1b). Specifically, after the cultivation of O. raphanipes, the number of OTUs in the casing soil decreased from 2738 to 2639, the Shannon–wiener index significantly decreased from 6.79 to 6.59 (P < 0.05), and the evenness significantly decreased from 0.86 to 0.84 (P < 0.01).

The bacterial community structure in casing soil before and after the cultivation of O. raphanipes was analyzed by PCoA. Results showed that bacterial community structure shifted significantly in CSBACO with the first axis explained 71.77%. Moreover, the bacterial communities in the casing soil before the cultivation of O. raphanipes were more similar and distributed together, while those after the cultivation of O. raphanipes were more dispersed, indicating that the cultivation of O. raphanipes may increase the differentiation of bacterial community in casing soil (Fig. 1c).

3.3 Composition Shifts of the Bacterial Community in Casing Soil

All 3871 OTUs were further classified into 41 phyla, 129 classes, 278 orders, 420 families, and 737 genera, including some unclassified groups. The bacterial community compositions in CSBACO were analyzed from different taxonomic levels. At the phylum level, the relative abundances of most phyla were low, and only 10 known phyla with their relative abundance greater than 1.0% (Fig. 2a). Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria were dominant phyla in casing soil of O. raphanipes with their accumulated relative abundance 77.32%. The total relative abundance of the top eight predominant phyla accounted for more than 90% of the population, with significantly higher abundance of Actinobacteriota and Myxococcota in the CSBC samples (Fig. S1a). At the class level, the relative abundance of Alphaproteobacteria, Actinobacteria, Vicinamibacteria, Gammaproteobacteria, Thermoleophilia, Bacilli, Acidimicrobiia, and the candidate class MB-A2-108 accounted for about 60% of the population, with Actinobacteria and the candidate class MB-A2-108 significantly varied in CSBACO. After the cultivation of O. raphanipes, the relative abundance of Actinobacteria sharply decreased from 16.18 to 8.42%, while the candidate class MB-A2-108 abruptly increased from 3.10 to 5.37% (Fig. S1b).

Fig. 2
figure 2

Composition of bacterial community in the casing soil before and after O. raphanipes cultivation. a Bacterial community composition in phylum level. b Biomarkers of the bacterial community with linear discriminant analysis score ≥ 3.5. c Bacteria abundance on the genus level in the casing soil before and after O. raphanipes cultivation. The y-axis represented the genus at the taxonomic level. The x-axis represented the average relative abundance in different soil samples. *P < 0.05, **P < 0.01, ***P < 0.001

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The shift of bacterial community in CSBACO was further analyzed via LEfSe method. Results showed that when the LDA value was greater than 3.5, 37 biomarkers were obtained with their relative abundance significantly varied in CSBACO (Fig. 2b), among which 16 and 19 biomarkers were respectively recognized in CSBS and CSAS. Furthermore, the relative abundance of phyla Actinobacteriota and Myxococcota; the class Actinobacteria, Acidimicrobia, and Polyangia; and the family Nocardioidaceae, Sphingomonas, and Pseudonocardiaceae, as well as the genus Marmoricola, Nocardioides, Sphingomonas, and Streptomyces, was significantly higher in bacterial community of CSBC, while that of the phylum Chloroflexi and the candidate classes KD4-96, MB-A2-108, and Methylomirabilis, as well as the genus Bacillus and Ensifer, was significantly higher in bacterial community of CSAC. In addition, relative abundances of two unknown genera belonged to the candidate classes MB-A2-108 and KD4-96 increased significantly after the cultivation (Fig. 2b, c; Fig. S2b). Furthermore, the CSBC samples had higher relative abundances of Nocardioidaceae (P < 0.001), Sphingomonadaceae (P < 0.01), Nitrosomonadaceae, Streptomycetaceae, Micromonosporaceae, Geodermatophilaeae, Pseudonocardiaceae, Intrasporangiaceae, and Caldilineaceae (P < 0.05), but lower abundance of Gemmatimonadaceae, Solirubrobacteraceae, and Roseiflexaceae at the family level, compared with that in the CSAC samples (Fig. S2a, Fig. S3).

3.4 Effect of Physicochemical Properties of Casing Soil on Bacterial Community

Correlations between physicochemical properties of casing soil and the structure of bacterial community were investigated to determine the effect of physicochemical properties of casing soil on bacterial community. Results showed that the concentrations of total nitrogen, organic matter, ammonium nitrogen, nitrate nitrogen, and available phosphorus were positively correlated with the alpha diversity indices of casing bacterial community, while the contents of available potassium and pH were negatively correlated with the alpha diversity of casing bacterial community (Table S1). What is more, partial mantel test revealed that among eight soil physicochemical properties, available potassium and pH posed the most effects on the casing bacterial community structure (r = 0.67, P = 0.02 and r = 0.51, P = 0.02) (Table S2).

The top 30 genera in relative abundance were selected for the Spearman’s rank correlation analysis and visualized by heatmap (Fig. 3a). Fourteen genera were significantly correlated with the eight physicochemical properties of casing soil. The genus Ensifer was positively correlated with available potassium and pH, and negatively correlated with available phosphorus and NO3−-N. The genus Bacillus showed strong negative correlation with available phosphorus and total nitrogen. The genus Gaiella, Sphingomonas, and Streptomyces were positive correlated with organic matters and NH4+-N. The genus Marmoricola was negatively correlated with pH and positively correlated with NO3−-N. Furthermore, four major properties including organic matter, available potassium, ammonium nitrogen, and pH were chosen for the final RDA model (Fig. 3b), and results revealed the importance of these factors and their effect on bacterial community assembly with the first two canonical axes explained 71.11% and 15.12%. Specifically, pH and available potassium showed greater effect on the bacterial community after the cultivation of O. raphanipes, while organic matters and ammonium nitrogen were identified as predominant factors shaping bacterial community before the cultivation of O. raphanipes.

Fig. 3
figure 3

Correlations between the soil physicochemical properties and the dominant bacteria in casing soil on the genus level. a Heatmap of Spearman’s rank correlations between the soil physicochemical properties and the dominant bacteria in the casing soil samples on genus level. Positive correlations were showed in red, while negative correlations were in blue. *P < 0.05, **P < 0.01, ***P < 0.001. b Redundancy analysis showed the correlations between the soil physicochemical properties and the bacterial community structure in the casing soil before and after O. raphanipes cultivation. OM, organic matter; TS, total salt; TN, total nitrogen; AP, available phosphorus; AK, available potassium; NH4+-N, ammonium nitrogen; NO3−-N, nitrate nitrogen

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3.5 Predictive Functions for Bacteria in Casing Soil of O. raphanipes

Functions of bacteria in CSBACO were predicted based on three different methods including FAPROTAX, Bugbase, and PICRUSt2, and results showed that after the cultivation of O. raphanipes, relative abundances of bacteria with specific functions significantly changed (Fig. 4). Specifically, results from FAPROTAX showed that the relative abundance of the bacteria with the function of cellular metabolism decreased in CSAC, and on the contrary, the relative abundance of the bacteria with the function of nitrate reduction, animal parasites, symbionts, or human pathogens increased sharply after the cultivation of O. raphanipes (Fig. 4a). Meanwhile, results from Bugbase also showed that the relative abundance of the aerobic bacteria was lower and that of bacteria functional as potential pathogens was higher significantly in CSAC than that in CSBC (Fig. 4b). Furthermore, results based on the PICRUSt2 showed that four prevalent function types were metabolism, cellular processes, genetic information processing, and environmental information processing, in which 20 KEGG pathways were dominant in CSBACO. The relative abundance of bacterial function related to amino acid and lipid metabolism, as well as metabolism of terpenoides and polyketides, obviously decreased, while membrane transport significantly increased in bacterial community in CSAC (Table S3).

Fig. 4
figure 4

a Function predictions of bacterial communities in the casing soil before and after O. raphanipes cultivation using FAPROTAX. The y-axis represented the function types. The x-axis represented the average relative abundance in different samples. b Function predictions using Bugbase. *P < 0.05, **P < 0.01, ***P < 0.001

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4 Discussion

The dynamic changes of microorganism diversity and abundance significantly affect the growth and development of edible fungi (McGee 2018), and the growth and development of edible fungi also affect the diversity and structure of bacterial community in the casing soil. In this study, alpha diversity indices of bacterial community decreased after the cultivation of O. raphanipes, which may be due to the consumption of soil nutrient and the increase of secondary metabolites produced by fruiting bodies. The decrease of bacterial community diversity in casing soil is recognized as an important factor for the occurrence of continuous cropping obstacle of edible fungi (Ke et al. 2019; Ren et al. 2020) or other crops (Wang et al. 2019). Therefore, it is very important to maintain high diversity coupling with functional redundancy of bacterial community for the healthy growth and high yield of edible fungi and other crops.

Actinobacteria, Proteobacteria, Chloroflexi, and Acidobacteria were dominant in the bacterial community in CSBACO. However, after the cultivation of O. raphanipes, the composition and structure of bacterial community changed dramatically (Fig. 1c), and the relative abundances of Chloroflexi and Acidobacteria increased, while those of Actinobacteria and Proteobacteria showed opposite trends. As one of the largest bacteria groups, Actinobacteria plays significant roles in the biodegradation and recycling of organic matter (Trujillo 2016). Chloroflexi constitutes a specialized group of filamentous bacteria, which are only active under aerobic conditions and could consume primarily carbohydrates and grow on complex polysaccharides (Kragelund et al. 2007). Hence, the composition change of bacterial community indirectly reflects the functional changes of the bacterial community.

Compared with the bacterial community in casing soil before the cultivation of O. raphanipes, the relative abundance of the genus Marmoricola, Nocardioides, Sphingomonas, and Streptomyces in bacterial community after the cultivation of O. raphanipes decreased. The genus Marmoricola was the core soil bacterial genera in incubated soils (Lupwayi et al. 2021), and tolerant to alkaline-polluted soil (Li et al. 2020), and was a good candidate for the production of bioactive compounds to resist pathogenic fungi in soil (Jiang et al. 2018). Nocardioides and Streptomyces also benefit in the growth of mushroom mycelia (Wang et al. 2021); Nocardioides spp., usually considered phosphorus-solubilizing bacteria, can metabolize phenanthrene through dioxygenase (Saito et al. 1999) and enhance the alkaline phosphatase activity (Wang et al. 2021). Some strains in the genus Sphingomonas were able to fix nitrogen, degrade aromatic and xenobiotic compounds, and inhibit plant pathogens as well as human opportunistic pathogens (Videira et al. 2010). Furthermore, the above mentioned bacteria were positively or negatively correlated with the physicochemical properties (Fig. 3a). Decrease of the relative abundance of the above mentioned beneficial bacteria may partly explain the reduction of soil nutrients and the unsuitable soil environment. Megyes et al. (2021) found that the candidate class MB-A2-108 adapts to soil with low nutrient content, and have a high ability to tolerate adverse environment. Meantime, the relative abundance of the candidate class MB-A2-108 was significantly higher in bacterial community of CSAC than that of CSBC, but its ecological roles and functions in casing soil merit further research.

Soil properties dynamically changed with the growth of edible fungi, and significantly affected the microbial community structure associated with edible fungi, and finally influenced the yield and quality of edible fungi (Zhang et al. 2018; Ke et al. 2019). Zhou et al. (2017) studied the influencing factors during the development of fruiting bodies of A. sinodeliciosus, and found that dissolved organic carbon, total nitrogen, and C/N ratio affected the fruiting body morphology and microbial community structure. Similarly, soil properties changed after the cultivation of O. raphanipes, and significantly affected the casing bacterial community structure. Compared with the bacterial community in casing soil before the cultivation of O. raphanipes, AP, TN, NH4+-N, and NO3−-N concentrations decreased, similar to the results found in Ganoderma lucidum (Ke et al. 2019). Interestingly, compared with CSBC, the content of available potassium in the casing soil increased after the cultivation of O. raphanipes. This finding is consistent with the previous research results of G. lucidum and Morchella rufobrunnea (Ke et al. 2019; Cao et al. 2021). It is speculated that the large demand for potassium by O. raphanipes at the fruiting stage decomposed the unavailable potassium into available potassium. Meanwhile, some secretions or metabolites of O. raphanipes can increase the pH of casing soil and promote the decomposition of mineralized potassium, so as to increase the content of available potassium (Li et al. 2018; Ke et al. 2019; Cao et al. 2021). The change of casing soil properties reflected the loss of soil nutrients; therefore, paying attention to soil nutrients and selecting appropriate casing soil are conducive to the efficient and safe production of O. raphanipes in the production.

It is worth noting that functional prediction results are different when different methods based on different databases are used. There are multiple copies of the marker gene 16S rRNA in many bacteria genomes, only the taxa with homologous reference genome sequences in the Greengenes database can be predicted via PICRUSt2, and PICRUSt2 can avoid the overestimation of bacterial abundance (Hariharan et al. 2017). FAPROTAX is based on the culturable bacteria with better prediction accuracy but lower predicted coverage compared with that of PICRUSt2; Bugbase predicts organism-level microbiome phenotypes. To better reflect the function change in CSBACO, the combined function prediction methods were used in this study. Results showed that the relative abundance of the aerobic bacteria functional in amino acid and lipid metabolism, as well as metabolism of terpenoides and polyketides, obviously decreased, while that of bacteria with the function of nitrate reduction, animal parasites, symbionts or human pathogens increased sharply after the cultivation of O. raphanipes (Fig. 4a). As the growth of O. raphanipes is also an aerobic process, it is speculated that due to the competition for oxygen, the relative abundance of aerobic bacteria in CSAC decreased compared with that in CSBC. With the growth of O. raphanipes, the nutrients in the rod or casing soil decreased, resulting in weakening capacity of disease resistance, metabolism disorder, growth and development obstruction, and accumulation of harmful metabolites of O. raphanipes, as well as increase of pathogenic bacteria, which may be important reasons for the obstacle of continuous cropping of O. raphanipes. Furthermore, it is valuable to further investigate whether such changes result from the direct effects of edible fungus cultivation, or indirect effects through the bacterial community in casing soil, or both. There is an increasing interest in studying bacterial-fungal interactions associated with edible fungi. It is reported that microbial community possesses the protective functions against competing fungi without affecting P. ostreatus growth (Renáta et al. 2021). Therefore, the growth-promoting bacteria in bacterial community should be explored and their roles in casing soil exchange between the edible fungi and bacteria should be tested. Community reconstruction from casing soil as well as casing soil-independent cultivated process should be the focus in the future.

5 Conclusions

This is the first study to examine the structure and potential function shifts of bacterial community in casing soil before and after the cultivation of O. raphanipes. After the cultivation of O. raphanipes, the alpha diversity indices and the relative abundance of some beneficial genera decreased, nutrients of casing soil lost, and the adverse environment-tolerant bacteria increased. Furthermore, the relative abundance of bacteria with the function of amino acid and lipid metabolism decreased. Conversely, that of bacteria function as parasites or pathogens increased. The above results revealed the significance of bacterial community in casing soil. Shifts of bacterial community may be the reasons for the obstacle of continuous cropping of O. raphanipes.