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Article

Microbiome Dynamics in Four Different Casing Materials Used for Milky Mushroom (Calocybe indica) Cultivation

by
Jia-Ling Zhao
1,2,
Yuan-Yuan Shao
3 and
Bin Liu
1,*
1
Institute of Applied Microbiology, College of Agriculture, Guangxi University, Nanning 530004, China
2
College of Intelligent Transportation and Modern Industry, Anhui Sanlian University, Hefei 230601, China
3
Key Laboratory of Environment Change and Resources Use in Beibu Gulf, Nanning Normal University, Nanning 530001, China
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(6), 667; https://doi.org/10.3390/horticulturae11060667
Submission received: 7 April 2025 / Revised: 4 June 2025 / Accepted: 6 June 2025 / Published: 11 June 2025
(This article belongs to the Section Vegetable Production Systems)
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Abstract

:
Calocybe indica (milky mushroom), an edible mushroom with significant nutritional value, shows potential for cultivation in subtropical regions. Investigating the composition and diversity of the microbial community structure of the casing materials of C. indica is of great significance for understanding the stable yield of the mushroom. This study evaluated four casing materials—loamy soil (LS), loamy soil + cow dung (LS + CD), loamy soil + sand (LS + S), and plant ash (PA)—for their effects on mushroom yield, soil physicochemical properties, and microbial dynamics. The results demonstrated that LS + CD significantly enhanced the yield (2078.50 g) and fruiting body quality, with the shortest pinhead formation time (7.67 days) and superior morphological traits (e.g., cap diameter: 10.10 cm). Physicochemical analysis revealed LS + CD’s elevated moisture retention (19.7%), nutrient availability (e.g., available P: 59.63 mg/kg), and microbial biomass (C: 399.22 mg/kg), alongside a distinct microbial community dominated by Basidiomycota and Actinobacteria. Conversely, LS + S exhibited poor performance due to low water retention and nutrient deficiencies. Redundancy analysis highlighted strong correlations between soil nutrients (nitrogen, potassium, phosphorus) and microbial composition, with LS + CD fostering a microbiome conducive to mushroom growth. These findings underscore LS + CD as the optimal casing material for C. indica cultivation, improving both yield and soil health. Future studies should explore the functional roles of key microbes and refine organic amendments for sustainable practices.

1. Introduction

Calocybe indica (milk mushroom) is a tropical edible mushroom originating from India. Its fruiting bodies are milky white, large in appearance, and hard in texture [1]. Calocybe indica has long been used due to its nutritional and medicinal value [2]. Guangxi Zhuang Autonomous Region is located in the south of China, which has the largest and most famous karst areas in the world; most regions of Guangxi experience a subtropical monsoon climate with high temperature and rainfall. This is very advantageous for the growth of C. indica. Therefore, it is highly desirable to introduce and cultivate C. indica in China. Guangxi University has pioneered the first systematic research on C. indica cultivation techniques in China [3]. Owing to its extended shelf life, high biological efficiency, and heat tolerance, C. indica represents an ideal candidate for high-temperature cultivation. Furthermore, it can be integrated into annual crop rotation cycles with other mushroom species [4]. Casing soil involves applying a layer of soil-based material on the growth substrate of mushrooms. This layer helps to retain moisture and stimulates the development of fruiting bodies [5]. The casing material has a significant impact on the yield and quality of mushrooms [6]. It can assist mycelium in transitioning from nutritional growth to the formation of fruiting bodies [7]. There are various options for mushroom casing materials; for example, a mixture of peat, sand, and ash can serve as an excellent covering medium [8]. Studies have demonstrated that a 2 cm soil cover layer in C. indica cultivation can optimize primordia formation, enhance fruiting body size, and achieve maximum biological efficiency (100.76%) [9]. However, layers exceeding this thickness may result in delayed fruiting and reduced yield. According to experiments conducted by Amin et al., a mixture of loamy soil and cow dung performs optimally at a thickness of 3 cm, while sandy soil mixtures may require a thinner layer of 2 cm to maintain an appropriate balance between water retention and aeration [10].
In mushroom cultivation, field soil serves as the predominant casing material, and appropriate casing soil can markedly enhance both the quality and yield of mushrooms [11]. Attempts have been made to replace field soil with other materials [12], but thus far field soil has been indispensable. Investigating shifts in the casing layer’s microbial community can help clarify its effects. It has effects on pathogenic bacteria and the development of fruiting bodies [13]. These microbial dynamics, in turn, influence the yield and quality of edible mushroom fruiting bodies; additionally, the carbon and nitrogen content of the casing soil modulates bacterial and fungal community composition [14]. Microorganisms present in the soil layer facilitate the degradation of lignocellulose by mycelia through the secretion of specific metabolic products, such as chitinase and nitrogen-fixing enzymes, while at the same time suppressing the enzymatic activities of pathogenic bacteria, like protease and hyaluronidase [15].
Using loamy soil (LS), loamy soil + cow dung (LS + CD), loamy soil + sand (LS + S), and plant ash (PA) as casing materials, the aim of this study was to investigate the effects of these different casing materials on the growth parameters and yield of C. indica. Furthermore, we analyzed the physical and chemical properties of these casing materials as well as their effects on microbial diversity. Ultimately, we aimed to select the most suitable soil covering material for the cultivation of C. indica in China.

2. Material and Methods

2.1. Strains and Substrate

The C. indica strain was obtained from India in 2014 and deposited in the Institute of Edible Fungi, Guangxi University. The liquid medium consisted of 2% soybean powder, 1.5% glucose, 0.2% potassium dihydrogen phosphate, 0.5% wheat bran, 1% corn flour, 0.01% vitamin, and 0.1% magnesium sulfate in 1 L of water. The medium was sterilized at 121 °C for 30 min and then inoculated with C. indica mycelium when the temperature dropped below 30 °C. Incubation at 28 °C and 120 r/min on a magnetic stirrer for 5 days before inoculation.
The substrate in this study was a 67% spent mushroom substrate of Pleurotus eryngii mixed with 30% corn flour, 2% CaCO3, and 1% gypsum. The cultivation methods with casing materials have been described in our previous studies [3]. The moisture content was maintained at approximately 65% and the pH was adjusted to 7.5–8.0 with composting for 24 h before filling polypropylene bags (17 × 33 cm) with the substrate (1000 g). Four treatments were established, with three replicates of 12 bags per replicate. The bags were sterilized at 120 °C for 2 h and then transferred to a sterile room to cool. Subsequently, 25 mL of liquid inoculum was aseptically introduced into each bag within a laminar-flow inoculation chamber, and the inoculated bags were incubated in the culture room.

2.2. Preparation of Casing Mixture and Casing

Field soil, cattle manure, sand, and plant ash were supplied by the Experimental Base of the Agricultural College, Guangxi University. Four casing treatments were employed: loamy soil (LS); loamy soil amended with cow dung at a volume-to-volume ratio of 3:1 (LS + CD); loamy soil amended with sand at a volume-to-volume ratio of 3:1 (LS + S); and plant ash (PA).
Pre-treatment of casing materials was carried out as previously described [3]. Preliminary studies indicated that when using mushroom residue as the covering material for C. indica cultivation, it resulted in soil with a poor water-retention capacity. Pre-experiments were conducted with mixing field soil + cow dung at ratios of 1:3, 1:2, 1:1, 2:1, and 3:1. The results indicated no significant difference in yield among the tested ratios; however, because ratios below 3:1 exhibited a higher contamination risk, a 3:1 ratio was selected for this study. The sand proportion was optimized using an identical procedure.

2.3. Cultivation and Sampling

To prepare the soil covering components, lime and water were added to adjust the humidity to the given percentage and the pH to between 8.0 and 9.0. Upon the observation of primordia, twelve pre-inoculated bags were placed in each basket and covered with a 3 cm layer of soil. The mushroom growth chamber was maintained at 30 °C and 70–80% relative humidity, with regular cleaning and adequate ventilation.
Sampling commenced once the newly emerged mushrooms reached 4–5 cm in length. Briefly, samples were collected from each basket using a five-point sampling method [16]. The samples were dried and passed through a 2 mm sieve. Each homogenized sample was then divided into three aliquots, one of which was used for physicochemical analysis. The second aliquot was stored at 4 °C for microbial isolation and soil microbial biomass determination, while the third aliquot was kept at −80 °C for microbial community structure analysis.
During the fruiting body harvest period, standardized methods were employed to quantify the yield, and the following parameters were recorded: pinhead initiation, fresh weight, number, size, stipe length, and diameter of pileus and stipe. The yield and biological efficiency of the mushrooms were calculated. For each treatment, three bags were randomly selected as replicates. The data represent the cumulative values from the first and second flushes, expressed as “mean ± standard deviation”.

2.4. Determination of Physicochemical Properties of Casing Materials

The contents of nitrogen, available phosphorus, potassium, calcium, magnesium, and trace copper were determined according to the method described by Kumar et al. [17]. A soil-to-water ratio of 2.5:1 (w/v) was maintained, and after 30 min of ultrasonication, the pH was measured using a PHS-3C precision pH meter.
Water content determination was carried out during the early growth stages of C. indica, and soil samples were collected on the third day after sprinkling water. The soil samples were weighed using the drying method [18]. Samples were dried in covered aluminum boxes at 105 °C for 6 h, cooled in a vacuum dryer for 30 min, and then weighed.

2.5. Estimation of Microbial Population in Casing Material

The population of microorganisms in the casing was estimated using the dilution-to-extinction technique [19]. Soil samples were diluted 1:64 in 1% carboxymethyl cellulose solution in 48-well microplates, whereas bacterial isolation employed 96-well microplates, with beef extract peptone medium for bacteria and Martin’s medium for fungi. After serial dilution, 10 µL of each dilution was dispensed into wells containing the appropriate culture medium. Bacterial cultures were incubated at 37 °C for 2–3 days, and colonies from the 10−7 dilution were selected for enumeration. The isolated fungi were cultured at 25 °C for 3–5 days, and colonies from the 10−4 dilution were selected. Actinomycetes were incubated at 28 °C for 4 days, with colonies from the 10−6 dilution chosen. All dilutions were performed in triplicate.

2.6. Determination of Microbiomass C, N, and P

Microbial biomass carbon was determined using the chloroform fumigation volumetric method [20]. Microbial biomass nitrogen was analyzed using chloroform fumigation extraction–indotrione colorimetry [21] at 570 nm. Microbial biomass phosphorous was determined using the chloroform fumigation phosphomolybdenum blue colorimetric method at 882 nm [22].

2.7. Illumina Genome Analyzer IIx

A high-throughput Illumina instrument (Shanghai Meiji Biomedical Technology Co., Ltd., Nanning, China) was used to sequence fungi and bacteria. DNA was extracted using a kit (E. Z. N. A. Soil DNA Kit, Omega Bio-tek, Nanning, China). The quality of the total DNA was detected using 1% agarose gel electrophoresis, and the purity was detected using ultraviolet spectrophotometry. Qualified DNA after quality inspection was stored at −80 °C. The bacterial primers used were 341F (5′-ACTCCTACGGAGGCAGCAGCAG-3′) and 806R (5′-GGACTACHGGGTWTCTAAT-3′). The fungi primers were 2045F (5′-GCATCGATAGAACGCAGC-3′) and 2390R (5′-TCCCGCTTATTATGC-3′). The average length of bacterial sequence reads was 253 bp, and for fungi, it was 300 bp. Finally, Illumina MiSeq PE300 was used for online data analysis and identification.

2.8. Statistical Analysis

Statistical and variance analyses were performed using Excel 2013 and SPAA 18.0. Online data processing was conducted via the Meiji Biology Company cloud data platform. Species prevalence in the samples was initially determined based on OTU abundance. Community richness and diversity were evaluated using the ACE, Simpson, Shannon, and Chao indices. A Venn diagram was constructed to illustrate the number of OTUs shared among—and unique to—the different covering materials.

3. Results

3.1. Effect of Different Casing Materials on Yield

The data in Figure 1 indicate that different casing materials significantly affect the yield and growth parameters of C. indica. Specifically, variations in casing materials lead to distinct changes in both yield and physiological growth metrics. The field soil + cow dung treatment exhibited the best performance across all key indicators, with a yield of 2078.50 g (Figure 1a)—significantly higher than that of the other treatments. Moreover, individual fruiting body weight was the greatest (107.80 g, as shown in Figure 1b), and time to primordia emergence was the shortest (7.67 days, as shown in Figure 1c). The stipe diameter (3.30 cm), stipe length (18.70 cm), cap diameter (10.10 cm), and cap thickness (2.43 cm) were all significantly greater than those of the other treatment. These findings suggest that cow dung amendment significantly improved soil fertility, thereby promoting the rapid growth and development of high-quality C. indica fruiting bodies.
The wood ash treatment ranked second, yielding 1866.34 g with an average mushroom weight of 99.16 g (as shown in Figure 1b). Stipe and cap morphological parameters were comparable to those of the cow dung treatment, although the time to primordia emergence was slightly longer (10.69 days). These results indicate that wood ash can improve soil properties and promote fruiting body development. However, its overall efficacy is slightly inferior to that of cow dung.
In contrast, the field soil treatment alone resulted in a lower yield (1568.83 g, as shown in Figure 1a) and individual mushroom weight (97.17 g). The time to primordia emergence was intermediate (9.50 days), and the morphological parameters were similarly moderate. The field soil + sand treatment exhibited the poorest performance across all parameters, yielding only 1209.50 g with an average mushroom weight of 85.95 g. It also produced the smallest cap diameter (7.20 cm, as shown in Figure 1d) and the lowest mushroom number. These results suggest that sand addition is detrimental to C. indica cultivation, likely due to its poor water-retention capacity and limited nutrient availability.

3.2. Physical and Chemical Properties of Different Casing Materials

The physical and chemical properties of the various casing materials are summarized in Table 1. All casing materials exhibited pH values above 7.0, whereas the optimal pH range for C. indica cultivation is 6.5–8.5. The LS + CD treatment exhibited the highest moisture content (19.7%), indicating superior water retention capacity, whereas the LS + S treatment showed the lowest moisture content (5.3%), reflecting the poorest retention among all treatments.
The nitrate-N content was highest in the LS + S treatment (36.35 mg kg−1), representing a 24.36% increase over the control, whereas the PA treatment exhibited the lowest nitrate-N content (10.73 mg kg−1). The available K in the PA and LS + CD treatments was 334.77 mg kg−1 and 255.20 mg kg−1, respectively—both significantly higher than the control—whereas the LS + S treatment contained significantly less available K. The LS + CD treatment also recorded the highest levels of available P (59.63 mg kg−1), Cu (54.83 mg kg−1), Mg (46.36 mg kg−1), and Ca (212.68 mg kg−1).

3.3. Effects of Casing Materials on Microflora

As shown in Table 2, the bacterial population in the LS treatment was the highest, while in LS + CD, it was the lowest. The LS + CD treatment yielded the highest actinomycete counts, whereas the LS + S and PA treatments exhibited significantly lower counts compared to both the control and LS + CD treatments. The total fungal counts were highest in the LS + CD treatment, approximately 3.47-fold greater than the control. The culturable fungal numbers in the LS + CD and PA treatments were 1.87-fold and 4.5-fold higher than those of the control, respectively.

3.4. Effects of Different Casing Materials on Microbial Biomass C, N, and P

As shown in Figure 2, among the four treatments, the microbial biomass C was highest in LS + CD, followed by PA, LS, and LS + S. The LS + CD treatment exhibited a microbial biomass C content of 399.22 mg kg−1—35.37% higher than that of the control. The PA treatment exhibited the second highest microbial biomass C content, whereas the LS + S treatment had the lowest (177.85 mg kg−1), significantly lower than the control. The trend in the microbial biomass P across the soil covering materials mirrored that of the microbial biomass C. By contrast, the microbial biomass N exhibited a distinct pattern, with the highest content in the LS + CD treatment, followed by PA, LS + S, and CK. Overall, the microbial biomass in the LS + CD treatment was significantly greater than in the other treatments.

3.5. High-Throughput Sequencing of Bacteria and Fungi

The results of high-throughput sequencing are summarized in Table 3. In total, 191,722 optimized sequences of bacteria and 257,776 optimized sequences of fungi were obtained. The optimized baseline counts for bacteria and fungi were 103,521,448 and 83,520,289, respectively. Operational taxonomic units (OTUs) were analyzed in each sample to assess the abundance of different microorganisms. At 97% similarity, 477 bacterial OTUs and 11 fungal OTUs were recovered. Sequence alignment enabled the identification of 245 bacterial taxa at the genus level and 346 at the species level, whereas 10 fungal taxa were resolved to both genus and species.

3.6. Alpha Diversity Analysis of Different Soil Covering Materials

Alpha diversity, also referred to as within-sample diversity, is employed to assess the prevalence and diversity of microbial communities [23]. The metrics used to assess community prevalence were the Sobs, Chao, ACE, Jackknife, and Bootstrap indices [24]. Community evenness was assessed using the Simpson evenness index, the Shannon evenness index, the Heip index, and the Smith–Wilson index. Community diversity was evaluated using the Shannon index, the Simpson index, the NP-Shannon index, the Berger–Parker index, the inverse Simpson index, and the qstat index (reflecting community coverage) [25,26,27]. Data from Table 4 and Table 5 show that the Chao, ACE, and Shannon indices were highest in the LS + S treatment. The bacterial Shannon evenness index under LS + CD was the highest, whereas the corresponding index for fungi was the lowest. Overall, bacterial alpha diversity in the soil covering materials exceeded fungal alpha diversity.

3.7. Classification of Bacteria and Fungi in Different Soil Casing Materials at Phylum Level

The community structure of fungi and bacteria at the phylum level is depicted in Figure 3. A total of 17 bacterial phyla were identified, including Actinobacteria, Proteobacteria, Bacteroidetes, and Firmicutes. Proteus and actinomycetes were the most dominant taxa, comprising the following proportions of the total microbial population in each experimental group: LS + CD (66.73 %), PA (80.22 %), LS + S (62.27 %), and LS (63.39 %). Verrucomicrobia was detected exclusively in the LS + S and LS treatments. Overall, the LS + S treatment group exhibited the highest number of identified bacterial phyla. Soil fungi were classified into two phyla: Basidiomycota and Ascomycota. Basidiomycota was the dominant fungal group across treatments, with the highest relative abundance in the LS + CD treatment, followed by LS + S, PA, and LS. In contrast, Ascomycota showed the greatest prevalence in the LS treatment, followed by PA, LS + S, and LS + CD.

3.8. RDA of Microbial Community Composition and Environmental Factors in Different Casing Materials

We compared shared and unique taxa across samples using a Venn diagram (Figure 4). A total of 172 bacterial OTUs were identified, representing 36.06% of all OTUs; the LS + CD treatment harbored 37 unique OTUs (7.76% of total OTUs). No treatment group contained unique fungal OTUs, and ten fungal OTUs were shared by all treatments. Excluding the PA treatment, one OTU was common to the other three treatment groups.
Redundancy analysis (RDA) was employed to elucidate the relationships between bacterial communities and environmental factors. In the resulting biplot, red arrows represent environmental variables, while blue arrows denote the top ten OTU-ranked species. Arrow length reflects the influence of environmental factors on species abundance or OTU richness: longer arrows indicate a stronger effect. The angle between arrows denotes correlation: acute angles signify positive correlations, obtuse angles indicate negative correlations, and right angles imply no correlation. The proximity of a sample point to an environmental factor arrow represents the magnitude of that factor’s impact on community distribution—the shorter the distance, the greater the influence.
Figure 5 presents an RDA of OTU-level community composition for fungi and bacteria in relation to environmental factors across the different soil covering materials. Furthermore, all six environmental indicators were strongly correlated with the LS + CD treatment. In the bacterial RDA ordination, the longest arrows—reflecting the greatest influence—aligned with Proteobacteria and Actinobacteria (as shown in Figure 5a), in agreement with previous findings. Richness was lowest in the LS + CD treatment group. Actinobacteria abundance was negatively correlated with soil nitrate-nitrogen content and positively correlated with the other five indicators, indicating that soil nitrate-nitrogen, available potassium, available phosphorus, available calcium, magnesium, and copper significantly influence bacterial community composition. At the OTU level, Ascomycota and Basidiomycota exhibited high richness (as shown in Figure 5b). Ascomycota abundance was independent of available nitrogen but negatively correlated with available phosphorus, copper, and calcium and positively correlated with available potassium and magnesium. Ascomycota exhibited the lowest abundance in the LS + CD treatment, whereas Basidiomycota was significantly correlated with all six indicators and was most abundant under LS + CD.
In conclusion, field soil amended with cow dung is the most suitable casing material for C. indica cultivation, significantly enhancing both yield and fruiting body quality. Wood ash may serve as a viable alternative, whereas the use of field soil combined with sand should be avoided. Future studies should investigate the optimal ratios of cow dung and other organic amendments to further enhance cultivation efficiency.

4. Discussion

4.1. Effects of Different Casing Materials on the Yield of Calocybe indica

This study evaluated the effects of different casing materials on the yield, fruiting body quality, and growth mechanisms of C. indica. The field soil + cow dung treatment yielded the highest output (2078.50 g); it also produced the greatest individual fruiting body weight (107.80 g) and exhibited the most favorable morphological characteristics.
These findings corroborate recent studies demonstrating that organic amendments markedly enhance mushroom growth. Cow dung, rich in nitrogen, phosphorus, and trace elements, promotes C. indica mycelial differentiation. Previous research has confirmed that incorporating cow dung during cultivation increases C. indica yield [28], a trend consistently observed in this study. Additionally, cow dung enhances soil water retention and aeration [29]. This likely contributed to the shortest time to fruiting (7.67 days) observed in the field soil + cow dung treatment. These data indicate that cow dung effectively optimizes the substrate microenvironment. The wood ash treatment ranked second in yield (1866.34 g). The alkalinity of wood ash (pH 9–11) can neutralize the acidity of the substrate layer, thereby promoting the growth of C. indica mycelium [30]. Wood ash is also rich in potassium, which enhances the synthesis of cell walls in fruiting bodies [28], so the cap diameter (9.9 cm) was comparable to that of the cow dung group. However, the time of primordia formation for the wood ash treatment was longer (10.69 days); this may potentially be due to an excessively high C/N ratio. Some studies have indicated that a high C/N ratio may indirectly inhibit carbon utilization by mycelium through nitrogen limitation [21]. The field soil + sand treatment exhibited the poorest performance, yielding the lowest output (1209.50 g) and the smallest cap diameter (7.20 cm). This inferior result is likely due to sand’s poor water-retention capacity, which reduces substrate layer humidity. Previous studies have demonstrated that higher humidity promotes cap expansion [31]. Notably, inadequate moisture retention in the substrate layer directly impacts mushroom development.

4.2. Differences in the Physical and Chemical Properties of Different Soil Covering Materials

pH is a critical factor in microbial growth as it directly influences fungal development by altering nutrient availability [32]. The pH range suitable for fungus growth is relatively wide, spanning from 3.0 to 8.0 [33]. However, mycelial growth thrives under neutral to slightly acidic conditions (pH 5.0–7.0). In contrast, conidial production is supported over a broader range, from pH 5.0 to 8.0 [34]. The suitable pH range for the growth of C. indica mycelium is 6.5–8.5 [30], and the pH value of the casing soil is higher than 7. Under identical conditions, the loamy soil + cow dung treatment exhibited the highest casing moisture (19.7%), indicating its strong capacity to retard water loss. Previous studies have demonstrated a positive correlation between casing soil moisture and Agaricus bisporus yield [35]. N, P, and K are essential nutrients in crop nutrition, playing critical roles in plant growth. The soil N and P contents directly affect soil fertility [36] and also determine the structure and function of the surrounding ecosystem [37]. Specifically, nitrate-N, available phosphorus (P), and available potassium (K) serve as reliable indicators of soil N, P, and K availability. In this study, the loamy soil + cow dung treatment showed significantly higher contents of nitrate-N and available P, along with elevated available K content. Therefore, such nutrient enrichment likely enhances C. indica mycelial activity, promotes rapid growth, and supports fruiting body development.
Previous studies showed that the content of Cu in the same soil correlated with the change in pH [38]. Mycelial growth rates generally exhibited a positive correlation with Ca concentrations across the 0–300 mg/kg range [39]. The loamy soil + cow dung treatment exhibited a calcium content of 212.68 mg kg−1, significantly higher than that of the other treatments. Magnesium (Mg), a mobile nutrient, has been shown to influence soil nitrogen red C. indica istribution when its concentration varies [40]. The loamy soil + cow dung (LS + CD) treatment exhibited the highest available magnesium content, whereas both loamy soil (LS) alone and loamy soil + sand (LS + S) showed lower levels; accordingly, fruit bodies achieved their optimal growth under the LS + CD treatment.

4.3. Effects of Different Soil Casing Materials on Soil Microbial Biomass and Soil Microflora Microorganisms

Soil microorganisms constitute over 95% of total soil biomass and play crucial roles in maintaining a healthy soil ecosystem, including pedogenesis, soil organic matter dynamics, and nutrient cycling [41]. Thus, soil chemical composition significantly affects both the abundance and diversity of soil microorganisms [42]. In this study, the loamy soil + cow dung (LS + CD) treatment yielded higher concentrations of N, P, and K and supported the greatest abundance of fungi and actinomycetes, yet the lowest bacterial count. The dominant bacterial phylum was Proteobacteria, whose relative abundance was negatively correlated with nitrate-N, available P, available Ca, Mg, and Cu. Furthermore, microbial biomass and activity were directly related to soil organic matter content and thus serve as reliable indicators of soil fertility [43]. Incorporating organic matter into soil enhances its physicochemical properties and nutrient status. It increases microbial biomass carbon content, stimulates microbial growth and activity, and elevates the fungi-to-bacteria ratio. Moreover, it augments both microbial community structure and functional diversity [44]. Soil organic matter plays a pivotal role in soil health by stabilizing soil aggregates, enhancing water-holding capacity, buffering pH fluctuations, and serving as a nutrient reservoir [45]. Under the condition of poor soil aeration, soil pH as well as soil microbial activity and soil microbial biomass will decrease [46]. The loamy soil + cow dung (LS + CD) treatment elevated soil pH, improved aeration, and enhanced nutrient status, thereby promoting microbial activity and yielding the highest microbial biomass C, N, and P of all treatments. In contrast, the loamy soil + sand (LS + S) treatment—although it also increased soil pH—suffered from poor permeability, inadequate water retention, lower nutrient content, and reduced microbial activity.

4.4. Effects of Different Casing Materials on Composition and Structure of Microbial Community

The results of alpha diversity analysis showed that loamy soil + sand had the highest bacterial richness and diversity, and loamy soil + cow dung had the lowest fungal diversity. RDA showed that the bacterial OTUs in the loamy soil and loamy soil + sand treatment groups were very similar, while the other two treatment groups were dissimilar. The fungi OTUs of plant ash and loamy soil + sand showed similarities, and the other two treatments were unrelated. In addition, soil nitrate nitrogen, soil available potassium, and soil available phosphorus were selected to analyze the influence of environmental factors on the OTU composition. We found that these three indices had a great impact on the composition of bacterial and fungal communities. Furthermore, the OTU component of the loamy soil + cow dung treatment was positively correlated with the three previously mentioned indices. Microbial community variability can arise from environmental factors such as soil type, geographic location, and physicochemical soil properties [47]. In this study, the experimental treatments exhibited a marked influence on the structure of bacterial and fungal communities. Notably, the loamy soil + cow dung treatment demonstrated a significant positive correlation with environmental factors. Using high-throughput sequencing technology, 477 bacterial OTUs and 11 fungal OTUs were identified at a 97% similarity threshold. The observed OTU richness values were relatively low compared with those reported in previous studies [48]. We hypothesize that this effect arises because the covering material was sterilized before application.
Alpha diversity analysis demonstrated that the loamy soil + sand treatment exhibited the highest bacterial richness and diversity, whereas the loamy soil + cow dung treatment showed the lowest fungal diversity. Redundancy analysis (RDA) indicated that the bacterial OTU compositions of the loamy soil and loamy soil + sand treatments were highly similar. In contrast, the compositions of the other two treatments were distinct. The fungal OTU compositions of plant ash and loamy soil + sand treatments were similar, while those of the remaining treatments differed significantly. Soil nitrate-N, available K, and available P were chosen to assess the influence of environmental factors on OTU composition. These three parameters markedly shaped both bacterial and fungal community structures. Furthermore, the OTU profile of the loamy soil + cow dung treatment was positively correlated with all three indices.
At the phylum level, Proteobacteria and Actinobacteria were the predominant bacterial taxa, with Proteobacteria exhibiting the highest abundance across all treatments according to RDA. Moreover, RDA revealed that Proteobacteria abundance was negatively correlated with soil nitrate-N, available phosphorus, calcium, magnesium, and copper concentrations but showed no correlation with soil available potassium. In the loamy soil + cow dung treatment, these environmental parameters exerted their greatest influence, yielding the lowest Proteobacteria abundance. Moreover, certain Proteobacteria taxa have been reported to facilitate nitrogen fixation and to thrive under varied environmental conditions [49]. The total bacterial abundance was lowest in the loamy soil + cow dung treatment, indicating that this environment was less conducive to bacterial growth. At the phylum level, only Ascomycota and Basidiomycota were detected among fungi, with Basidiomycota representing the dominant group. The loamy soil + cow dung treatment exhibited the lowest total bacterial abundance, suggesting that this environment was less conducive to bacterial proliferation. At the phylum level, only Ascomycota and Basidiomycota were detected, with Basidiomycota being the predominant group. Ascomycota abundance followed as loamy soil > plant ash > loamy soil + sand > loamy soil + cow dung. Redundancy analysis revealed that Basidiomycota abundance was positively correlated with nitrate-N, available K, P, Ca, Mg, and Cu, and reached its maximum under the loamy soil + cow dung treatment. Conversely, Ascomycota abundance was negatively correlated with available P, Cu, and Ca, but positively correlated with available K and Mg.

5. Conclusions

This study systematically evaluated the effects of various casing materials on C. indica yield, soil physicochemical properties, and associated microbial communities. The findings indicated that the loamy soil + cow dung treatment was the most effective casing material for C. indica cultivation, significantly enhancing both yield and fruiting body quality. Wood ash may serve as a viable alternative, whereas the use of field soil combined with sand should be avoided. Soil analysis demonstrated significantly enhanced moisture, available potassium, phosphorus, and calcium, indicating improved fertility and water retention. Microbiological profiling demonstrated a profound shift in microbial community dynamics, with microbial biomass carbon reaching 399.22 mg/kg and fungal propagule density increasing 3.47-fold relative to control substrates. Taxonomic characterization identified Basidiomycota as the dominant fungal phylum, accompanied by significant actinomycete proliferation. These findings collectively demonstrate a synergistic enhancement of yield parameters, edaphic conditions, and microbiome composition. The results establish a robust foundation for sustainable C. indica production, while highlighting key areas for future research, including optimization of organic amendment ratios and elucidation of microbial nutrient cycling pathways.

Author Contributions

J.-L.Z.: Conceived and conducted the experiments, collected and analyzed the data, prepared and revised the manuscript. Y.-Y.S.: Assisted in experiments and writing; B.L.: Supervised the project, conceived and designed the study, edited and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Key Research and Development Program of Guangxi (AB18221047) and the Guangxi Mushroom Science and Technology Vanguard (GNKM202404-2, GNKM202504-2).

Data Availability Statement

All data needed to evaluate the conclusions in the paper are included in the article, further inquiries can be directed to the authors.

Acknowledgments

We thank Deng-Qi Ou for technical help, and Usman Rasheed for discussion in the writing.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Horticulturae 11 00667 g001
Figure 1. Effect of different casing materials on the yield-contributing characteristics of Calocybe indica. Different lowercase letters at the top of the same type of column indicate significant differences (p < 0.05, Duncan’s method). (a): Effects of different casing materials on yield; (b): Effects of different casing materials on weight of individual fruiting bodies; (c): Effects of different casing materials on days to primordium formation; (d): Effects of different casing materials on morphological characteristics of mushrooms.
Figure 1. Effect of different casing materials on the yield-contributing characteristics of Calocybe indica. Different lowercase letters at the top of the same type of column indicate significant differences (p < 0.05, Duncan’s method). (a): Effects of different casing materials on yield; (b): Effects of different casing materials on weight of individual fruiting bodies; (c): Effects of different casing materials on days to primordium formation; (d): Effects of different casing materials on morphological characteristics of mushrooms.
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Figure 2. Effect of different casing materials on soil biomass C, N, and P. Different lowercase letters at the top of the same type of column indicate significant differences (p < 0.05, Duncan’s method).
Figure 2. Effect of different casing materials on soil biomass C, N, and P. Different lowercase letters at the top of the same type of column indicate significant differences (p < 0.05, Duncan’s method).
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Figure 3. Classification of soil fungi and bacteria retrieved from different casing materials at the phylum level.
Figure 3. Classification of soil fungi and bacteria retrieved from different casing materials at the phylum level.
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Figure 4. Venn diagram showing shared OTUs among different casing materials (OTUs0.03) between fungi and bacteria.
Figure 4. Venn diagram showing shared OTUs among different casing materials (OTUs0.03) between fungi and bacteria.
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Figure 5. Redundancy analysis of the relationship between the bacterial (a) and fungal (b) communities and soil environmental factors in different casing materials. LS: loamy soil; LS + CD: loamy soil + cow dung; PA: plant ash; LS + S: loamy soil and sand; SNN: soil nitrate nitrogen; SAO: soil available potassium; SAP: soil available phosphorus; SAC: soil active copper; SACA: soil available calcium; SAM: soil available magnesium.
Figure 5. Redundancy analysis of the relationship between the bacterial (a) and fungal (b) communities and soil environmental factors in different casing materials. LS: loamy soil; LS + CD: loamy soil + cow dung; PA: plant ash; LS + S: loamy soil and sand; SNN: soil nitrate nitrogen; SAO: soil available potassium; SAP: soil available phosphorus; SAC: soil active copper; SACA: soil available calcium; SAM: soil available magnesium.
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Table 1. Statistical analysis of soil physical and chemical properties.
Table 1. Statistical analysis of soil physical and chemical properties.
Soil EnvironmentalLS (CK)LS + CDPALS + S
pH7.68 ± 0.01 d8.36 ± 0.04 a7.93 ± 0.01 c8.24 ± 0.02 b
Soil water content (%)10.50 ± 0.55 b19.70 ± 0.01 a10.30 ± 0.12 b5.30 ± 0.03 c
Soil nitrate nitrogen (mg/kg)27.63 ± 2.53 b36.35 ± 5.61 a10.73 ± 1.86 d23.19 ± 3.14 c
Soil available potassium (mg/kg)59.27 ± 0.36 c255.20 ± 2.80 b334.77 ± 2.55 a47.75 ± 0.17 d
Soil available phosphorus (mg/kg)38.21 ± 0.03 b59.63 ± 0.61 a25.20 ± 0.15 d34.06 ± 0.53 c
Soil active copper (mg/kg)40.98 ± 0.14 b54.83 ± 0.07 a40.73 ± 0.04 b29.83 ± 0.03 c
Soil available calcium (mg/kg)133.25 ± 0.59 c212.68 ± 1.15 a156.37 ± 1.70 b133.27 ± 1.44 c
Soil available magnesium (mg/kg)17.78 ± 0.15 d46.36 ± 0.26 a38.08 ± 0.07 b19.50 ± 0.11 c
Note: Different lowercase letters in the same line indicate significant difference (p < 0.05, Duncan’s method). Abbreviations: LS: loamy soil; LS + CD: loamy soil + cow dung; PA: plant ash; LS + S: loamy soil and sand.
Table 2. Effect of different casing materials on microflora.
Table 2. Effect of different casing materials on microflora.
TreatmentBacteria (106 cfu/g)Fungi (104 cfu/g)Actinobacteria (106 cfu/g)
LS (CK)29.33 ± 2.69 a3.55 ± 0.98 c5.41 ± 1.21 a
LS + CD18.73 ± 1.89 c12.33 ± 1.92 a6.15 ± 3.31 a
LS + S27.80 ± 1.32 b6.59 ± 2.67 b1.52 ± 1.09 b
PA19.70 ± 5.24 c2.74 ± 1.01 c1.07 ± 2.45 b
Different lowercase letters in the same column indicate significant difference (p < 0.05, Duncan’s method).
Table 3. Sequencing results for bacteria and fungi.
Table 3. Sequencing results for bacteria and fungi.
NameNumber
OTUGenusSpeciesSequencesBase Pairs (bp)
Bacteria477245346191,722103,521,448
Fungal111010257,77683,520,289
Table 4. Mean α-diversity of the bacterial communities in the different casing materials (species level).
Table 4. Mean α-diversity of the bacterial communities in the different casing materials (species level).
Treatmentα-Diversity Index
Chao Ace ShannonevenShannon
LS(CK)126.00 ± 4.12 b125.37 ± 9.24 c0.77 ± 0.24 b3.70 ± 0.68 b
LS + CD126.25 ± 11.45 b126.38 ± 13.01 b0.79 ± 0.06 a3.29 ± 0.55 d
PA119.88 ± 6.78 c120.76 ± 5.65 d0.73 ± 0.12 c3.51 ± 1.41 c
LS + S128.00 ± 11.56 a127.53 ± 3.21 a0.68 ± 0.34 d3.85 ± 1.05 a
Note: Different lowercase letters in the same line indicate significant difference (p < 0.05, Duncan’s method).
Table 5. Mean α-diversity of the fungal communities in the different casing materials (species level).
Table 5. Mean α-diversity of the fungal communities in the different casing materials (species level).
Treatmentα-Diversity Index
ChaoAceShannonevenShannon
LS(CK)18.00 ± 2.12 c18.00 ± 1.24 c0.58 ± 0.14 a1.67 ± 0.58 a
LS + CD16.00 ± 1.35 d16.50 ± 3.06 d0.38 ± 0.07 d1.15 ± 0.57 c
PA21.00 ± 0.78 b21.48 ± 2.65 b0.47 ± 0.02 c1.29 ± 0.43 b
LS + S31.00 ± 1.66 a32.00 ± 1.21 a0.49 ± 0.04 b1.65 ± 0.05 a
Note: Different lowercase letters in the same line indicate significant difference (p < 0.05, Duncan’s method).
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Zhao, J.-L.; Shao, Y.-Y.; Liu, B. Microbiome Dynamics in Four Different Casing Materials Used for Milky Mushroom (Calocybe indica) Cultivation. Horticulturae 2025, 11, 667. https://doi.org/10.3390/horticulturae11060667

AMA Style

Zhao J-L, Shao Y-Y, Liu B. Microbiome Dynamics in Four Different Casing Materials Used for Milky Mushroom (Calocybe indica) Cultivation. Horticulturae. 2025; 11(6):667. https://doi.org/10.3390/horticulturae11060667

Chicago/Turabian Style

Zhao, Jia-Ling, Yuan-Yuan Shao, and Bin Liu. 2025. "Microbiome Dynamics in Four Different Casing Materials Used for Milky Mushroom (Calocybe indica) Cultivation" Horticulturae 11, no. 6: 667. https://doi.org/10.3390/horticulturae11060667

APA Style

Zhao, J.-L., Shao, Y.-Y., & Liu, B. (2025). Microbiome Dynamics in Four Different Casing Materials Used for Milky Mushroom (Calocybe indica) Cultivation. Horticulturae, 11(6), 667. https://doi.org/10.3390/horticulturae11060667

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