Effects of short-term application of Moutai lees biochar on nutrients and fungal community structure in yellow soil of Guizhou

In order to realize the utilization of Moutai lees and the improvement of soil fertility of yellow soil in Guizhou, a field experiment was carried out to study the effects of short-term application of Moutai lees biochar on nutrients and fungal community structure diversity of yellow soil. The results showed that the application of Moutai lees biochar increased the pH, soil organic matter (SOM), total nitrogen (TN), ammonium nitrogen (NH4+-N), nitrate nitrogen (NO3−-N), available phosphorus (AP), and available potassium (AK), while the microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN) were reduced. The application of biochar significantly reduced the number of fungal OTU and community diversity. The application of biochar increased the relative abundances of Chytridiomycota and Mortierellomycota, while the relative abundance of Ascomycota was significantly reduced. Redundancy analysis (RDA) suggested that SOM, NH4+-N and NO3−-N were the key factors correlated with changes in microbial community structure. Overall, the short-term application of lees biochar can not only improve the nutrient content of yellow soil, but also change the structure and diversity of soil fungal communities. More importantly, Moutai lees biochar can reduce the relative abundance of some pathogenic fungi and play the role of inhibiting the growth and reproduction of harmful plant pathogens.


Introduction
The dry-land yellow soil in Guizhou occupies more than 4.6 million ha, accounting for about 46% of the dry land area in the province Luo et al. 2020). The yellow soil is not only sticky in texture and strongly acidic, but also readily leaches nutrients due to the shallow soil layer. Most of the dryland yellow soil in Guizhou is planted with high value crops such as pepper, sorghum, and flue-cured tobacco. Due to continuous crop cultivation over the years, the soil quality has deteriorated, negatively affecting crop yield and quality. Therefore, determining how to improve the quality of yellow soil is an important problem that presently needs to be solved (Cheng et al. 2020;Ding et al. 2019). Additionally, as a byproduct of liquor production, the annual production of lees is about 2 million tons. However, less than 1 million tons of lees are processed each year with about 50% being left idle or abandoned . Therefore, the comprehensive utilization of lees has become a major problem faced by the developing liquor industry in Guizhou, and is also a key development task and research direction for the utilization of agricultural organic waste.
Biochar is prepared from solid agricultural and forestry waste by anaerobic high-temperature carbonization and can be used in agricultural production. Not only can biochar be used to realize the rational utilization of waste resources, and can also be used to improve and regulate the microbial composition of soils. Green, ecologically sound, and sustainable agricultural development was made possible with the emergence of biochar (Dai et al. 2017;Kumuduni et al. 2019;Zheng et al. 2019). Biochar plays an important role in regulating the structural diversity of soil microbial communities because its special physicochemical properties directly provide soil microbes with a quality habitat and essential nutrients Responsible Editor: Zhihong Xu for growth (Sheng and Zhu 2018;Yu et al. 2018;Zheng et al. 2016). As an important component of the soil microbiome, fungi plays critical decompositional roles in driving energy flow and material cycling in soil ecosystems (Nguyen et al. 2018). It has been found that the addition of 3.0 or 6.0% biochar can increase the activity of nitrogen mineralizing enzymes, and can influence the response of fungal communities to alfalfa phytoremediation (Zhang et al. 2018). It has also been shown that biochar application increased the pH and nutrient content of soils in which rubber trees were grown and also affected the associated soil bacterial and fungal communities, having a greater impact on fungal communities than bacterial communities (Herrmann et al. 2019). Chen et al. (2020) found that biochar application could effectively control bacterial wilt by trapping more carbon and nitrogen, enriching specific beneficial bacteria, and reducing the abundance of pathogenic bacteria in the soil. It was found that continuous application of biochar over 4 years significantly increased the alpha diversity of bacterial communities in rice paddy fields, while decreasing the fungal community structure. Changes in soil chemical properties (such as pH, SOM, and C/N) caused by biochar are important factors contributing to changes in the soil microbial composition (Zheng et al. 2016). Thus, biochar has shown obvious utility for soil fertility enhancement and microcosm improvement.
The beneficial effects of biochar on soil microenvironments are apparent, but current research on the effects of biochar on soil microorganisms primarily focuses on the structural diversity of bacterial communities (Abujabhah et al. 2018;Gao et al. 2019;Senbayram et al. 2019). Biochar's influence on fungal community structure and its inherent relationship with soil physical and chemical properties needs further exploration. In this study, we qualitatively investigated the effects of different application rates of Moutai lees biochar on soil nutrients and fungal community structure and diversity through short-term field culture experiments, and assessed the relationship between soil nutrients and fungal community diversity. This study provides a theoretical framework for quality improvement of yellow soil and the rational utilization of Moutai lees resources in Guizhou.

Experimental site
This research was conducted at the experimental base of Guizhou Academy of Agricultural Sciences from April to June in 2019. The daily average temperature during the course of the experiment was 20.4°C and daily average rainfall was 2.89 mm. The soil tested was the typical zonal yellow soil in Guizhou. The test biochar used was Moutai distillers grain biochar, which was prepared at 550°C using special carbonization equipment. The basic physical and chemical properties of test soil and biochar are shown in Table 1.

Experimental design and management
The experiment was conducted using open field cultivation methods, using experimental setup made from PVC pipe with a diameter of 20 cm and a height of 35 cm (Fig. 1), filled with a 3-5-cm layer of quartz sand at the bottom and yellow soil amended with different ratios of biochar layered on top. The bottom of the PVC pipe was wrapped in gauze to prevent the loss of soil and quartz sand. After adding the quartz sand layer, the yellow soil was mixed thoroughly with Mouotai lees biochar and carefully poured into the PVC pipes to avoid spillage. For each treatment, 10 kg of soil was used, and the biochar ratios tested were 0% (MB0%), 0.5% (MB0.5%), 1.0% (MB1.0%), 2.0% (MB2.0%), and 4.0% (MB4.0%). The experimental setups were placed in the field and the soil was collected for analysis after 60 days of continuous incubation.

Collection of soil samples
At the end of the experiment, the soil in each PVC pipe was mixed thoroughly and collected. The soil samples were divided into three portions. One portion of each soil sample was wrapped in aluminum foil and quickly packed into centrifuge tubes, frozen in liquid nitrogen and stored at −80°C, then later used for high throughput sequencing of associated soil microorganisms. Another portion of each sample was stored at 4°C prior and used to determine the NH 4 + -N, NO 3 − -N, MBC, and MBN content. The remaining portion of each sample was airdried, ground, and sieved for determination of soil pH, SOM, TN, AP, and AK

Determination of soil physical and chemical properties
Soil was mixed with water in a 1:2.5 ratio (w/v) and pH determined using a pH meter (FE20K, Mettler Toledo, Switzerland). SOM was determined using a wet combustion method. TN was determined using the Kjeldahl method. NH 4 + -N and NO 3 − -N was determined by immersing soil in a 1 mol L −1 KCl solution then measured with a continuous flow analyzer. MBC and MBN were determined using a chloroform fumigation-K 2 SO 4 extraction method. AP was determined using a sodium bicarbonate method. AK was extracted with ammonium acetate and boiling nitric acid, and determined with a flame photometer (FP640, Jingke, Shanghai, China) (Bao 2000).

Soil microbial DNA extraction and high-throughput sequencing
Microbial genomic DNA was extracted from soil samples using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer's instructions. The quality of extracted DNA extract was checked on a 1% agarose gel, and DNA concentration and purity were determined using a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, USA). The hypervariable region of the fungal rRNA gene ITS2 was PCR which were amplified using the primer pairs ITS3F (5′-GCAT CGATGAAGAACGCAGC-3′) and ITS4R (5′-TCCT CCGCTTATTGATATGC-3′) on an ABI GeneAmp® 9700 PCR thermocycler (ABI, CA, USA). The PCR mixture contained 4 μL5x TransStart FastPfu buffer, 2 μL of dNTP mix (2.5 mM of each dNTP), 0.8 ul each of forward and reverse primers (5 μM), 0.4 μL of TransStart FastPfu DNA Polymerase, 10 ng of template DNA, and ddH 2 O to 20 μL. The thermocycling conditions were as follows: 95°C for 3 min, followed by 27 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s, followed by a final extension of 72°C for 10 min and a final hold at 4°C. PCR reactions were performed in triplicate. PCR products were run on a 2% agarose gel, purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions and quantified using a Quantus™ Fluorometer (Promega, USA).
Purified amplicons were pooled in equimolar amounts and paired-end sequenced (2×300) was performed on an Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Raw reads were deposited into the NCBI Sequence Read Archive (SRA) database.

Statistics and analysis
Sequences analysis was performed by usearch software (V10). Sequences with ≥97% similarity were assigned to the same operational taxonomic unit (OTU). An OTU was thought to possibly represent a species. The α-diversity was applied for analyzing complexity of fungal species diversity of a sample including Coverage, Ace, Chao1, Shannon, and Simpson index, which were calculated with QIIME (V1.9.1) and displayed with R software (V2.15.3). Two-dimensional NMDS (function metaMDS from R package Vegan) was used to explore variation in community composition. Redundancy analysis (RDA) using Canoco 4.5 software was performed to determine significant relationships between the fungal communities and soil environmental variables. Analysis of variance was performed using the ANOVA method with Duncan multiple comparisons (P<0.05) in SPSS 20.0 software. Graphs were plotted using Origin 8.0.

Fungal community composition
After screening and filtering the fungal sequences from the soil samples, we found that the average base length was 315.0 bp, and the average sequencing coverage rate reached 99.91%. In addition, we also found that the fungal dilution curve gradually flattened, indicating that the number of measured OTU became saturated (Fig. 1), and the number of OTU decreased as the biochar increased (Fig. 2).
The test results showed that a total of 3234 OTU were detected in the soil samples (Fig. 3)

NMDS analysis of fungal community structure
The non-metric multidimensional scaling analysis (NMDS) of the fungal community composition in yellow soil (Fig. 4) showed a NMDS stress coefficient value of 0.22, which indicates that the NMDS analysis accurately reflected the degree of variation among the samples. The MB0% treatment was skewed towards the left side of the NMDS and could be completely separated from the fungal communities of other treatments, indicating that the application of Moutai lees biochar could significantly affect the fungal community structure of the yellow soil. However, the sample spacing of the MB0.5%, MB1.0%, MB2.0%, and MB4.0% treatments was similar and there was partial overlap, indicating the similarity coefficient of soil fungal species after biochar application was high and suggesting growth promotion of some of the same species across fungal communities in different treatments.

Fungal community diversity index
Compared with MB0% treatment, the application of lees biochar reduced the Ace and Chao1 indexes by 5.12-0.23% and 5.29-11.59%, respectively (Table 3). The Simpson index increased along with increased biochar application, while the Shannon index was not significantly different. Compared against the MB0% treatment, the Simpson index of MB0.5%, MB1.0%, MB2.0%, and MB4.0% treatment increased by 10.25-91.30%, with changes induced by the
Increasing amounts of applied biochar significantly reduced the relative abundance of Ascomycota by 23.86-29.06%, while the relative abundance of Mortierellomycota increased significantly, being 5.27-11.27 times higher than that of the MB0% treatment. It is noteworthy that the application of lees biochar also increased the relative abundances of Basidiomycota and Glomeromycota in some fungal phyla having small relative abundance.

Genus level fungal community structure
We detected a total of 444 fungal species at the genus level. The MB0%, MB0.5%, MB1.0%, MB2.0%, and MB4.0% treatments contained 287, 277, 254, 257, and 271 species, respectively. The results showed that the relative abundances o f U n a s s i g n e d f u n g i , A s p e r g i l l u s , unclassified_Chaetomiaceae, Mortierella, Spizellomyces, Penicillium, Fusarium, unclassified_Chromista, and Chaetomium increased with increased biochar application, and their combined relative abundance was 87.08-92.03% (Fig. 6). Compared with the MB0% treatment, the application of lees biochar reduced the relative abundances of Aspergillus and Fusarium by 8.80-22.11% and 1.77-12.44%, respectively, while the relative abundances of Mortierella and Spizellomyces increased significantly. In addition, it is noteworthy that the relative abundances of Wallemia and Purpureocillium in the MB4.0% treatment were significantly reduced, while the relative abundances of Chaetomium, Geomyces, and unclassified_Trichocomaceae showed a significant increases with increasing biochar application.

Richness and diversity of fungal communities
We subjected the relative abundance data from the 15 dominant genera identified in the fungal communities to detrended correspondence analysis (DCA), which showed that the maximum value of the gradient was 2.42 in the four axes (<3). We further subjected the 15 dominant genera to redundancy analysis (RDA) with the soil nutrient indicators, and found that SOM, NH 4 + -N, and NO 3 − -N appeared to be the main factors influencing fungal community structure in yellow soil under different biochar applications (Fig. 8).

Effect of biochar on soil nutrient effectiveness
Biochar is produced from agricultural organic waste through high temperature charring. Most biochar is alkaline, loose in texture, and rich in major elements such as N, P, and K, and also rich in a variety of mineral nutrients such as Ca, Mg, Zn, and other trace elements. Biochar application to soils not only is capable of changing the physical properties and improving porosity of the soil, but it can also increase soil nutrient content, which is conducive to the balanced availability of multiple nutrients Zhang et al. 2019aZhang et al. , 2019b. The results of this study showed that short-term application of Moutai lees biochar could significantly improve the pH, SOM, TN, NH 4 + -N, NO 3 − -N, AP, and AK content of yellow soil in Guizhou, which is similar to the results of previous studies Zhang et al. 2019aZhang et al. , 2019b. Moutai lees are by-products of by high temperature fermentation of sorghum and other crops. The pH of Moutai lees is weakly acidic and is not only rich in nutrients but also contains large amounts of residual fat, protein, cellulose, vitamins, trace elements, and N-free leachable elements (Dai et al. 2020). Moutai lees biochar is made by high temperature charring of Moutai lees, which is alkaline in pH (8.8), and rich in SOC, TN, and AK. Application of Moutai lees biochar can improve the fertility of yellow soil. However, short-term application of lees biochar reduced the MBC and MBN contents. This may be attributed to the fact that biochar has a high C/N ratio. When biochar is applied to the soil, the excess carbon source disrupts the balance between the microbial produced carbon and nitrogen, which can ultimately inhibit the microbial population and activity (Xu et al. 2014;Zhu et al. 2017). Alternatively, the application of organic materials with containing high levels of carbon can increase the content of MBC and MBN (Foster et al. 2016). Biochar is rich in inactive organic carbon which is stable in nature. Although the application of biochar directly increases the SOC content, it reduces the proportion of activated carbon available to microorganisms, leading to a decrease in MBC and MBN (Johannes et al. 2006;Zwieten et al. 2010).
MBC/MBN is commonly used to assess the structure of soil microbial communities. The C/N ratios of bacteria ranged from 3 to 6, while those of fungi ranged from 7 to 12 (Vries et al. 2006). In this study, we found that the MBC/MBN for the treatments of MB0%, MB0.5%, MB1.0%, MB2.0%, and MB4.0% were 4.50, 5.65, 10.07, 8.23, and 7.01, respectively. This indicates that the soil microbial communities are dominated by bacterial when yellow soil is exposed to (MB0%) or low (MB0.5%) biochar application rates, whereas they are dominated by fungi in soils exposed to high application rates (MB1.0%, MB2.0%, MB4.0%). It has been shown that the application of nitrogen fertilizer along with biochar can increase the proportion of bacteria in the microbial community. Note: Different lowercase letters in the same column indicate significant differences between treatments (P<0.05) Fig. 5 The relative abundance of fungi at phylum level in soil When the total soil microflora remained unchanged, the accelerated growth and reproduction rate of bacteria led to a decrease in MBC/MBN (Wang et al. 2010). In this study, when the biochar application rate exceeded 1.0%, the MBC/ MBN experienced a decreasing trend. Although the soil microbial community was still dominated by fungi, the growth and reproduction rate of bacteria gradually increased, leading to a decrease in MBC/MBN and an eventual shift form a "fungal dominant" to a "bacterial dominant" soil microbial community (Chen et al. 2012).

Effect of biochar on soil fungal community diversity
Fungi are important members of the soil microbial community which can promote the energy flow and material cycling in soil ecosystems and play important roles in promoting soil-plant interaction. The results of this study showed that the short-term application of lees biochar significantly increased the Simpson index of fungal communities but had no significant effect on Ace, Chao1, and Shannon indices. This indicates that the application of Moutai lees biochar can reduce the fungal diversity in yellow soil, but has no effect on species composition, which is consistent with the results of previous studies (Hu et al. 2014). It has been shown that the application of biochar-based fertilizers can significantly reduce the abundance and diversity indices of soil fungal communities, which may be related to the different pathways involved in decomposition of soil organic matter employed by soil microorganisms. Biochar application not only affects the diversity of soil fungal communities, but also improves the fungal community structure and reduces the number of potentially pathogenic species, resulting in soil microbial communities dominated by beneficial microflora (Bai et al. 2019). In this study, we found that the application of lees biochar significantly reduced the relative abundance of Fusarium fungi and increased the relative abundance of Mortierella, similar to results of previous studies (Yao et al. 2017). Fusarium oxysporum is among the primary fungal pathogens causing soil-borne diseases in crops. The application of biochar can significantly reduce the gene copy number and relative abundance of Fusarium in the soil, which may relate to the increased the soil pH and decreased effectiveness of some phenolic acids after the application of biochar (Jaiswal et al. 2017;Wu et al. 2020). Meanwhile, Mortierella fungi were able to promote increases of SOM and nutrients, which ultimately promotes the growth and development of plant roots (Curran et al. 2000). Additionally, the application of lees biochar increased the relative abundance of some functional fungi such as Chaetomium. It has been found that the enhancement of Chaetomium not only promotes the uptake of active substances in the soil by plants, but also produces antibiotics and cell wall degrading enzymes which ultimately act to inhibit the occurrence of soil-borne pathogens (Ingrid et al. 2015).

Relationship between fungal community structure and soil environmental factors
Soil microbial community structure is influenced by physical factors such as soil moisture and aeration conditions, and chemical factors such as pH, SOM, and TN that also play important roles in influencing the soil microbial community structure . The results of this study showed that SOM, NH 4 + -N, and NO 3 − -N may be important factors influencing structural changes in the fungal communities of yellow soil. It is known that fungi prefer to grow in acidic environments. Biochar application increased the soil pH, which is a primary factor affecting the soil microbial community structure (Chen et al. 2015;Nielsen et al. 2014). Conversely, it has also been found that soil pH is the dominant factor affecting fungal community structure, while the soil fungal community structure is much more influenced by the soil nutrients than the soil pH (Dai et al. 2016). Some studies also confirmed a significant correlation between soil nutrients and fungal community composition (Zhang et al. 2019a(Zhang et al. , 2019b. Therefore, the relationship between the environmental factors and fungal community structure may vary depending on the soil, crop and biochar type. It is worth noting that there are few studies on the effect of Moutai lees biochar on the fungal diversity of yellow soils, and the soil microbial environment is bound to change with the time of biochar application and the aging process of biochar itself. Therefore, more experiments are needed to explore the short-and longterm effects of lees biochar application on the diversity of fungal communities in yellow soil, which can provide a theoretical framework for the enhancement of soil fertility and the rational utilization of lees.

Conclusion
Moutai lees biochar had strongly improved the nutrient content and fungal diversity of yellow soil on a short time scale. The results indicate that application of Moutai lees biochar could increase the SOM content, improve the fungal community structure, and inhibit the growth and reproduction of harmful plant pathogens. However, more field studies are needed to evaluate the long-term effects of Moutai less biochar on the nutrient availability and microbial communities.
Author contribution All authors contributed to the study conception and design. Meng Zhang conceived the experiments; Meng Zhang and Yanling Liu performed the experiments and analyzed the data; Quanquan Wei contributed materials; Meng Zhang wrote the paper; and Jiulan Gou revised the paper. Data availability All data generated or analyzed during this study are included in this published article.

Declarations
Ethical approval Not applicable Consent to participate Not applicable Consent to publish All authors have read the manuscript and approve of its submission to Environmental Science and Pollution Research.

Competing interests
The authors declare no competing interests.