Biochar’s role in improving pakchoi quality and microbial community structure in rhizosphere soil

Experimental materials

In this study, the soil was obtained from the experimental base of the Agronomy College of Heilongjiang Bayi Agricultural University. The soil is weakly alkaline chernozem with the following physical and chemical properties: contained 156 mg kg^-1 alkali-hydrolyzable nitrogen, 14.99 mg kg^-1 phosphorus, 100 mg kg^-1 potassium, and 0.478 mg kg^-1 heavy metal cadmium, with 3.78% organic matter, and the soil’s pH was 8.3. This research acquired the pakchoi variety of “ Siji Xiaobaicai” (Year-round plantable pakchoi). The biochar was prepared with corn stalk (preparation temperature was 450 °C, provided by Anhui Xiangzheng Biological Engineering Co., Ltd., pH 8.41, EC 1.07 mS cm⁻¹, NH₄⁺-N 7.61 mg kg⁻¹, NO₃⁻-N 30.82 mg kg⁻¹, available P 97.12 mg kg⁻¹, available K 3,012.00 mg kg⁻¹, bulk density 0.35 g cm⁻³.

Experimental design

The experiment was conducted in the Heilongjiang Bayi Agricultural University greenhouse from May to June 2019. CK1, without biochar, was used as a control. Four treatments (Table 1) with 1% (T1), 3% (T2), 5% (T3), and 7% (T4) biochar were repeated thrice in the test facility. The cultivated soils were filled into a plastic basin with a 22 cm height and an 18 cm diameter. The same compound fertilizer and chicken manure were added to each pot. The sowing and thinning management were kept constant for all experiments. Samples were collected after 30 days (seedling stage) of growth to determine the relevant indexes of pakchoi.

Experimental methods

When pakchoi samples were collected after 30 days (seedling stage) of growth, plant height and root length were measured using a meter ruler. The aboveground and underground parts of pakchoi were rinsed, leaf moisture was dried using filter paper, and the plant was weighed on a scale to obtain the fresh weight. The plants were wrapped in paper, placed in an oven at 105 °C for 30 min to remove water, and dried at 75 °C to obtain dry weight.

This study took 0.1 g from the fifth leaf of fresh pakchoi in a leaching solution (alcohol: acetone = 1:1) for 24 h, and colorimetry was performed at 663 and 645 nm to calculate the contents of chlorophyll a, chlorophyll b, and the overall amount of chlorophylls (Zhang, 1992). A 10 g sample of pakchoi and 5 ml of 2% oxalic acid solution were ground in a mortar. The ground sample was diluted to a 100 ml volumetric flask with 2% oxalic acid, filter, and a 10 ml of the filtrate was placed in an evaporating dish. It was titrated with a calibrated 2,6-dichlorophenol indophenol solution until it turned pink and did not fade within 30 s. The amount of dye used was recorded and the content of vitamin C was calculated (Li, 2010). The method established by Li (2010) was used to determine the soluble sugar content. A total of 0.2 g of the sample to 20 ml of distilled water was added and extracted in a water bath for 30 minutes. Then, 1.5 ml of distilled water was added to 0.5 ml of the extraction solution. Then, 0.5 ml of ethyl acetate and 5 ml of concentrated sulfuric acid were introduced into the test tube in sequence. After vigorous shaking, the mixture was subjected to a boiling water bath for 1 min. After cooling, absorbance at 630 nm was measured to calculate the soluble sugar content (Li, 2010). A total of 2 g of the sample was ground into a homogenate and extracted in 45 °C water bath for 1 h. A total of 0.2 ml of the extraction solution was added to a test tube containing 5 ml of 5% salicylic acid sulfate solution for 30 min, then 19 ml of NaOH solution was added absorbance was measured at 410 nm, and nitrate content was calculated (Li, 2010). A total of 0.5 g of fresh sample was ground into a homogenate with 5 ml of distilled water, then centrifuged at 10,000 r min⁻¹ for 10 min; then, 1.0 ml of the supernatant was taken and placed in a test tube, 5 ml of Coomassie Brilliant Blue G-250 solution was added, mixed and placed for 2 min at 595 nm for colorimetry. The absorbance was measured, and the protein content was calculated (Li, 2010). A total of 0.2 g of dry sample was placed into a beaker, 100 ml of 60% H₂SO₄ was added, shaken well, and filtered with a Buchner funnel into another beaker. A total of 2 ml of the filtrate was taken into a stopper tube, 0.5 ml of 2% anthrone reagent was added, and 5 ml of concentrated H₂SO₄ was added. The stopper was sealed and shaken well, and let to stand for 12 min. The absorbance was measured at a wavelength of 620 nm and the cellulose content was calculated (Xiong, Zuo & Zhu, 2005).

For the experimental soil samples in 2019, the soil was sampled by shaking the roots (Wang et al., 2009) after 30 days of growth. For each sampling, the roots from five tomato seedlings were collected and pooled together as one sample with three samples per treatment (n = 3), and sieved using a 20 mesh. Some of these soil samples were stored at 4 °C to evaluate soil enzyme activities. The phenol-sodium hypochlorite colorimetric and disodium phenyl phosphate methods were used to determine soil urease and alkaline phosphatase activities. In addition, 3,5-dinitrosalicylic acid colorimetric, TTC staining, and pyrogallol colorimetric determination of sucrase activity, dehydrogenase activity, and soil polyphenol oxidase activity, respectively, were performed. Soil cellulase activity was determined using 3,5-dinitrosalicylic acid (Zhang & Li, 2005). Another portion of these soil samples was naturally air-dried to determine the alkali-hydrolyzable nitrogen by the alkaline hydrolysis-diffusion-titration procedure in a diffusion disk, the available phosphorus by molybdenum blue colorimetry using a spectrophotometer, and the available potassium by ammonium acetate extraction-flame photometry. The pH and EC values were determined using an acidimeter and a conductivity meter, respectively, after leaching and filtering the soil-water mixture (soil: water = 1:5) (Bao, 2000). Then, 0.5 g of the pulverized dry plant samples were digested with the HNO³-HClO⁴ (V:V = 4:1) mixed acid system and analyzed using an atomic absorption spectrometer (Emmett, Buckley & Drinkwater, 2020).

For the experimental soil samples in 2019, adopting the MolPure^® Soil DNA Kit soil DNA extraction kit (18815ES50) can increase recovery yield by: (1) preheating the eluent at 65 °C; (2) adding the filtrate to the column again, let it sit at room temperature for 3 min, and elute. The sample DNA can be stored at −20 °C for short-term storage and −80 °C for long-term storage. The soil microorganism MiSeq was sequenced by the Shanghai Meiji Biological Company (Shanghai, China). Polymerase chain reaction (PCR) amplification as described by Magoč & Salzberg (2011) then the bacterial v3–v4 and fungal regions were amplified. The primers employed in this investigation were 338f/518r. The PCR products were analyzed using quantifluor™. Each PCR reaction, with a total volume of 20 µL, consisted of 9 µL of 2 × Real SYBR Mixture, 0.2 µL of each primer (10 µM), 2.5 µL of template DNA, and 8.1 µL of ddH2O. The qPCR reaction conditions are as follows: (1) Annealing: Following denaturation, the reaction proceeded to an annealing step. The annealing temperature was set at 65 °C and lasted for 30 s. During this step, the primers bind to their complementary sequences on the DNA template. (2) Extension: After annealing, the reaction underwent an extension step. The extension temperature was set at 72 °C and lasted for 1 min. During this step, the DNA polymerase extends the primers, synthesizing new DNA strands. (3) Final extension: The amplification process was repeated for a total of 22 cycles, followed by a final step of elongation. The temperature was set at 72 °C and lasted for 10 min. This step ensures that any remaining DNA strands are fully extended. (4) Fragment length: The amplified fragment length obtained in this study was approximately 230 base pairs (bp). This length corresponds to the targeted DNA region amplified by the primers. To analyze the quantitative data, a ST Blue Fluorescence Quantitative System (Promega, Madison, WI, USA) was used. Additionally, the MiSeq library was constructed and sequenced to obtain the sequence of DNA fragments (Xu, Wang & Wu, 2015).

Data processing

All collected data were analyzed using IBM SPSS Statistics ver. 18 (SPSS Inc., Chicago, IL, USA). Duncan’s Multiple Range tests (DMRT) and post hoc tests were used to compare the means of the treatments. The significance level was set at P <0.05. The biological information analysis method statistically and visually analyzed the sequencing data to obtain optimized effective sequence statistics, OTU distribution statistics, diversity index analysis, and sample community composition analysis. Finally, a heatmap diagram was plotted (Xu, Wang & Wu, 2015).

Effects of different treatments on the growth of pakchoi

The results demonstrated that pakchoi root length, shoot dry weight, and root dry weight significantly increased under 3% biochar treatment (T2) compared to CK. Compared with CK and 3% biochar treatment (T2), the fresh and root dry weight of pakchoi significantly increased under 5% (T3) and 7% biochar treatments (T4). Therefore, the growth of pakchoi can be promoted by adding biochar at a concentration of at least 3% (Table 2).

Effects of different cultivation modes on the quality of pakchoi

The experiments proved that the soluble sugar, soluble protein, VC, and cellulose contents in pakchoi leaves were significantly increased after biochar treatment compared with CK. The application of biochar at 3% (T2) and 7% (T4) treatments significantly increased the chlorophyll a, chlorophyll b, and total chlorophyll content in pakchoi leaves compared to the control without biochar application. Nitrate reductase activity in pakchoi leaves was significantly higher than in control and 1% biochar treatments when the amount of biochar added was more than 3%. With an increase in biochar content, nitrate content first decreased and then increased, and it was significantly reduced in the 3% biochar treatment. Therefore, the quality of pakchoi under the 3% biochar treatment was significantly better than that under the other treatments (Table 3).

Effects of different concentrations of biochar on soil nutrient and enzyme activities

The urease, phosphatase, sucrase, catalase activity, and dehydrogenase of pakchoirhizosphere soil increased after adding biochar (Table 4). The dehydrogenase activity of pakchoi in the rhizosphere soil increased significantly with biochar application rate. Compared with the soil under control conditions, the urease, phosphatase, sucrase, and catalase activities in the soil first increased and then decreased with increased biochar added. These properties peaked in the soiltreated with 3% biochar, which was significantly higher than that of CK.

The pH value and contents of organic matter, alkali-hydrolyzable nitrogen, phosphorus, potassium, and EC values in the rhizosphere soil of pakchoi were enhanced after applying biochar (Table 5). Compared with the soil under control conditions, the contents of phosphorus, potassium, EC value, and organic matter in the rhizosphere soil of pakchoi gradually increased with the increase in biochar content. The highest values were found in the soil treated with 7% biochar and were significantly higher than in the control soil. The alkali-hydrolyzable nitrogen content and pH value were first enhanced and then reduced. Peak values were found in the soil under the 3% biochar treatment and were significantly higher than those without biochar addition.

Effects of different concentrations of biochar on soil bacterial community structure of pakchoi

After adding biochar, the structure of bacterial flora in the rhizosphere soil of pakchoi changed (Fig. 1). Among the relatively abundant dominant phyla Anaerolineae, the relative abundances of Gemmatimonadetes and Deltaproteobacteria were reduced, and Alphaproteobacteria, Gammaproteobacteria, Bacteroidia, Acidimicrobiia were increased. The microbiot structure changed significantly when the biochar content exceeded 3%.

Adding different biochar impacted the bacterial diversity in the rhizosphere soil of pakchoi significantly (Fig. 2), while adding 1% biochar (T1) had no significant change compared with the control, which was clustered together. The structure of the bacterial community in the rhizosphere soil of white pakchoi was significantly changed after adding 3% biochar (T2). The structure of the microbial community in the rhizosphere soil of white pakchoi treated with 3% biochar (T2), 5% biochar (T3), and 7% biochar (T4) was similar and clustered together. Microbial communities in the rhizosphere soil of pakchoi were classified into two groups. The first group was comprised Ilumatobacter, Luteolibacter, Lysobacter, Arthrobacter, Mesorhizobium, Pontibacter, Lactobacillus, Faecalibacterium, and Bifidobacterium, and the relative abundance of these bacteria increased significantly. The second group comprises the sweet and sour bacteria Gemmatimonas, Bacillus, Stenotrophobacter, Ramlibacter, Bryobacter, Actinoplanes, Comamonas, Flavisoib, Megamonas, Steroldobacter, Ohtaekwangia, and Luteitalon. These were clustered together, and the relative abundance of these bacteria was significantly reduced. Adding 3% biochar significantly changed the microbial community structure in the rhizosphere soil of pakchoi plants.

Cluster analysis depicted that 1% biochar (T1) clustered together with the control group withsimilar bacterial community structures. A biochar content of >3% (T2) was also clustered. However, the structure of the bacterial community differed from that of the control group. All treatments were grouped into two types: (i) the control (CK) and 1% (T1) biochar, and (ii) the treatment above 3% biochar (T2, T3, and T4), among which the abundance of Gemmatimonas was significantly reduced after treatment with more than 3% biochar (T2) (Fig. 3). Analysis of similarities (ANOSIM) compared the difference in beta diversity between samples from different groups. The boxplot results revealed significant differences between the T2 and T3 treatment groups and the control group (Fig. 4A). Principal component analysis demonstrated that CK and T1 significantly differed from T2, T3, and T4 in the PC1 axis when they were clustered together three times. Adding 1% biochar had no significant effect on the structure of the soil microbial community, and adding more than 3% led to significant changes in the structure of the soil microbial community (Fig. 4B).

There was a significant positive correlation between Gemmatimonas and nitrate content and a significant negative correlation between Gemmatimonas and pH, NR, SDW, and RDW. There was a significant negative correlation between Ilumatobacter and nitrate content and a significant positive correlation between Ilumatobacter and pH, NR, SDW, and RDW. The control, without biochar (CK), was positively correlated with nitrate and VC. Biochar treatment (T2) was positively correlated with soil pH and NR, and biochar treatments (T3 and T4) were also positively correlated with COSS, nr, and urease activities. Therefore, biochar application is closely related to dry weight, nitrate accumulation, and root DW. In addition, COSS, NR, pH, and urease activity were the main influencing factors (Fig. 5).

The control rhizosphere soil had significantly higher carbohydrate transport and metabolism, amino acid transport and metabolism, general function prediction, energy production and conversion, post-translational modification, protein turnover, and chaperones than T2 treatment. The T2 rhizosphere soil had significantly higher lipid transport and metabolism, transcription, cell motility, signal transduction metabolism, intracellular trafficking, secretion, and vesicular transport than CK (Fig. 6).

The control rhizosphere soil had significantly higher carbohydrate transport and metabolism,amino acid transport and metabolism, and general function predictions than T3 treatment. The T3 rhizosphere soil had significantly higher intracellular trafficking, secretion, vesicular transport, secondary metabolite biosynthesis, transport and catabolism, cell motility, lipid transport and metabolism, and transcription than the control (CK) (Fig. 7).

Carbohydrate metabolism, membrane transport, cellular community-prokaryotes, energymetabolism, biosynthesis of other secondary metabolites, xenobiotic biodegradation and metabolism, lipid metabolism, and global and overview map control (CK) were significantly higher than those in treatment T4 (7%). The following metabolic processes were significantly higher in T4 (7%)-treated rhizosphere soil than in control soil (CK): nucleotide metabolism, glycan biosynthesis and metabolism, cell motility, replication and repair, cell growth and death, signal transduction, amino acid metabolism, metabolism of cofactors and vitamins, metabolism of other amino acids, and translation (Fig. 8). Therefore, carbohydrate transport and metabolism in the rhizosphere soil of pakchoi decreased, and lipid transport and metabolism increased after applying biochar. These metabolic changes may explain the increase in soil nutrients after applying biochar.

Biochar promotes the growth of pakchoi and improves its quality

Several studies have focused on the effect of biochar on vegetable plant growth, yield, andquality. A study demonstrated that the combination of 35 t ha⁻¹ biochar and 200 kg ha⁻¹ N fertilizers significantly increased tomato yield, VC content, sugar-acid ratio, and economic benefits (Guo et al., 2021). Wood chip biochar enhanced the growth of ice plants in coastal soil. Adding wood chip biochar improved the nutritional quality of the plants, regardless of whether chemical fertilizer was applied (You et al., 2021). The results of this study confirmed the hypothesis that the application of 3% biochar enhanced the growth and chlorophyll content of pakchoi, improved the nutritional quality of pakchoi (Table 3). The positive effect of biochar amendment on pakchoi growth and quality may be attributed to improvements in soil properties (Zheng et al., 2018), improved soil bulk density and water retention (Wang et al., 2022), enhanced nutrient availability (Amin, 2020), alterations in the soil microbial community (Lu et al., 2020). Research has shown that biochar can promote the development of plant roots, and then enhance the photosynthesis of leaves, so that plants can grow healthily, which is conducive to the plant production and the cultivation of soil (Ren et al., 2021). The results of this study show that indicate that 3% biochar significantly increased root length and chlorophyll content of pakchoi (Tables 2 and 3), this may be one of the main reasons for promoting the growth and improving the quality of pakchoi.

Biochar enhances soil nutrient availability and reduces nitrate in pakchoi

Biochar has a loose and porous structure, and its addition to the soil can improve the soil structure and enhance soil fertility (Gorovtsov et al., 2020), fix nutrients in the soil, and increase the content of available nutrients (Zhang et al., 2019). The high carbon content of biochar can replenish the organic carbon in the soil and increase the soil organic matter content; biochar applied at certain concentrations gradually absorbed the nitrogen released from the N, P, and K compound fertilizers, reducing the nitrogen amount absorbed by plants and decreasing the nitrate content in plants (Yousaf et al., 2016). Because biochar is alkaline, its application to the soil can increase soil pH (Liu, Liu & Zhang, 2014). Moreover, the relatively large surface area of biochar addition based on biochar in the short term can improve soil quality by neutralizing soil pH, increasing soil nutrient contents (Kong et al., 2021), and promoting plant growth. In this study, under weakly alkaline soil conditions, adding 3% biochar to the rhizosphere soil of pakchoi resulted in a peak alkaline nitrogen content, a significant increase in soil pH (Tables 3 and 5), and nutrient balance, which was beneficial for the growth and quality improvement of pakchoi. Biochar can significantly promote the growth of plant roots (Table 2), and an increase in root biomass can increase the secretion of secretions (Wang et al., 2018), thereby altering the abundance of rhizosphere soil microorganisms and increasing soil enzyme activity (Feng et al., 2021). In this study, 3% biochar significantly increased soil urease activity (Table 4) and nitrogen availability in the soil (Table 5). Studies have illustrated the combined action of biochar and nitrogen-fixing bacteria on microbial and enzymatic activities of soil N cycling (Gou et al., 2022). Application of biochar accelerates the metabolism of soil biota, turning more nitrogen from fertilizers into organic forms; hence, less mineral nitrogen is left for plant intake, which reduces nitrate levels in red beet (Maroušek et al., 2018). It can increase the utilization rate of nitrogen in the soil and reduce nitrate accumulation in the leaves of pakchoi (Table 3). However, the improved soil nutrient metabolism and microbial community structure caused by adding biochar require further in-depth research.

Correlation analysis between biochar changes in soil nutrient metabolism and microbial community structure

Biochar amendments can alter plant rhizosphere microbiome, which profoundly affects plant growth and fitness (Jin et al., 2023). Regulation of C/N ratio by adding biochar and N fertilizer affects the composition and diversity of soil bacterial communities (Kong et al., 2021). This study demonstrated that the relative abundance of the bacterial community in pakchoi rhizosphere soil was changed by applying more than 3% biochar. Among the relatively abundant dominant phyla, Anaerolineae, Gemmatimonadetes, Deltaproteobacteria, and Verrucomicrobiae were reduced, and 303 Alphaproteobacteria, Gammaproteobacteria, Bacteroidia, and Acidimicrobiia relative abundance increased (Fig. 1). Biochar affects the symbiotic pattern of microorganisms, increasing the proportion of positive interactions in the microbial community (He et al., 2021), the advantages of these changes will be verified in the future.

Previous research has revealed that biochar amendment significantly increases the relativeabundance of the potential biocontrol bacteria Bacillus and Lysobacter (Feng et al., 2021). Bacteria, including species within the genera Arthrobacter and Bacillus, can solubilize phosphate or mitigate salinity stress (Jiang et al., 2019; Tchakounté et al., 2020). The abundance of some potentially beneficial bacteria, such as Luteolibacter, Glycomyces, Flavobacterium, and Flavihumibacter, increases in organic fertilizer-amended soil (Huang et al., 2022a; Huang et al., 2022b). The application of both types of biochar increases the nodule number by 52% under well-watered and drought conditions by improving the symbiotic performance of chickpeas with Mesorhizobium ciceri (Egamberdieva et al., 2019). Research displays that Gemmatimonas possess unique characteristics for assimilative and dissimilative N processes, with new implications for cultivation strategies to better assess the metabolic abilities of Gemmatimonadetes (Chee-Sanford, Tian & Sanford, 2019). In addition, the relative abundance of Gemmatimonas was reduced, whereas the relative abundances of Ilumatobacter, Luteolibacter, Lysobacter, Arthrobacter, Mesorhizobium, and other bacteria in soil supplemented with 3% biochar were significantly increased (Fig. 2). The increase in these five (Ilumatobacter, Luteolibacter, Lysobacter, Arthrobacter, and Mesorhizobium) beneficial bacteria after adding biochar might be related to increased soil available nutrients and improved pakchoi quality. Gemmatimonas species carry genes that promote mineralization, nitrification, dissimilatory/assimilatory nitrate reduction, denitrification, anammox reactions, and N fixation. The functional microbes Gemmatimonas reduce various N conversion processes at different rates in biochar-added soil, which might reduce nitrate accumulation in pakchoi (Zhou et al., 2022a; Zhou et al., 2022b). The bacterial diversity was significantly improved, growth-promoting bacteria, such as Gemmatimonadetes and Proteobacteria, became more abundant, and Acidobacteria and Actinobacteria became less abundant after adding biochar (Feng et al., 2021). Xu et al. (2016) demonstrated that under biochar application conditions, the relative abundance of Acidobacteria, Chloroflexi and Gemmatimonadetes decreased, while Proteobacteria, Bacteroidetes and Actinobacteri a increased. The results were not nearly identical and may be related to the soil type and original flora of the test site.

Adding modified biochar to the soil changed the relative abundance of dominant bacterialcommunities and increased the relative abundance of some functional bacterial communities (Hua et al., 2021). In this study, the nitrate content was positively correlated with the abundance of Gemmatimonadetes (Fig. 5A), and the reduction in nitrate accumulation by biochar addition was closely related to the decrease in Gemmatimonadetes abundance. Biochar enhances the retention capacity of nitrogen fertilizers and affects the diversity of nitrifying functional microbial communities in soil (Zhang et al., 2021). However, the role of microorganisms in soil metabolism requires further verification. Ilumatobacter is a new genus of acid microorganisms that have been isolated from the sea by scholars (Matsumoto et al., 2013). In this study, the nitrate content was significantly negatively correlated with the relative abundance of Ilumatobacte and positively correlated with aboveground dry matter accumulation and soluble sugar content (Fig. 5A). This indicates that the increase in the abundance of this genus is closely related to the growth and quality improvement of pakchoi.