Addition of hydrothermal biochar significantly alters growth of forage oats and rhizosphere microbiota

Summary

There are few reports on the application of biochar in forage oat cultivation. Therefore, we compared the effects of adding different amounts of hydrothermal biochar on the growth of forage oats and the properties of rhizosphere soil. Results indicated that biochar significantly enhanced forage oats biomass, crude protein (CP) content, and chlorophyll content, with optimal effects at a 10 t/ha application rate. The C and N content and the C/N ratio in rhizosphere soil increased with biochar addition. Excessive addition of biochar reduced the diversity of rhizosphere bacteria. The addition of biochar increased the abundance of Patescibacteria and Ascomycota and decreased the complexity and stability of bacterial communities, while the fungal communities showed the opposite trend. Moderate addition of biochar can promote microbial processes such as the degradation of aromatic compounds and nitrogen cycling. This study provides a scientific basis for the application of hydrothermal biochar in forage oat cultivation.

Graphical abstract

Highlights

• •Hydrothermal biochar can effectively promote the growth of forage oats
• •Hydrothermal biochar altered the rhizosphere microbial community
• •Excessive addition of hydrothermal biochar can have negative impacts

Soil science; Microbiology; Plant Biology; Plant nutrition; Soil chemistry; Soil biology

Introduction

Forage oats (Avena sativa L.) are characterized by their high crude protein (CP) content, high digestibility, and richness in carbohydrates, along with a substantial quantity of trace elements, minerals, vitamins, and other nutrients. These characteristics render it a highly valuable feed ingredient for livestock due to its nutritional benefits.¹ Due to the rapid development of the livestock industry in China, there is an escalating challenge in the supply of high-quality forage. At the same time, the salinization and alkalization of soils worldwide, as well as the degradation of soil quality caused by long-term intensive management, have already threatened soil ecological security and food security. The frequent occurrence of extreme climatic events caused by global climate change also poses challenges to agricultural production.^2,3 Therefore, it is necessary to take appropriate measures to improve land productivity and enhance the production capacity of high-quality forages such as oats, so as to alleviate the contradiction between human and animal feed.

As a soil amendment, biochar has been repeatedly demonstrated to improve soil structure, regulate soil pH, retain soil moisture, reduce nutrient leaching, and enhance plant stress resistance.^4,5,6 However, biochars produced by different preparation methods vary in properties, leading to variable effects on soil fertility and crop productivity enhancement. Pyrolysis is the primary method currently used for the production of biochar. Due to the presence of alkaline functional groups in biochar, the pyrolysis process can retain inorganic minerals and alkaline components (nitrates, carbonates, and hydroxides), explaining why the majority of biochar samples are alkaline. Therefore, adding pyrolysis-derived biochar to saline-alkali soils may not substantially modify the pH and could, in fact, exacerbate the increase in soil pH.⁷ Hydrothermal carbonization (HTC) is a thermochemical process that converts high-moisture biomass into hydrochars at relatively low temperatures (180°C–375°C) with short residence times ranging from minutes to hours under self-generated pressure (26 MPa) in a subcritical or supercritical water environment.⁸ Compared with pyrolysis, HTC has the advantages of high conversion efficiency, no need for predrying, and relatively lower heating temperature, making the production process more energy-saving.⁹ However, it should be noted that the HTC method has higher requirements for equipment, which to some extent increases the cost of purchasing equipment. Therefore, when choosing the type of biochar, it is necessary to select the appropriate type of biochar according to the soil properties of the region. Additionally, the preparation of biochar through the HTC method results in a substantial generation of acidic functional groups, such as carboxyl and phenolic hydroxyl groups. The presence of these functional groups endows hydrochar with a relatively strong acidity, which can effectively reduce the pH of alkaline soils.¹⁰ Jager et al. found that the pH value in Chernozem decreased from 7.9 to 7.2 after the addition of 5% (w/w) hydrothermal biochar.¹¹ Furthermore, due to the reduced trace element content in biochar prepared by the HTC method, hydrothermal biochar may possess a greater capacity to reduce the electrical conductivity (EC) of soils.¹² For instance, a study by Zhou et al. found that the addition of hydrothermal biochar, produced from corn stalks, to saline-alkali soils resulted in a decrease of 1.0 pH units and an 18.14% reduction in salinity by the harvest period. Concurrently, there was a significant increase in sorghum yield, with an average enhancement of 32.98%.¹³ However, to date, there is a lack of studies evaluating the effects of hydrothermal biochar application under saline-alkali conditions on the growth and development of forage oats.

In addition, the effectiveness of biochar in promoting plant growth is closely related to the amount applied. An overabundance of biochar, when introduced to the soil, may unexpectedly induce adverse effects on the growth dynamics of crops. Zaid et al. found that the addition of 15 and 30 t/ha of biochar significantly improved the growth and yield of rapeseed, as well as increased the content of nutrients such as CP and soluble sugars. However, an increase in the application rate to 60 t/ha had adverse effects.¹⁴ Currently, the existing literature is scarce regarding the impact of varying biochar application rates on the growth of oats. Consequently, ascertaining the optimal rate of biochar application to maximize its growth-promoting effects on oat holds significant implications for subsequent research and practical applications.

Furthermore, soil microorganisms are crucial for driving biogeochemical cycles, suppressing pathogens, maintaining soil quality and health, and are essential for plant development, ultimately affecting the sustainable productivity of agricultural fields.^15,16,17 Over the years, numerous researchers have discovered that the application of biochar can alter the structure of soil microbial communities and enhance the functional activities of microorganisms.^18,19,20 Therefore, understanding the impact of biochar application on soil biota and soil health is crucial for exploring how biochar can improve land productivity under future environmental changes. Based on the previous discussion, this study hypothesizes that the addition of hydrothermal biochar under saline-alkali conditions will have a significant impact on the growth of forage oats and the rhizosphere microbial community. The experimental material for this study comprised biochar produced from corn stalks via the HTC method, with the aim of clarifying the growth of forage oats and the changes in the structure of their rhizosphere microbial communities under different application rates of biochar. The findings of the study provide a reliable theoretical basis for the promotion and application of biochar in the cultivation and management of forage oats.

Results and discussion

Changes in oat growth after addition of biochar

The effects of biochar amendment on the biomass of oats, as well as the SPAD values and nitrogen content of their leaves, are depicted in Figure 1. The biomass of each part of the oats, as well as the total aboveground biomass, exhibited significant variations with increasing application rates of biochar (p < 0.05). After biochar application, the biomass of the oats’ stems, leaves, spikes, and aboveground parts increased by 11.21%–36.11%, 11.10%–36.97%, 9.74%–52.72%, and 15.75%–29.64%, respectively, compared to the B0. The biomass of stems, leaves, and the total biomass of the aboveground parts reached their maximum values in the B10 group, at 16.02 g, 9.21 g, and 38.04 g, respectively. As the amount of biochar added continued to increase, the biomass of stems and leaves began to decrease significantly. By B30, the biomass of stems and leaves had decreased to 13.09 g and 7.47 g, respectively. The biomass of spikes increased continuously with the increasing amount of biochar added, reaching the maximum value of 16.56 g at B50. Biochar, as a widely used soil amendment, has been consistently shown to enhance the productivity of various crops. Han et al. used a meta-analysis to evaluate the impact of biochar on plant productivity and found that the application of biochar can increase plant productivity by 14.28%.²¹ Choudhary et al. found that reducing the amount of fertilizer by 25% and applying 10 t/ha of biochar could significantly increase hay yield by 84.66%, a result that is similar to the findings of this study.²² It should also be noted that due to the highly stable nature of biochar, it maintains its effects for a long period after being added to the soil. Jiang et al. found that biochar can persistently and significantly increase crop yields, with a 15% increase still observed six years after application.²³ Although the initial addition of biochar leads to a significant increase in costs, in the long term, the application of biochar can significantly enhance the economic benefits of growing forage oats.

Figure 1.

The impact of biochar on the growth and quality of forage oats

(A–F) The dry matter weight of forage oat stems (A), leaves (B), spike (C), above-ground biomass (D), and as well as leaf relative chlorophyll content (SPAD) (E), and nitrogen content (F).

(G–I) ADF (G), NDF (H), and CP (I) content of above-ground parts of forage oats.

B0, B5, B10, B20, B30, and B50 represent the addition of 0, 5, 10, 20, 30, and 50 t/ha of biochar, respectively. Different letters in the figure indicate significant differences in the mean values of variables among different treatment groups (p < 0.05). Data are represented as mean ± SEM.

Due to its unique physicochemical properties, biochar can effectively enhance the availability of macro and micronutrients as well as the water-holding capacity of soils, improve plant photosynthesis, and consequently increase biological yield.^24,25 The results of this study indicate that as the amount of biochar added increased, the SPAD values of the oat leaves initially increased, followed by a plateau or slight decrease. The SPAD values in the biochar-amended groups increased by 9.62%–32.34% compared to the B0 group, with the B20 treatment group showing the highest SPAD values, reaching 38.71. The SPAD values of B30 and B50 were marginally lower than those of B20, but the difference was not significant (p > 0.05). This suggests that indiscriminately increasing the amount of biochar does not always promote the synthesis of plant photosynthetic pigments and the enhancement of photosynthesis. Some researchers believe that excessive biochar can lead to excessive porosity and air space in the soil, which may, to some extent, reduce the availability of magnesium and adversely affect the synthesis of chlorophyll.²⁶ Similar to the SPAD values, the leaf nitrogen content also followed a similar pattern after the addition of biochar. The B20 treatment exhibited the highest nitrogen content, reaching 14.91 mg/g, while the B0 group had the lowest, with a leaf nitrogen content of only 11.91 mg/g, which was significantly different from the other treatment groups (p < 0.05). The improvement in the rhizosphere microenvironment due to biochar application has promoted the plant’s absorption of nutrients such as nitrogen.²⁷ The significant increase in CP content also demonstrates this. In this study, the CP content of B10 and B20 was significantly higher than that of B0, reaching 10.24% and 10.27%, respectively (p < 0.05). This finding is highly consistent with the results obtained by Choudhary et al. in their study on weed biochar. Unlike this, compared with B0, the acid detergent fiber (ADF) and neutral detergent fiber (NDF) of oats did not change significantly after the addition of biochar (p > 0.05). These results indicate that the addition of biochar can effectively promote the growth of oats and the absorption and utilization of nitrogen and other nutrients. Studies have shown that the main mechanism by which biochar improves plant growth is through enhancing soil properties (such as pH, water-holding capacity or nutrient retention capacity) and increasing the availability of soil nutrients, rather than relying on the release of its own nutrients. Therefore, we will further explore whether the application of biochar can replace part of the traditional fertilizers in the future, so as to reduce the use of chemical fertilizers while improving the productivity of forage oats, and form an environmentally friendly forage oat production model.

Carbon and nitrogen content of rhizosphere soil

The effects of biochar on the C and N content of oat rhizosphere soil are shown in Figure 2. The addition of biochar had a significant impact on the soil C and N content and the C/N ratio (p < 0.05). The C content and C/N ratio increased linearly with the increasing amount of biochar added. When the application rate reached 50 t/ha, the C content increased from 0.975% to 1.876%, and the C/N ratio increased from 14.49 to 22.31. The N content in the B30 and B50 treatment groups was significantly higher than that in B0 (p < 0.05), reaching 0.078% and 0.084%, respectively. The increase in soil C and N content was mainly due to the high C and N content of the biochar used in this study. Additionally, because biochar has abundant pore structures, strong ion adsorption capacity, and a large specific surface area, applying biochar to the soil can effectively reduce gaseous N loss and N leaching.²⁸ Liu et al. found that applying 9 t/ha of biochar in the field could reduce the N₂O emission flux by 14.94%–29.13%.²⁹ Zhang et al. also proved that after adding 20 t/ha of biochar, TN, AN, and NN decreased by 13.57%, 29.79%, and 13.9%, respectively.³⁰ The reduction in N loss is also the main reason for the increased N content after biochar addition in this study.

Figure 2.

The impact of biochar on the soil properties in the rhizosphere of forage oats

(A–C) The N content (A), C content (B), and C/N ratio (C) of the rhizosphere soil of forage oats.

B0, B5, B10,B20, B30, and B50 represent the addition of 0, 5, 10, 20, 30, and 50 t/ha of biochar, respectively. Different letters in the figure indicate significant differences in the mean values of variables among different treatment groups (p < 0.05). Data are represented as mean ± SEM.

Changes in rhizosphere bacterial and fungi diversity

Figures S1A and S1B show the alpha-diversity indices of oat rhizosphere bacteria and fungi. In this study, the Coverage index of bacteria and fungi did not change significantly with the addition of biochar (p > 0.05), and all treatment groups were above 0.99. When the biochar application rate was between 5 and 20 t/ha, the Chao, ACE, and Sobs indices of bacteria were slightly higher than those of B0, but the differences were not significant (p > 0.05). The indices began to decrease when the application rate increased to 30 t/ha. B50 had the lowest Chao, ACE, and Sobs indices (p < 0.05). Biochar affects the growth, activity, and community of soil microbes by directly providing growth promoters for soil biota (such as substrates and porous structures) or indirectly altering the basic properties of soil (such as pH).^31,32 Meanwhile, the improvement of soil fertility may also inhibit the growth and reproduction of oligotrophic microbial taxa, thereby causing changes in microbial diversity. Different from previous studies,³³ in this study, when the biochar application rate continued to increase to 50 t/ha, the diversity of oat rhizosphere bacteria decreased significantly, even lower than that of the treatment group without biochar. One possibility is that when the biochar application rate is too high, it will increase the content of EPFR and derived hydroxyl radicals (·OH) in the soil, which are toxic to soil bacteria. In addition, EPFRs and ·OH promote the release of dissolved organic carbon and ammonium nitrogen. According to the ecological enzyme stoichiometric theory, the increase in C and N content inherently leads to a relative shortage of P, thereby causing resource competition among bacteria and a subsequent decrease in bacterial alpha-diversity.³⁴ However, the Chao, Sobs, and ACE indices of fungi did not change significantly after the addition of biochar (p > 0.05).

To assess the changes in β-diversity of oat rhizosphere microorganisms, NMDS (non-metric multidimensional scaling) analysis based on the Bray-Curtis distance matrix was applied. The bacteria in the collected soil samples were clearly divided into six clusters (Figures S1B, stress = 0.118, R = 0.7547), indicating that the bacterial community in the oat rhizosphere soil changed significantly after the addition of biochar. The bacterial communities in the B50 and B0 treatments were the most distant from each other, indicating that the addition of 50 t/ha of biochar led to significant changes in the soil bacteria of the oat rhizosphere. Soil bacterial communities and their assembly processes respond very clearly to cultivation methods and additives. When biochar is added to the soil, it significantly alters the soil structure and provides habitats for microorganisms, thus affecting the growth and reproduction of rhizosphere bacteria.^27,35 The NMDS analysis of the fungi also showed that the fungal communities were relatively similar between the treatment groups, suggesting that the changes in fungal communities following the addition of biochar were relatively small (Figure S1C). Although total soil carbon is considered a major determinant of soil fungal diversity, the carbon in biochar is very stable and has a low utilization rate by microbes, which may explain the relatively minor changes in the fungal community.^36,37

Changes in microbial composition

We then analyzed the composition of oat rhizosphere bacteria and fungi at the phylum level (Figures 3A and 3B). At the phylum level, bacteria were mainly composed of Actinobacteriota (30.18%–33.53%), Proteobacteria (19.37%–24.85), Chloroflexi (8.44%–19.52%), and Patescibacteria (2.46%–22.93%), accounting for more than 70% of the total abundance. Pang et al. found that Actinobacteriota (11.85%–30.09%), Proteobacteria (26.81%–47.18%), and Chloroflexi (7.75%–11.93%) are common bacterial taxa in soil after biochar application.³⁸ However, different from Pang et al.’s study, the abundance of Patescibacteria increased significantly after biochar application in this study, reaching 22.93% in B50, the highest among all treatment groups. In contrast, the abundance of Patescibacteria in B0 was only 2.46%. The increase in soil nitrogen fertilizer content may be the main reason for the increased abundance of Patescibacteria. Ren et al. also found that the abundance of Patescibacteria tends to increase gradually with the application of nitrogen fertilizer over a period of 5 years.³⁹ In this study, the N content of rhizosphere soil increased significantly from 0.067% in B0 to 0.084% in B50, which may have promoted the growth and reproduction of bacteria in Patescibacteria.

Figure 3.

The relative abundance of soil microbial phyla in the rhizosphere

(A and B) At the phylum level, the community composition of bacteria (A) and fungi (B) in the oat rhizosphere.

B0, B5, B10, B20, B30, and B50 represent the addition of 0, 5, 10, 20, 30, and 50 t/ha of biochar, respectively.

Ascomycota was the most abundant fungal group, accounting for 77.66%–90.87% of the total fungal abundance, a result similar to that found in studies by Zhang et al.⁴⁰ Following the addition of biochar, the relative abundance of Basidiomycota decreased. In the B0 treatment, the relative abundance of Basidiomycota was the highest, reaching 8.69%, while in the B50 treatment it was the lowest, at only 1.37%. Previous studies have also indicated that the addition of biochar can reduce the abundance of Basidiomycota.⁴¹ One possible explanation is the high content of mineralizable carbon (C) in the biochar used in this study, and previous research has found that a high content of mineralizable C could be the primary factor responsible for the reduced relative abundance of Basidiomycota.⁴² Therefore, further studies building on this research are needed to investigate the effects of biochar addition on changes in soil C components, to further elucidate the underlying mechanisms driving changes in microbial communities.

The results of LEfSe analysis showed that there were 42 and 15 taxa identified as significant biomarkers in the bacterial and fungal communities of oats, respectively (i.e., the taxa in the bacterial and fungal communities that were significantly different among different treatment groups, Figure 4). In the bacterial communities, the B50 treatment had the highest number of biomarkers, with 16 species. The B0 treatment followed, with 13 biomarkers. No biomarkers were found in the B30 treatment. Species from the Gemmatimonadota phylum and Thermoleophilia class were the main biomarkers in B0. Gemmatimonadota and Thermoleophilia have been shown to prefer drier soil conditions, and their abundance tends to increase under drought conditions.^43,44 Biochar, with its abundant porous structure, has been shown to effectively enhance soil water retention when added to soil, alleviating drought stress.^45,46 Therefore, in this study, it was found that bacteria from Gemmatimonadota and Thermoleophilia were significantly enriched in the treatment group without biochar addition. This also indirectly suggests that the addition of biochar may partially improve soil moisture and other environmental conditions.

Figure 4.

The impact of biochar on the composition of microbial communities in the rhizosphere of forage oats

LEfSe analysis of oat rhizosphere bacteria (A) and fungi (B).

B0, B5, B10, B20, B30, and B50 represent the addition of 0, 5, 10, 20, 30, and 50 t/ha of biochar, respectively.

Fungi had a total of 16 biomarkers, with none found in the B5 treatment group. Bolbitiaceae, Conocybe, and Podospora were significantly enriched in B0. Unclassified_f__Stachybotryaceae, unclassified_f__Microascaceae, Pithoascus, and Onygenales were the biomarkers for B10. Stachybotryaceae is typically found in moist, nutrient-rich soil environments.⁴⁷ Glomeraceae was significantly enriched in the B20 treatment group. Members of Glomeraceae include many arbuscular mycorrhizal fungi that form symbiotic relationships with plant roots. These fungi provide nutrients for plants and enhance plant tolerance to environmental stresses.^48,49 F__Clavicipitaceae, F__Hypocreaceae, and G__Metarhizium are the biomarkers in the B30 treatment group. Among these, Metarhizium is a genus of entomopathogenic fungi that can have certain effects in preventing pest infestations during plant growth.⁵⁰ Gibberella and Alternaria are biomarkers that are significantly enriched in the B50 treatment group. Some studies have found that certain secondary metabolites produced by Gibberella and Alternaria can be harmful to plants, causing plant diseases.^51,52 Therefore, we speculate that the addition of an excessive amount of biochar may have some potential negative effects, such as the enrichment of certain fungi that are harmful to plants. Further research is needed to explore the impact of adding high doses of biochar on soil and plant health.

Functional prediction of microbial community structure

Here, we predicted the bacterial and fungal functions by functional annotation of prokaryotic taxa (FAPROTAX) and FUNGuild databases. The FAPROTAX analysis was performed to predict potential functional profiles based on microbiome composition (Figure 5). After conducting a one-way ANOVA on the 40 most abundant functional groups, the analysis revealed that 14 functional groups changed significantly after the addition of biochar (p < 0.05). Chemoheterotrophy (36.61%–40.32%) and aerobic_chemoheterotrophy (34.78%–38.68%) were the two most abundant functional groups, and both showed a similar trend, with their relative abundances initially increasing and then decreasing as the amount of biochar added increased, although this difference was not significant (p > 0.05). In this study, the relative abundance of aromatic_compound_degradation was notably high and exhibited significant changes after the addition of biochar (p < 0.05). The main reason is likely that biochar itself contains certain amounts of phenolic, ketone, aldehyde, and other aromatic compounds, which affected the bacterial community’s degradation process of aromatic compounds after the addition of biochar.⁵³ Relative abundance of ureolysis continuously increased with the increasing addition of biochar, reaching its maximum value of 4.51% at an application rate of 50 t/ha, which is a 255.12% increase compared to B0 (p < 0.05). Additionally, the relative abundances of nitrite_respiration, nitrate_respiration, and nitrogen_respiration all exhibited a pattern of initial decrease followed by an increase with the increasing amount of biochar added, reaching their minimum values at B20. This suggests that the addition of biochar is beneficial to the N cycle in the plant rhizosphere soil, which is similar to the findings of Li et al.⁵⁴ However, it is important to note that this study also found that the excessive addition of biochar might weaken or even inhibit this effect. The research results also indicate that the appropriate addition of biochar can reduce the relative abundance of denitrification, nitrite_denitrification, nitrate_denitrification, and nitrous_oxide_denitrification (p < 0.05). This suggests that biochar can inhibit the denitrification process of bacteria, reduce the loss of nitrogen fertilizers, and mitigate the emissions of N₂O.⁵⁵ Relative abundance of nitrogen_fixation also significantly changed after the addition of biochar (p < 0.05), reaching its maximum value at an application rate of 5 t/ha, which is a 56.95% increase compared to B0. However, when the biochar application rate continued to increase to 10 t/ha, the relative abundance of nitrogen_fixation significantly decreased, reaching its minimum value at 50 t/ha, at which point it was 76.42% lower than B0. Mia et al. also found that the addition of 10 t/ha of biochar could maximize the biological nitrogen fixation of red clover, but when the application rate was increased to 50 t/ha, the nitrogen-fixing effect significantly decreased.⁵⁶ In addition to the N cycle-related functional groups, photoheterotrophy, human_pathogens_pneumonia, and dark_oxidation_of_sulfur_compounds also significantly changed after the addition of biochar (p < 0.05).

Figure 5.

The relative abundance of bacterial function assigned by FAPROTAX

B0, B5, B10, B20, B30, and B50 represent the addition of 0, 5, 10, 20, 30, and 50 t/ha of biochar, respectively. “∗” and “∗∗” indicate significance at p < 0.05 and p < 0.01, respectively.

Figure 6 illustrates the ecological guild and trophic mode of the fungal community by the FUNGuild database. In this study, a total of 10 trophic modes were identified among the fungi, with Saprotrophs and Pathotrophs being the dominant ones. The B30 treatment group had the highest relative abundance of Saprotrophs and the lowest of Pathotrophs. The composition of guilds revealed that the relative abundance of plant pathogens in the B30 treatment was also the lowest. This suggests that the addition of 30 t/ha of biochar could improve soil health and mitigate the risk of pathogenic fungi. Yao et al. also found that in the third year of adding biochar to black soil, the relative abundance of OTUs of potential crop pathogens in the soil decreased with the addition of biochar, indicating that biochar may have the effect of suppressing the occurrence of crop diseases in the long term.⁵⁷ Some researchers believe that the potential mechanisms by which biochar suppresses plant diseases may be achieved through three pathways: altering the composition of plant root exudates, changing soil microbial and biochemical properties, and inducing plant systemic defense mechanisms.⁵⁸ In addition, the effects of biochar on the rhizosphere microbial community of crops also vary at different growth stages. However, this study only compared the rhizosphere microbial community of forage oats at harvest among different treatment groups. In future related studies, the impact of crop growth stages on the effects of biochar should be taken into account, and multiple time points should be selected for analysis as far as possible to provide more sufficient evidence for revealing the mechanism of biochar.

Figure 6.

Functional prediction results of FUNGuild

(A and B) The relative abundance of Trophic modes (A) and mainly Guilds (B) predicted by FUNGuild.

B0, B5, B10, B20, B30, and B50 represent the addition of 0, 5, 10, 20, 30, and 50 t/ha of biochar, respectively.

Microbial community co-occurrence networks

To demonstrate the co-occurrence of soil microbial communities and niche partitioning in the B0, B5, B10, B20, B30, and B50 treatments, we next constructed microbial networks (Figures 7 and 8). In this study, the co-occurrence networks and their topological properties of oat rhizosphere bacteria and fungi responded differently to the addition of biochar. The number of edges in the bacterial co-occurrence network decreased after the addition of biochar, with the B0 treatment having the highest number of edges and the B10 treatment having the fewest. Previous studies have shown that networks with more nodes and links display higher complexity among microbes.⁵⁹ At the same time, the change in the average degree of the network followed the same pattern as the number of network edges. B0 had the highest average degree, while B10 had the lowest. As the amount of biochar added increased, the average degree of the network first decreased and then increased. The average path length of the bacterial co-occurrence network also increased after the addition of biochar (Table S2). Generally, a network with a higher average degree and a shorter average path length is considered to be more tightly connected and more complex.⁶⁰ Therefore, it is believed that the complexity of the oat rhizosphere bacterial community in this study was reduced to varying degrees after the addition of biochar. This differs from previous research findings. Zhai et al. found that the complexity of soil bacterial communities and the interactions among them were reduced after the addition of 50 t/ha of biochar, but this phenomenon did not occur when the application rate was between 10 and 30 t/ha.⁶¹ The cause of this difference may be due to the varying characteristics of biochar resulting from differences in the raw materials and preparation methods used to produce the biochar. In symbiotic networks, a higher number of positively correlated edges indicate stronger synergistic interactions among microbial communities, while a higher number of negatively correlated edges suggests stronger antagonistic interactions.⁶² In this study, except for the B30 treatment, the proportion of positive edges in the other biochar-amended groups was lower than in the B0 group. This suggests that the addition of a specific amount of biochar can enhance the synergistic interactions within the oat rhizosphere bacterial community. Modularity is an important attribute of ecological networks, indicating the degree of spatial segregation, resource partitioning, and ecological niche differentiation.⁶³ Within a certain range, the addition of biochar increases the modularity of bacterial networks, but an excessive amount can lead to a decrease in network modularity.

Figure 7.

Bacteria co-occurrence networks under different biochar application rates

Network nodes represent the individual taxa colored by microbial phyla, and network links (edges) express the pairwise correlations between network nodes. The size of each node is proportional to the number of links. The green and red links indicate negative and positive interactions between two nodes, respectively (A). The distribution of core microbial communities based on the topological properties of the co-occurrence networks is shown using Zi-Pi plots in the B0 (G), B5 (H), B10 (I), B20 (J), B30 (K), and B50 (L) (the threshold values were 2.5 and 0.62 for Zi and Pi, respectively) (B).

B0, B5, B10, B20, B30, and B50 represent the addition of 0, 5, 10, 20, 30, and 50 t/ha of biochar, respectively.

Figure 8.

Fungi co-occurrence networks under different biochar application rates

Network nodes represent the individual taxa colored by microbial phyla, and network links (edges) express the pairwise correlations between network nodes. The size of each node is proportional to the number of links. The green and red links indicate negative and positive interactions between two nodes, respectively (A). The distribution of core microbial communities based on the topological properties of the co-occurrence networks is shown using Zi-Pi plots in the B0 (G), B5 (H), B10 (I), B20 (J), B30 (K), and B50 (L) (the threshold values were 2.5 and 0.62 for Zi and Pi, respectively) (B).

B0, B5, B10, B20, B30, and B50 represent the addition of 0, 5, 10, 20, 30, and 50 t/ha of biochar, respectively.

The topological role of each node in the network is investigated by analyzing the properties of both within-module connectivity (Zi) and among-module connectivity (Pi) values. The analysis results are shown in the figure. Number of module hubs and connectors in the B5 and B30 treatment groups both increased compared to B0. In the B10 and B50 groups, only the number of connectors significantly increased. Similar to B0, there were no connectors in the bacterial networks of B20, but the number of module hubs in B20 greatly increased, reaching 15, which is the highest value among all treatment groups. The number of connectors reflects the degree of connectivity between different modules. A higher number of connectors indicates closer connections between modules, allowing for smoother transmission of information and materials within the network. Module hubs are nodes with the strongest connectivity within a specific module and serve as hubs. A greater number of module hubs suggests the presence of more relatively independent modules within the network, with tighter internal connections.⁶⁴ The results of this study indicate that, except for the B20 treatment group, the number of connectors in the bacterial networks of the other biochar-amended groups increased. This suggests that adding biochar at low levels (5–10 t/ha) or high levels (30–50 t/ha) leads to closer connections between different modules of the oat rhizosphere bacterial network, which is beneficial for the coordination and feedback among various parts of the network, thereby enhancing its overall stability.⁶⁵ In contrast, the addition of 20 t/ha of biochar significantly enhanced the stability within modules, leading to more specialized functions. However, due to less tight connections between modules, the resilience of the entire bacterial network to disturbances may be somewhat reduced.⁶⁶

Unlike bacterial communities, after the addition of biochar at both low and high levels, the number of edges in the oat rhizosphere fungal network increased, and the average degree of the network also increased (Figure 8). The B20 treatment, however, showed the same number of nodes as B0, but with a decrease in the number of edges and a reduction in average degree. This suggests that the addition of biochar generally leads to the formation of a more tightly knit and complex network structure in fungi, but under specific application rates, the opposite outcome may occur. Furthermore, the proportion of positive edges in all treatment groups was above 80%, indicating that the interactions within the oat rhizosphere fungal community are predominantly synergistic, and the addition of biochar has further strengthened these synergistic interactions.⁶² Additionally, it was observed that the inter-module connections in the fungal networks were generally not very tight, and no connectors were found in the networks of all treatment groups except for B5.

Conclusion

The results show that the addition of biochar significantly increased the C content, N content, and C/N ratio in the rhizosphere soil. The production performance of forage oats was most significantly improved when 10 t/ha of biochar was added, which also increased the SPAD and N content of the leaves, and the CP content of the aboveground parts was relatively high. However, excessive addition of biochar can have negative effects. This is also reflected in the diversity of the root-associated bacterial community. When the addition reached 50 t/ha, the diversity of the rhizosphere bacterial community was significantly reduced, but the fungal community did not change significantly. The addition of biochar significantly changed the community composition of bacteria and fungi in the oat rhizosphere, especially increasing the abundance of certain specific phyla, such as Patescibacteria in bacteria and Ascomycota in fungi. It also enhanced microbial processes beneficial to plant growth, such as the degradation of aromatic compounds and nitrogen cycling processes, but excessive biochar can also inhibit these microbial functions. It is concluded that the addition of 10 t/ha of hydrothermal biochar can effectively promote the growth of forage oats and improve the structure and function of the rhizosphere microbial community when forage oats are planted on saline-alkali land.

This study provides a theoretical basis for the rational application of hydrothermal biochar in forage oat cultivation, points out the appropriate range of biochar addition, and reveals the possible mechanism by which biochar promotes plant growth by affecting the structure and function of the rhizosphere microbial community. Future research should further explore the optimal balance between plant growth and soil health after the addition of hydrothermal biochar in the field, as well as the long-term effects of hydrothermal biochar in different soil types and agricultural systems.

Limitations of the study

The climatic conditions outdoors are significantly different from those in greenhouses, and these differences can have a major impact on crop growth. This study only investigated the effects of adding hydrothermal biochar on the growth of forage oats through a pot experiment conducted indoors. Further research is needed to explore the application effects of hydrothermal biochar in the field based on the findings of this study. Additionally, the dynamic changes of rhizosphere microorganisms at different growth stages after the addition of biochar also need further investigation.

Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Gentu Ge (gegentu@163.com).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• •The raw 16S rRNA gene sequence data for this study have been deposited in National Center for Biotechnology Information (NCBI) under accession number (Bioproject: PRJNA1286924). The raw ITS gene sequence data for this study have been deposited in the NCBI with accession number (Bioproject: PRJNA1286937).
• •This paper does not report original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

This study was financially supported by National Center of Pratacultural Technology Innovation (under preparation) (CCPTZX2023B07), the earmarked fund for CARS (CARS-34-25) and National Natural Science Foundation of China (32260346).

Author contributions

X.Y.: methodology, conceptualization, validation, formal analysis, writing – original draft, writing – review and editing. Z.L.: formal analysis. M.Z.: methodology, validation, writing – review and editing. J.B.: methodology, data curation. M.W.: writing – review and editing. J.L.: software and validation. Y.J.: writing – review and editing. Z.W.: methodology, conceptualization, investigation. G.G.: resources, methodology, conceptualization, validation, writing – review and editing, supervision, project administration, funding acquisition.

Declaration of interests

The authors declare no competing interests.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Hydrothermal Biochar	Zhengzhou Haosen Environmental Protection Technology Co., Ltd.	Corn stover hydrothermal biochar
Critical commercial assays
FastDNA® Spin Kit for Soil	MP Biomedicals	N/A
Deposited data
The raw 16S rRNA gene sequence data	National Center for Biotechnology Information	PRJNA1286924
The raw ITS gene sequence data	National Center for Biotechnology Information	PRJNA1286937

Experimental model and study participant details

Experimental design and plant growth

The pot experiment was carried out in the greenhouse of Inner Mongolia Agricultural University in Hohhot, Inner Mongolia, China (111°43'37.038" E, 40°48'45.076" N) from July 27, 2023, for approximately three months. The soil used in the pot experiment was collected from the Hailiutu Science and Technology Park of Inner Mongolia Agricultural University. After air - drying, the soil was passed through a 2 - mm sieve for later use. The specific characteristics of the soil are shown in Table S1. The biochar used in the study was purchased from Zhengzhou Haosen Environmental Protection Technology Co., Ltd. It was made from corn straw by the HTC method. It has a pH of 6.5, organic matter content of 742 g/kg, total nutrient content of 6.69%, specific surface area of 323.5 m²/g, and ash content of 10.7%. Hydrothermal biochar generally has a lower pyrolysis temperature than pyrolysis biochar. Compared with pyrolysis biochar, it retains more organic matter and has lower ash content, making it more suitable for use in saline - alkali soils.⁸ Before planting, the same weight of soil was added to plastic pots (42 cm long, 30 cm wide, and 25 cm high), and 7.56 g of compound fertilizer (N - P₂O₅ - K₂O: 22 - 11 - 10) was applied to each pot. Then biochar was added to each group according to different addition ratios. A total of six biochar application rates were set, namely 0 t/ha (B0), 5 t/ha (B5), 10 t/ha (B10), 20 t/ha (B20), 30 t/ha (B30), and 50 t/ha (B50). After adding the biochar to the soil in proportion (0 g, 63 g, 126 g, 252 g, 378 g and 630 g of biochar were added to the pots), it was thoroughly mixed with the soil to ensure even distribution. The forage oat variety used was Beile, purchased from Beijing Zhengdao Seed Co., Ltd. One week after emergence, thinning was carried out to ensure that there were about 45 seedlings of forage oats in each pot. During the experiment, the ambient temperature was controlled between 20°C and 28°C, the relative humidity was between 50% and 60%, with a daily photoperiod of 14 h and a dark period of 10 h.

Method details

Measurement of forage oat growth and nutritional quality

When the oats grew to the heading stage, three plants were randomly selected from each pot. A portable chlorophyll meter SPAD-502 Plus (Konica Minolta, Japan) was used to measure the relative chlorophyll content (SPAD) and leaf nitrogen content of the plants in different treatment groups. The flag leaves with the same light exposure direction were selected for measurement. The SPAD value and nitrogen content of each leaf were calculated as the average of three points: the leaf tip, the middle part of the leaf, and the base. Then, the aboveground parts of the oats were cut off and divided into three parts: leaves, stems, and panicles. After drying at 65°C for 48 hours, the dry weight of each part was measured, and the total weight of the aerial parts was calculated. After weighing, the stems, panicles, and leaves of each pot were mixed together, ground, and used to determine the nutritional quality indicators. Crude protein (CP) was determined using the Kjeldahl method (Alva Instruments, China, fully automatic Kjeldahl apparatus - NKD6280). Acid detergent fiber (ADF) and neutral detergent fiber (NDF) were measured using the ANKOM fully automatic fiber analysis system (ANKOM, USA).

Soil collection and measurement

After cutting the aboveground parts of the forage oats, rhizosphere soil was collected. The root system of the oats was excavated and loose soil was shaken off. Then, soil still attached to the roots was collected into a sterile centrifuge tube using a sterile soft - bristled brush. The collected soil was divided into two parts. One part was air - dried and then analyzed for soil C content, N content, and C/N ratio using a FlashSmart elemental analyzer (ThermoFisher, USA). The other part was sealed and stored at - 80°C for later use.

DNA extraction and high-throughput sequencing

DNA was extracted from oat rhizosphere soil samples using the FastDNA® Spin Kit for Soil (MP Biomedicals, USA), and the quality of the extraction was assessed using a NanoDrop 2000 spectrophotometer. The extracted DNA was amplified using polymerase chain reaction (PCR). The primers 806R (5′-GGACTACHVGG GTATCTAAT-3′) and 338F (5′-ACTCCT ACGGGAGGCAGCAG-3′) were used to amplify the bacterial V3–V4 region, and the primers ITS1-F (5′-CTTGGTCATTTAGA GGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′) were used to amplify the fungal internal transcribed spacer (ITS) regions , followed by a polymerase chain reaction. PCR was conducted as follows: 95°C for 3 min, 27 cycles (95°C, 30 s; 55°C, 30 s; 72°C, 45 s), then an extension at 72°C for 10 min, ending at 4°C. Subsequently, the PCR products were extracted from a 2% agarose gel and purified. Finally, purified amplicons were sequenced using the Illumina MiSeq 2 × 300 bp platform by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).

Quantification and statistical analysis

The data are presented as the mean ± the standard deviation. SPSS 26 software was used to perform a one-way analysis of variance (ANOVA) on the biomass, SPAD values, and leaf nitrogen content of the forage oats, followed by Duncan's multiple range test (P < 0.05) as the statistical method for determining significant differences.

Sequence data analysis was mainly conducted using the Majorbio Cloud Platform (https://www.majorbio.com/tools). Alpha-diversity indices based on the ASV level, including the Chao1 richness estimator, Sobs index, ACE index, and Good’s coverage, were all extracted from the ASV table using the Majorbio Cloud Platform. Beta diversity analysis was performed to evaluate structural variation in microbial communities across samples, employing Bray-Curtis metrics and visualized using nonmetric multidimensional scaling (NMDS).⁶⁷ Linear discriminant analysis effect size (LEfSe) was employed to identify biomarkers associated with different growth patterns (Wilcoxon p-value < 0.05, linear discriminant analysis (LDA) score > 3).⁶⁸ Functional predictions for bacterial and fungal communities in the oat rhizosphere were conducted using the FAPROTAX 1.2.4 and FUNGuild databases, respectively.^69,70

Published: July 22, 2025