Effect of Biochar and Green Manure on Soil Nutrients and Rhizosphere Soil Fungal Community Structure

Introduction

Soil microorganisms are an indispensable active component of the soil ecosystem, a driving force for nutrient cycling and transformation, and a potential indicator for evaluating soil quality (Mariangela and Francesco 2010; Wang, et al. 2013). As a biological population in the soil second only to bacteria, fungi were the main component of soil microbes and constitute most of the microbial biomass of the soil. They were functioning as decomposing organic matter, promoting nutrient circulation, providing nutrients for plants, and regulating the composition of plant communities, while soil fungi could also form symbiosis with plants, driven changes in soil productivity together (Henrik, et al. 2019). Meanwhile, The diversity of soil fungi and the composition of their community structure would also have a profound impact on the ecosystem and play an important role in the balance of the ecosystem (Wang and BAU 2014).

Biochar was a carbon-rich solid stable substance produced through pyrolysis of agricultural and forestry wastes under anaerobic conditions, which was generally used as a soil amendment (Johannes, et al. 2011). Biochar was considered to be a substance which could improve soil fertility and slow climate change. Yan et al. (2018) found that biochar could increase soil nutrient content, reduce soil bulk density, and significantly change the structure of soil fungal community by applying biochar in cinnamon soil. Feng et al. (2021a) believed that biochar mainly affected soil microbiological characteristics by changing the rate of soil carbon and nitrogen mineralization. Biochar has been shown to change the composition and abundance of soil biological communities. These changes might affect nutrient cycling and give an impact to the soil structure, leading to changes in the communities and abundance of bacteria and fungi in the rhizosphere, which indirectly affected plant growth (Liang, et al. 2010). The application of biochar in the soil was conducive to the retention of soil carbon, which might change the microbial community, not only affecting nutrient cycling and plant growth, but also the cycle of soil organic matter (Yun, et al.). In addition, the application of biochar could reduce emissions of CO₂, CH₄ and N₂O (Wang, et al. 2019).

Green manure was an important measure to effectively improve the ecological environment, soil quality, and fertility. Studies have shown that green manure can conserve water and soil (Wang, et al. 2016), reduce nutrient loss, improve soil physical and chemical properties and increase soil fertility (Gómez-Munoz, et al. 2014), indirectly affect the number, activity and community structure of microorganisms to a certain extent (Greg, et al. 2018; Whitelaw-Weckert, et al. 2007), reduce the occurrence of diseases (Yim, et al. 2016), and improve the yield and fruit quality of fruit trees (Srivastava, et al. 2007; Sweet and Schreiner 2007). In 2017, the Ministry of Agriculture and Rural Affairs of China formulated the Action Plan for Carrying out Organic Fertilizers for Fruits and Vegetables to Replace Chemical Fertilizers. "Natural grass + green manure" was one of the technical models implemented in orchards, and the area of green manure planting was increasing day by day. This experiment studied the effects of combined application of biochar and green manure on the fungal community structure of citrus rhizosphere soil, and explored the effect of soil-microbe interaction on soil health, in order to provide a theoretical basis for the regulation of soil micro-ecological control in orchards in China.

Materials And Methods

Test varieties

The experiment was carried out in a citrus orchard in Jinhu Village, Liuyang City, Hunan Province. The area was in a humid mid-subtropical monsoon climate, moderate light and heat, and abundant rain. The annual average temperature was 17.4 ℃, the average number of sunshine was 1650 h, and the annual average precipitation was 1680 mm. The precipitation was mostly concentrated in April to July. The soil type was quaternary red loam, with soil pH of the cultivated layer as 5.10, the organic matter as 36.10 g·kg^− 1, the alkaline nitrogen as 163.00 mg·kg^− 1, the available phosphorus as 79.80 mg·kg^− 1, and the available potassium as 169.00 mg·kg^− 1. The tested citrus variety was Orah, with tree age of 4 ~ 5 years, the crown width was 2 m×1.5 m, the tree height was 2.0 m, and the tree vigor was medium.

The experimental biochar was the raw material of peanut shell, provided by Henan Biochar Engineering & Technology Research Center. The carbonization process was prepared by low oxygen and continuous carbonization at 380 ~ 400 ℃ for 20 min. The basic indicators were as following: biochar particle size 250 ~ 500 µm, pH value 8.37, total carbon 409.73 g·kg^− 1, total nitrogen 16.25 g·kg^− 1, C/N value of 25.29, specific surface area of 23.49 m²·g^− 1, pore volume of 0.034 cm³·g^− 1, and pore size of 4.38 nm。

The green manure planting species was Vicia villosa Roth. The grass seeds were provided by Hunan Academy of Agricultural Sciences. On 15th October 2019, the fruit trees were sown between the rows, with a sowing amount of 45 kg·hm^− 2. Fertilizers were applied twice on 15th December 2019 and 15th March 2020, with a single application of 50% of the total fertilizer. Green manure crops were returned to the field on 10th May 2020.

Test design

There were 3 treatments in the experiment, which were CK: local conventional fertilization, no green manure, no biochar, T1: local conventional fertilization, no green manure, 5 kg of biochar per tree (30 cm ± 5 cm away from the tree), and T2: green manure, each tree was applied circularly (at a distance of 30 cm ± 5 cm from the tree) with 5 kg of biochar, and the fertilizer nitrogen was reduced by 10%. The green manure was turned back into the field, and the conventional fertilization rates for citrus were N, P₂O₅, and K₂O fertilization, with rates of 222.22, 111.11 and 155.56 kg·hm^− 2, respectively. The experiment was set to be repeated 3 times, and the plot area was 300 m². The field management of fruit trees was carried out in accordance with local conventional management measures, and all plot areas were kept consistent.

Test methods

Sample collection and pretreatment

Sampling points were determined according to the 5-point sampling method. For each treatment, 3 citrus plants were determined. Use a shovel to dig 10 cm of soil around the 3 citrus plants to a depth of 30 cm, cut any lateral roots of the plants in the soil, and dig out 3 plants Citrus plant with whole root. Most of the soil was shaken off with hands, then 3 root balls were put into the pot, shake the roots and remove the soil from the roots with a shovel, shake the soil in the collection pot to mix it, and collect no broken pieces in the collection pot block soil 5 ~ 10 g. Plant roots, animal remains and other impurities were removed after that, then mix well, pass through a 2 mm sieve, store in a 10 mL sterile centrifuge tube, store with dry ice, and collected samples were sent to Shanghai Meiji Biomedical Technology Co., Ltd. for microbial diversity testing。

Soil fungi detection

The total DNA was extracted with the EZNA ®soil kit (Omega Bio-tek, Norcross, GA, US). The concentration and purity of the DNA were detected by NanoDrop 2000 ultra-micro spectrophotometer (Thermo Fisher Scientific). The quality of DNA extraction was determined by 1% Agarose gel electrophoresis for detection; fungus 18S was PCR amplified with SSU0817F (5'-TTAGCATGGAATAATRRAATAGGA-3') and 1196R (5'-TCTGGACCTGGTGAGTTTCC-3') primers for the V5-V7 variable region, using Illumina's Sequencing on Miseq PE 300 platform (Shanghai Meiji Biomedical Technology Co., Ltd.).

Data analysis

Microsoft excel 2016 was used to analyze the data, the analysis of variance used the least significant difference method, the DPS 7.0 software was used to analyze and process the data, and the principal component analysis used SPSS 22.0 software. The algorithm of the four indices was as following:

\({D}_{simpson}\) =\(\frac{\sum \begin{array}{c}sobs\\ i=1\end{array}{n}_{i}\left({n}_{i}-1\right)}{N(N-1)}\)、\({H}_{shannon}=-\sum _{i=1}^{sobs}\frac{{n}_{i}}{N}ln\frac{{n}_{i}}{N}\)、\({S}_{chao1}\)=\({S}_{obs}+\frac{{n}_{1}({n}_{1}-1)}{2({n}_{2}+1)}\)、\(coverage=1-\frac{{n}_{1}}{N}\)

\({s}_{obs}\) =the actual number of OTUs observed, \({n}_{i}\)=the number of sequences contained in OTU of i, \(N\)=all sequence numbers, \({n}_{1}=\)the number of OTUs containing only one sequence。

Results And Analysis

The effect of biochar and green manure on soil physical and chemical properties

It could be seen from Table 1 that the application of biochar would significantly change the physical and chemical properties of the soil, and the combined application of biochar and green manure had a more significant impact on the physical and chemical properties of the soil. Compared with CK, both T1 and T2 treatments significantly increased the soil pH, porosity, alkali-hydrolyzable nitrogen, available phosphorus and available potassium content. Among them, in T2 treatment, soil pH, porosity, alkali-hydrolyzable nitrogen, available phosphorus and available potassium were increased by 19.09%, 19.74%, 9.38%, 19.13% and 14.30%, respectively, comparing to CK treatment. The soil bulk density of T1 and T2 treatments was reduced by 15.20% and 22.40%, respectively, compared with CK treatment, reaching to a significant level. The soil organic matter and soil cation exchange capacity of T1 and T2 treatments both increased, not reaching to a significant level yet.

Table 1

Effects of biochar and green manure on soil physical and chemical properties
Treatment	pH	Soil bulk density (g·cm^− 3)	Soil porosity (%)	Organic matter (g·kg^− 1)	Avail. N (mg·kg^− 1)	Avail. P (mg·kg^− 1)	Avail. K (mg·kg^− 1)	CEC (cmol·kg^− 1)
CK	5.76b	1.25a	52.68c	40.28a	194.50b	154.46b	326.78b	17.70a
T1	5.83a	1.06b	58.93b	41.05a	208.84a	172.36a	357.85a	18.25a
T2	5.87a	0.97b	63.08a	41.92a	212.74a	184.01a	373.52a	18.80a
Within each column, means followed by different letters are significantly different according to Tukey’s test at p ≤ 0.05

The effect of biochar and green manure on soil fungal α diversity

9 soil samples were tested, and a total of 428,819 valid sequences were obtained, with average sequence of a single sample as 47,647. Based on 97% sequence similarity, under cluster analysis of the tested sequences, the average number of OTUs for all samples was 270。The total number of OTUs in the three treatments was 174, of which the OTUs of the T2 treatment were significantly higher than that of the CK (Fig. 1)。

It could be seen from Table 2 that there was no significant difference in fungal α diversity between T1 treatment and CK treatment, but the Shannon index of T1 treatment was greater than CK, and the Ace index and Chao index were smaller than CK, indicating that the application of biochar could increase soil fungi Diversity, but reduce the abundance of fungi. The Shannon index, Ace index and Chao index of T2 treatment were significantly greater than CK treatment, and Simpson index was lower than CK treatment, indicating that applying biochar while tugging green manure can effectively increase the diversity and abundance of soil fungi。

Table 2

Sequencing sequence and of soil fungi α Diversity
Treatments	Sequencing numbei	Shannon index	Simpson index	Ace index	Chao index	Coverage(%)
CK	51140.00	2.73b	0.15a	183.68b	186.33b	99.97
T1	49312.33	2.83b	0.14a	170.59b	170.93b	99.97
T2	42487.33	3.02a	0.13a	201.68a	202.12a	99.95
Within each column, means followed by different letters are significantly different according to Tukey’s test at p ≤ 0.05

The effect of biochar and green manure on soil fungal community structure

Effect of biochar and green manure on fungal community structure in horizontal soil

The results of the species annotation showed that a total of 30 fungal floras were obtained at the phylum level in all samples, and the average relative abundance < 1% was classified as other groups, and 10 taxa were obtained, as shown in Fig. 2. Among them, the more abundant phylum species are Ascomycota, an unclassified supergroup (SAR_k__norank), an unclassified fungus (unclassified_k_norank), Basidiomycota and Mucoromycota. Compared with CK, the abundance of Ascomycota treated with T1 and T2 decreased by 7.54% and 22.85%. The relative abundance of Basidiomycota treated with T1 and T2 increased by 130.30% and 276.00%, respectively, compared with CK treatment. The relative abundance of Mucoromycota treated with T2 was 44.66% lower than that treated with CK.

The abundance of soil fungi showed differences at different treatment levels. As shown in Fig. 3, Ascomycota and Schizoplasmodiida showed significant differences among the three treatments, and Ascomycota was relatively abundant in CK treatment. The highest relative abundance of Schizoplasmodiida was oberved in T2 treatment. It could be seen from Fig. 3 that among the top 13 of relative abundance for fungi in phylum level, the relative abundance of 8 flora in T2 treatment was higher than that in CK treatment。

The effect of biochar and green manure on fungal community structure in horizontal soil

The results of the species annotation show that a total of 151 fungal floras were obtained at the genus level in all samples, and the groups with an average relative abundance of < 1% were classified as other groups, and 23 groups were obtained, as shown in Fig. 4. Among them, the more abundant floras were an unclassified genus (unclassified_k_Microascaceae), Chaetomium, Talaromyces, Colpoda, and Fusarium. The relative abundance of an unclassified genus (unclassified_k_Microascaceae) belonging to Microascaceae in T2 treatment increased by 9.33% compared with CK treatment, and the relative abundance of Chaetomium decreased by 58.67%. The relative abundance of Talaromyces in T1 and T2 treatments increased by 273.51% and 230.12% compared with CK treatment, and the relative abundance of Colpoda increased by 6.58% and 74.80%, respectively. The relative abundance of Fusarium decreased by 39.22% and 23.10%, respectively.

Effect of Biochar and Green Manure on the Principal Components of Soil Fungal Community

The principal component analysis of soil colony structure based on the abundance of OTUs is shown in Fig. 5. The contribution of the PC1 axis and the PC2 axis to the difference in sample composition are 41.28% and 27.29%, respectively. It could be seen from Fig. 5 that the biological repetition in each treatment group was not quite well, but the distance between CK treatment and T2 treatment was relatively long, indicating that there was a significant difference in soil fungal community composition between biochar and green manure and conventional fertilization. T1 treatment had a cross-over to both CK and T2 treatment, indicating that application of only biochar had a certain effect on soil fungi, but combined application of green manure on the basis of biochar could much significantly change the microbial community structure。

Correlation analysis between community structure of soil dominant fungi and environmental factors

The correlation between the top 20 fungal species with relative abundance at the phylum level and soil factors is shown in Fig. 6. pH, SBD, AN, AK and AP all had significant effects on soil bacterial community structure. There was a significant negative correlation between Ascomycota and AK (P < 0.05), and a negatively correlation(P < 0.05) between SBD with unclassified_k__norank, Schizoplasmodiida and Basidiomycota. There was a significant correlation between AK, pH, AN and AP with schizoplasmodiida, in which AK and schizoplasmodiida had a very significant positive correlation (P < 0.01). Aphelidea and Blastocladiomycota were negatively correlated with environmental factors. Among the top 20 fungal communities in relative abundance, about 1/2 were positively correlated with environmental factors. The effects of SOM, CEC, AK, pH, AN and AP on soil fungal community structure were consistent.

Discussion

The effect of biochar and green manure on soil nutrients

As an excellent soil conditioner, biochar had been analyzed in a lot of research and was widely used in agricultural production. It had excellent qualities such as improving crop agronomic traits and promoting increased production and income (Hong, et al. 2021). Green manure was a high-quality biological fertilizer that could provide nutrients for crops, improve the ecological environment of farmland and prevent soil pollution. Planting and pressing green manure was a sustainable way that could improve and maintain soil quality (Feng, et al. 2021b; Si, et al. 2014). By applying biochar and combined application of biochar and green manure in citrus orchards, it was found that the application of biochar and combined application of biochar and green manure could significantly increase soil pH, which was consistent to the results of Feng et al. (2021a). Hu et al. (2021) found that using green manure in mango orchards could increase the content of soil organic matter, available phosphorus and available potassium, and Gao et al. (2020) found that the combined return of green manure and rice straw to the field could significantly increase the soil organic matter and available potassium content, but reduce the soil alkalinity nitrogen content. It was found by this study that biochar and green manure significantly increased the content of available phosphorus and available potassium in the soil, but the organic matter content did not increase much accordingly. This was due to the high content of organic matter in the soil, and the soil organic matter did not change significantly after the green manure was compressed. Biochar had a rich microporous structure, which reduced soil bulk density and increase soil porosity, when applied to the soil. Green manure was a high-quality organic material that increased soil organic matter, after being pressed into the soil. Generally speaking, the low bulk density soil of organic matter containing higher content was more conducive to the release of nutrients and the storage of nutrients (Li, et al. 2021). The study by Liu et al. (2016) showed that the application of biochar could reduce soil bulk density, increase soil porosity, and increase soil water holding capacity. Soil cation exchange capacity was an index to evaluate the ability of soil to retain water and fertilizer, and was one of the important basis for soil improvement and rational fertilization. It was found by this study that the application of biochar and/or the combined application of biochar and green manure could increase the cation exchange capacity of the soil, which was consistent with the results of Liu et al. (2020) and Lü et al. (2020).

The effect of biochar and green manure on soil fungal diversity

Soil microorganisms were one of the main factors affecting the growth environment of crops and played a very critical role in the growth and development of plants. The number of fungi in soil microorganisms was second only to bacteria and actinomycetes. The diversity of fungi was of high value in maintaining the balance of the soil ecosystem and soil quality (Knelman and Nemergut 2014). A large number of studies have shown (Li, et al. 2021) that the application of biochar could increase the diversity of soil fungi and change the structure of fungal community. The results of this study show that the Shannon index of T1 treatment is greater than that of CK, but not reaching to a significant level yet, which was the same as the results of Li et al. (2020). Yin et al. (2021) found that soil nutrient content increased significantly after applying biochar to cinnamon soil for four consecutive years, and soil bulk density decreased significantly, but the fungal diversity did not change significantly. This might be due to the fact that biochar contained more inert carbon of which its nature was relatively stable, taking a certain amount of time to decompose.Compared with CK, T2 treatment had greater changes in soil fungal diversity and abundance than T1 treatment. This might be due to the accumulation of a large amount of organic matter in the soil after the green manure was pressed, which could be directly decomposed and utilized by microorganisms, similar to what was observed by Greg et al. (2018) and Fu et al. (2020). Li et al. (2021) found that if the amount of green manure returned to the field was too high and too late, it would reduce the abundance of wheat soil fungi. Thus, significance on crop growth of those methods should be studied further on choosing the appropriate quantity of green manure returning to the field and the proper time to return to the field.

The effect of biochar and green manure on the structure and function of soil fungal community

From the perspective of community composition, soil fungi promoted the carbon and nitrogen cycle processes in the soil by decomposing plant residues (Wang, et al. 2017). This study found that although there were certain differences in fungal communities at the phylum and genus levels in the three treatments, Ascomycota, Basidiomycota and Chaetomium were still the dominant microorganisms in the soil, similar to previous research results (Chang, et al.; Jin, et al.; Wan, et al. 2021). The results of this study showed that the application of both biochar and green manure reduced the relative abundance of the Ascomycota. The reason was probably that the Ascomycota is the most abundant type of microorganism in the soil, and the application of biochar increased the diversity of fungi and the competitiveness of other phyla, which reduced the relative abundance of Ascomycota.The main function of Basidiomycota in the soil was to decompose organic matter and cellulose. This study found that the application of only biochar and the combined application of both biochar and green manure significantly increased the Basidiomycota in the soil. Fusarium was a widespread soil-borne pathogen in the soil, while it was found that the application of only biochar and the combined application of both biochar and green manure could reduce the relative abundance of Fusarium in the soil, by this study. Wang et al. (2019) found that biochar could reduce the relative abundance of Fusarium oxysporum in the soil for continuous cropping daylily by adding biochar.

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Effect of Biochar and Green Manure on Soil Nutrients and Rhizosphere Soil Fungal Community Structure

Abstract

Highlights