Legacy Effects of Biochar and Compost Addition on Arbuscular Mycorrhizal Fungal Community and Co-Occurrence Network in Black Soil

Abstract

Compost and biochar are beneficial soil amendments which derived from agricultural waste, and their application was proven to be effective practices for promoting soil fertility. Arbuscular mycorrhizal (AM) fungi form symbiotic associations with most crop plant species, and are recognized as one group of the most important soil microorganisms to increase food security in sustainable agriculture. To understand the legacy effects of compost and biochar addition on AM fungal communities, a field study was conducted on the Songnen Plain, Northeast China. Two years after application, compost addition improved soil aggregate stability, but we did not detect a legacy effect of compost addition on AM fungal community. Our results indicated that AM fungal Shannon diversity and Pielou evenness indices were significantly increased by one-time biochar addition, but unaffected by compost addition after two year’s application. PERMANOVA analysis also revealed a legacy effect of biochar addition on AM fungal community. Network analysis revealed a dramatically simplified AM fungal co-occurrence network and small network size in biochar added soils, demonstrated by their topological properties (e.g., low connectedness and betweenness). However, AM fungal community did not differ among aggregate fractions, as confirmed by the PERMANOVA analysis as well as the fact that only a small number of AM fungal OTUs were shared among aggregate fractions. Consequently, the current study highlights a stronger legacy effect of biochar than compost addition on AM fungi, and have implications for agricultural practices.

1. Introduction

Black soil is regarded as one of the most fertile soils distributed in Northeast China [1]. It accounts for approximately 20% of the national arable land and plays a crucial role in ensuring national food security [2]. However, serious soil fertility deterioration has arisen in this region over the past several decades due to extensive agricultural practices [3], which is a huge threat to crop production. To mitigate these problems, effective agricultural practices should be taken in time to improve the quality of black soil.

The application of compost, derived from agricultural waste composting, is considered an effective practice for promoting soil fertility [4]. A wealth of research has shown that compost amendment can increase soil organic matter content [5,6], improve soil quality [7,8] and crop yield [9]. Biochar is another beneficial soil amendment, which is produced through the pyrolysis of agricultural wastes under limited oxygen [10,11]. Biochar addition could increase soil carbon sequestration, improve soil water holding capacity, and reduce nutrient loss due to their huge superficial area and porosity [5,6,7,8,12,13,14].

Even though, constraints still exist regarding the influences of biochar incorporation into agroecosystems [15,16]. One limitation is that biochar would contain some toxic substances (e.g., polycyclic aromatic hydrocarbons) that were produced as by-product from the pyrolysis process [16]. Another limitation is related to its high cost and inconvenience in application and operation [15]. However, compared with compost or other fertilizers, biochar was more recalcitrant due to its higher aromaticity and greater C condensation, and thus could be resident in soil for hundreds of years [17,18]. A long-running field study has indicated that the beneficial effects of biochar addition on soil fertility and crop productivity could be detected after 2–5 years of application [19,20]. Therefore, the legacy effect of biochar addition would make the application of biochar much more convenient because there is no need to apply annually.

Arbuscular mycorrhizae (AM) are symbiotic associations formed between 80% terrestrial plant roots and soil fungi of the Glomeromycota, and the most prevalent fungi in soil [21,22]. AM fungi can provide benefits to host plants in many ways: (1) act as an extension of roots to increase the soil volume for essential nutrient uptake [23,24]; (2) increase plant tolerance to environmental stress, and induce plant resistance to pathogens [25,26]; (3) develop soil aggregation and improve soil quality [27]. Consequently, AM fungi are receiving growing interests as biofertilizers and are recognized as one of the most important soil microorganisms to increase food security in future sustainable agriculture [28,29,30,31,32].

AM fungi generally benefit from application of compost [33,34,35]. Unlike chemical fertilizers, compost provides a sustained supply of nutrients for AM fungi without adverse impacting on soil pH [33]. Alternatively, it was reported that the humic substances in compost could directly stimulate AM fungal growth [36]. Therefore, previous studies reported that compost addition could enhance AM fungal growth, sporulation and diversity [22,33]. However, the studies that examined the effects of biochar addition on AM fungi yielded divergent results [37,38,39,40]. Some studies provided pieces of evidence that biochar addition would increase AM root colonization [38,39,40], while negative or neutral effects of biochar on AM fungi were also occasionally reported [37,38]. Furthermore, the studies mentioned above mainly focused on AM colonization in roots or soils, while rarely focused on AM fungal community composition and their co-occurrence networks [41].

Most of what we know about AM fungal community was obtained through research at the local, regional or global scale [28,33,42]. However, AM fungal community at the micro-scale such as at the soil aggregate level, is poorly understood [43]. In the current study, we explored the legacy effects of compost and biochar on the AM fungal community and network in different soil aggregate fractions in the soybean agroecosystem. We hypothesized as follows: (1) biochar would exhibit a stronger legacy effect than compost; (2) one-time biochar and compost addition would influence AM fungal community composition and networks; (3) soil aggregate fractions would be a strong determinant for AM fungal community.

2. Materials and Methods

2.1. Field Description and Environmental Design

This study was conducted at the **angyang experimental farm, Harbin, China (45°45’ N, 126°54’ E). This study site has a typical monsoon climate of 4–5.5 °C average annual temperature and precipitation of about 400–500 mm [44]. The soil is typical black soil and has a loamy texture (classified as Mollisols).

The field trial was established in 2018 with a two-way factorial design (compost addition and biochar addition). There were four treatments: (1) no-biochar addition and no-compost addition (CK); (2) biochar addition without compost addition (B), with biochar application rate of 10 t ha⁻¹; (3) compost addition without biochar addition (C), with application rate approximately equal to 180 kg N ha⁻¹ (ca. compost 10 t ha⁻¹); (4) compost mixed with 10% biochar (BC), with total application rate approximately equal to 180 kg N ha⁻¹. Each treatment was repeated four times, resulting in 16 randomly arranged plots (5 m × 5 m each and 2 m separated from each other) totally. The compost used in the present study was obtained through an on-farm composting of cow manure and maize straw. The biochar was produced using rice straw under slow pyrolysis of 450–500 °C and supplied by Sanli New Energy Company (Henan, China). The application rate of compost and biochar was equivalent to the recommended amount of fertilizer in this area. The biochar and compost addition were one-time addition, which was only applied in 2018, and discontinued to applicate in 2019. A detailed description of compost and biochar have been described in Bello et al. [10]. The plots were tilled to a depth of 20 cm before planting and were weeded manually during the growing season. All plots received no chemical fertilizer, herbicide or insecticide.

2.2. Soil Sampling, Aggregate Fractionation and Determination of Soil Variables

Soil sampling was collected on 15 September 2019, 1.5 years after one-time application. In detail, three soil cores (10 cm × 10 cm × 10 cm) were carefully collected using a spade and placed in one plastic box for each plot. In total, 16 undisturbed soil samples were collected and transported to the laboratory with no damage.

The aggregate fractionation procedure was in accordance with the description by Bach and Hofmockel [45]. Briefly, the field-moist soil samples were placed in the freezer(Haier, Qingdao, China) (4 °C) to achieve approximately 10% gravimetric water content. Then 500 g soils were placed on the top of a stack of sterile sieves, with 2 mm-sieve was above the 0.25-mm sieve. The set of sieves was placed onto a sieve shaker (Techang, **nxiang, China) and shaken at 200 rpms for 2 min. Then each sample was divided into three aggregate fractions: (1) large macroaggregate (>2 mm), (2) little macroaggregate (0.25 mm–2 mm), (3) microaggregate (<0.25 mm). Soils were weighed for each aggregate fraction and the proportion of each fraction was calculated. Therefore, there were 48 samples (4 treatments × 4 replicates × 3 fractions) after aggregate fractionation. Soil organic matter (SOM), total nitrogen (TN), total phosphorus (TP), available potassium (AK), available P (AP), and available N (AN) were determined for each sample, the method for determination was available in Yang et al. [44].

2.3. Miseq Sequencing and Bioinformatics

Soil DNA was extracted from 250 mg frozen soil samples using a PowerSoil DNA Isolation Kit. A ca. 340 bp of the 18S rDNA gene was amplified using a two-step PCR, and primer pairs GeoA-2 [46]/AML2 [47] and NS31 [48]/AMDGR [49] were used in the first and second PCR reaction, respectively. The primer NS31 was labeled with a unique 12 nt barcode at the 5′ end to discriminate different samples. The detail information about the PCR conditions and quality assessment can be found in Yang et al. [50]. The PCR products were purified, thoroughly mixed and sequenced at the Environmental Genome Platform of Chengdu Institute of Biology, Chinese Academy of Sciences. The raw sequence data have been deposited on the NCBI SRA database (accession No. PRJNA882223).

Low-quality sequences and potential chimeras were removed using QIIME Pipeline Version 1.8.0 (Boulder, CO, USA) [51] before further analysis. The remaining sequences were clustered into different operational taxonomic units (OTUs) with 97% similarity level using USEARCH v8.0 [52]. The representative sequences of each OTU were blasted against the NCBI nt database to remove non-AM fungal OTUs. The number of reads per sample was normalized to the smallest sample size using the ‘normalized.shared’ command in Mothur [53]. Then a neighbor joining tree was constructed in MEGA v5 [54] to precisely identify these OTUs. The tree was visualized with iTOL [55].

2.4. Data Analysis

Mean weight diameter (MWD) was used to represent for soil aggregate stability and calculated as follows:

$$\mathrm{MWD}={\displaystyle \sum _{i=1}^n\overline{Xi} \times }Wi$$

Here, Wi is the percentage of the ith aggregate; ** AM fungal community composition. Our findings are of significance for understanding the response of AM fungi to agricultural practices, and thus may offer an effective way to improve soil fertility through the management of AM fungal community.