Walnut (Juglans spp.) is a perennial deciduous angiosperm that has gained increasing attention in recent years due to its economic importance. It is primarily grown for nut and wood products but is also an attractive ornamental tree in parks (Mortier et al., 2020). Recently, many farmlands have been transformed into walnut orchards in an attempt to increase walnut production and profitability (Bai et al., 2020). The farmers found that growing maize, wheat, or vegetables beneath the walnut trees increased output and revenue (Zhang W. et al., 2014; Pardon et al., 2019). Walnut trees can also positively affect crops physiology (Zhang W. et al., 2014). It has been previously shown that a combination of forest trees and crops improves land-use efficiency, reduces weed pressure, optimizes soil temperature, and conserves soil moisture (Tsonkova et al., 2012; Zhang D. S. et al., 2014; Gebru, 2015; Torralba et al., 2016; Panozzo et al., 2019). Forest trees regulate the climate underneath them by providing shelter and limiting soil evaporation (Lasco et al., 2014; Pardon et al., 2017). It may result in changes in soil microclimatic factors, such as humidity and temperature, when compared to an open field (Lott et al., 2009). In China, tea (Camellia sinensis L.) is also widely intercropped with walnut trees in the southwest region and the Daba Mountains (Wang et al., 2014; Tang et al., 2021). Although walnut trees compete with tea for soil resources in intercropping systems, they can also improve the growing conditions of tea trees, especially by providing moderate shade and increasing humidity in spring (Kulasegaram and Kathiravetpillai, 1976). Several studies have examined intercropping between trees and annual crops (Zhang W. et al., 2014; Arenas-Corraliza et al., 2018; Pardon et al., 2019; Temani et al., 2021); however, very little research has been conducted on intercropping between perennial non-wood forest trees (walnut) and crops. Additionally, the effects of tree–crop interactions on soil microbial diversity and community composition are largely unknown.

Soil microbiota are an important component of both natural and managed ecosystems (Fierer, 2017). Crop management practices that shape soil microbial communities under field conditions have greatly improved our understanding of how management factors affect crop production, biogeochemical cycling, and disease progression (Berthrong et al., 2013; Hussain et al., 2018; Raza et al., 2019; Tao et al., 2020). Therefore, microbial communities in relation to management strategies have become an imperative aspect of research in sustainable agriculture (Lacombe et al., 2009; Bainard et al., 2012b; Zhang M. M. et al., 2019). Intercropping, growing two crops simultaneously in proximity, not only affects crop yield but may also change the structure and functions of the soil microbiota (Duchene et al., 2017; Yu et al., 2019). In intercropping systems, soil nutrients, enzymes, and microbes interact to enhance the micro-ecological conditions of the soil by increasing the microbial diversity and enzyme activity (Zhou et al., 2019). Microorganisms influence soil nutrient turnover by decomposing soil organic matter (OM), which in turn influences soil enzyme properties and enzyme secretion (Peng et al., 2015). Soil enzymes (e.g., catalase, urease, and invertase) catalyze biochemical reactions during the decomposition of microorganisms and plants debris, which provide the soil with nutrients that plants need to survive (Veres et al., 2015). Therefore, the soil enzyme activity is closely linked to microbiological characteristics for improving soil-plant health (Cleveland and Liptzin, 2007). In previous studies, intercropping peanuts with maize changed the abundance of nitrogen-fixing microbes in the rhizosphere (Chen et al., 2018). Cassava–peanut intercropping enriched for Actinomycetes in the rhizosphere of peanut that boosted soil available nutrients absorption, increasing the peanut yield (Chen et al., 2020). It is therefore necessary to conduct further research to understand how tree–crop intercropping affects soil nutrient content, enzyme activities, and microbial community diversity and structure that would be crucial for evaluating the nutrient status and energy outputs of soil microbes in forest ecosystems.

Since various intercropping patterns differentially affect soil physicochemical properties and microbial characteristics, this study examined the changes in the bacterial and fungal communities as a result of the walnut–tea intercropping forest system. We hypothesized that intercropping would greatly affect soil physicochemical properties, increase enzyme activities and microbial diversity, and change the microbial community structure. Thus, the objectives of this study were to (1) explore the effects of walnut–tea intercropping on soil physicochemical properties and soil enzyme activity; (2) compare the response of bacterial and fungal diversity and community composition to intercropping with monoculture plantations of walnut and tea; and (3) determine the relationship between soil microbial communities and soil physicochemical properties and soil enzyme activity.

Intercropping has become increasingly popular over the past decade as a way to maintain soil biodiversity, improve nutrient content, and minimize pest and disease problems (Dai et al., 2019). One of the greatest advantages of intercropping is the increase in land-use efficiency and positive interactions between crops, which contribute to their survival, growth, and fitness (Hauggaard-Nielsen and Jensen, 2005). Previous studies have demonstrated that trees within agroforestry systems are capable of improving soil physicochemical properties (Wang et al., 2022). Our results confirmed that soil nutrient contents (AN, AP, AK, and OM) were significantly higher in walnut–tea intercropped forests than in monoculture walnut and tea forests, indicating that growing two crops simultaneously could improve soil nutrient content. Surprisingly, the amount of AN was significantly lower than that of AK and AP in the intercropped forest, indicating the existence of nitrogen competition. The reduced amount of nitrogen contents may be linked to the walnut fruit development, which requires a large amount of nitrogen for the protein synthesis of nuts. On the other hand, a notable increase in AK and AP concentrations was observed in walnut–tea intercropped forests. This might be due to the increased abundance of associated microbial taxa involved in soil nutrient cycling, including rhizobia and phosphate- and potassium-solubilizing bacteria. Another reason for this might be the release of nutrients by agroforestry trees to meet crop demands (Palm, 1995). Potassium can promote nitrogen and phosphorus uptake, leading to lavish vegetative growth, phosphorus translocation, and stimulating photosynthesis that is beneficial to crop growth and biomass production, thereby leading to an increase in yield (Ahmed et al., 2020).

Soil enzyme activity is an important indicator of how the soils cycle nutrients and decompose OM (Nannipieri et al., 2012; Hussain et al., 2021). Soil physicochemical properties and soil microorganisms are known to affect soil enzyme activity (Gu et al., 2009). In this study, we found that intercropped forests had the highest sucrase enzyme activity than monoculture tea and walnut forests. In contrast, peroxidase enzymes activity in the intercropped forest was significantly lower than in monoculture tea and walnut forests. Soil urease activity was significantly lower in intercropped and walnut forests than in monoculture tea forests. Sucrase hydrolyzes sucrose and can reflect soil organic carbon conversion ability, while urease hydrolyzes urea can affect soil nitrogen metabolism (Cantarella et al., 2018). Peroxidase activity decreases with increasing inorganic N availability (Cantarella et al., 2018), which is in alignment with our results. Saccharose catabolism provides a carbon source for microbes, thereby promoting their growth (Ruan, 2014). It appears that intercropped forests can provide sufficient energy for soil microorganisms since they play a key role in the accumulation, decomposition, and transformation of soil organic carbon.

Another focus of our investigation was the comparison of soil microbial communities in walnut–tea intercropping versus monoculture systems. Microorganisms from distinct phylogenetic lineages differ in their response to environmental changes (Hussain et al., 2016; Tian et al., 2018; Zhang W. W. et al., 2019; Zhang et al., 2021); thus, intercropping practices may influence soil microbiota compositions (Bainard et al., 2012a). We observed a profound shift in soil microbial communities when walnut was intercropped with tea. Walnut–tea intercropping caused changes in soil physicochemical properties and enzyme activities, forcing a specific subset of functional bacteria and fungi to enrich in soil, as evidenced by the increase in bacterial and fungal diversity in the intercropping soil relative to tea plantation. Plant species diversity, soil chemistry, and litter quality affect microbial communities (Guo et al., 2018). Studies have demonstrated that microbial diversity increases with vegetation aboveground and with afforestation (Xiao et al., 2016; Liu D. et al., 2018; Liu J. L. et al., 2018; Gao et al., 2019). We speculate that with the extension of natural forest, shading intensifies, resulting in the decrease of canopy temperature and root exudates (Abbasi et al., 2020). Therefore, reasonable perennial non-wood production forests intercrop with crops (walnut/tea) will not only increase soil nutrient contents and enzyme activity but also increase microbial diversity, which will be more favorable for plant growth.

Although intercropped and monoculture walnut forests did not differ in α-diversity, the PCoA of soil bacterial and fungal communities revealed a clear separation between the two forest types, indicating that the microbiota composition differed between monoculture walnut forest and intercropped forest. In this study, Proteobacteria, Actinobacteria, Acidobacteria, Chloroflexi, Bacteroidetes, Firmicutes, and Planctomycetes were the dominant bacterial phyla in the different forest types, which roughly correspond to the results of previous studies investigating agricultural soils (Bai et al., 2020). The walnut–tea intercropped forest had a significantly higher abundance of phyla Proteobacteria, Bacteroidetes, and Firmicutes than the tea and walnut forest soil samples. In contrast, Acidobacteria and Chloroflexi were much less abundant in the intercropping forest than in the tea and walnut forest soil samples. Previous reports indicate that many members of the phyla of Proteobacteria and Bacteroidetes were closely associated with the C and N cycle (Leff et al., 2015; Pardon et al., 2017). As a result, the intercropping forest might increase soil C and N accumulation and nutrient utilization efficiency by promoting bacterial growth that is closely associated with N fixation or other C–N processes. In addition, we observed a high abundance of Firmicutes in the walnut–tea intercropping soil possibly due to their ability to produce endospore as a result of soil microclimate changes (Gomez-Montano et al., 2013). Firmicutes express transcripts for glycosylase hydrolases involved in cellulose and chitin breakdown (Wegner and Liesack, 2016). A striking pattern was that the abundance of Nitrospirae in the monoculture walnut forest was more than in the monoculture tea forest, which are known to be involved in ammonia-oxidizing and nitrite-oxidizing processes (Beeckman et al., 2018). According to previous studies, the decline in the RA of Nitrospirae is due to tea trees that prefer NH₄⁺, which reduces the ammonia-oxidizing and nitrite-oxidizing processes (Ruan et al., 2016). Gemmatimonadetes are facultative bacteria (Takaichi et al., 2010) and have been significantly correlated with soil OM and N content (Chen et al., 2016). Chloroflexi were found to be abundant because they produce energy by solar radiation and 3-hydroxypropionate bi-cycle (Berg, 2011; Klatt et al., 2013). Therefore, Chloroflexi are more abundant in individual forests due to open canopy that receives more sunlight than intercropping forest (Tripathi et al., 2016). Chloroflexi and Acidobacteria have previously been shown to be slow-growing bacteria (Davis et al., 2011) that prefer oligotrophic environments (Fierer et al., 2012). Because of the oligotrophic environment in the monocropping forest, the RA of Chloroflexi and Acidobacteria was higher than in intercropped forests (Zhu et al., 2019; Xu et al., 2020). Importantly, the significantly higher abundance of Proteobacteria in the intercropping forest compared to monocropping forests is indicative of eutrophic soils (Smit et al., 2001), pointing out that soil nutrient status was improved after intercropping.

In walnut–tea intercropping forest, we detected several enriched bacterial OTUs associated with phosphorus and potassium solubilization. They belonged to the bacterial genera Pseudomonas, Rhizobium, Arthrobacter, Sphingomonas, Bacillus, and Burkholderia, which are known for their functions such as plant growth promotion, phosphorus-solubilizing, nitrogen-fixing, degradation and biotransformation of organic compounds, and growth stimulation (Rodrıìguez and Fraga, 1999; Chen et al., 2006; Sharma et al., 2013; Panhwar et al., 2014; Hu et al., 2020; Morya et al., 2020; Paulitsch et al., 2020; Sadauskas et al., 2020; Tapia-García et al., 2020; Yang et al., 2020). Similarly, Bacillus and Burkholderia have demonstrated the ability to solubilize potassium (Basak and Biswas, 2009; Zhang and Kong, 2014). Moreover, we found a significant enrichment of Chujaibacter, Acidothermus, Acidipila, Micropepsaceae, Bryobacter, and Pseudolabrys in the intercropped forest. Chujaibacter is known to be involved in nitrogen cycling reaction–nitrification (Semenov et al., 2020), and its abundances were positively correlated with soil OM, nitrogen, and phosphorus content in the present study (Figure 8). Acidothermus is found to be arbuscular mycorrhizal fungi suppressive taxa (Svenningsen et al., 2018), and Bryobacter is plant growth-promoting rhizobacteria (Liu et al., 2019). Pseudolabrys is highlighted to be able to improve salinity stress (Lee et al., 2021). The intercropping forest also showed significant enrichment of Rhodanobacter. Members of Rhodanobacter are known as denitrifying bacteria that are used for bioremediation (Prakash et al., 2012).

In the fungal community, Ascomycota and Mortierellomycota were significantly more abundant in monoculture walnut forests than in monoculture tea forests. However, Glomeromycota were more abundant in tea soils than walnut soils and walnut–tea intercropping forests. The roots of tea trees form a symbiotic relationship with arbuscular mycorrhizal fungi in soils containing low levels of inorganic phosphorus (Öpik et al., 2010; Wu et al., 2019). As confirmed in this study, monoculture tea forest soils had a significantly lower amount of AP than in monoculture walnut and intercropping forest. Further research is needed to understand the reasons for the decrease in arbuscular mycorrhizal fungi in walnut forest land or intercropping forest land. Moreover, we found that soils in the intercropping forest contained a significantly higher abundance of Trechispora, Oidiodendron, Penicillium, Talaromyces, Trichoderma, and Pochonia than those in monoculture tea and walnut forests. The higher abundance of Trechispora in the soil has been positively correlated with the increased uptake of nitrogen and carbon sources by hosts (Vanegas-León et al., 2019) and litter decomposition (Midgley et al., 2015). Members of the genus Trichoderma are known for their ability to reduce the severity of plant diseases by inhibiting pathogenic organisms in the soil through their highly potent antagonistic and mycoparasitic activity (Schuster and Schmoll, 2010; Hermosa et al., 2012; Ghazanfar et al., 2018; Chen et al., 2021). In prior studies, Oidiodendron has been demonstrated to improve phosphorus and nitrogen uptake (Vohnfk and Albrechtová, 2005). Penicillium, Talaromyces, and Pochonia species are also shown to inhibit plant pathogens (Elsharkawy et al., 2012; Hamid et al., 2017; Mwaheb et al., 2017; Topalovic et al., 2020; Abdul et al., 2021).

Based on the above discussion, it becomes clear that intercropping walnut and tea positively affects soil nutrient contents, enzyme activity, and the composition of bacterial and fungal microbiota in the soil microbiome and that changes in microbial diversity and RA play an important role in nutrient cycling, which benefits both walnut and tea trees.

The results of our study suggest that intercropping practices in orchards had a significant impact on improving the soil’s nutritional conditions by increasing soil nitrogen, phosphorus, potassium, and OM. The intercropping practice also induced great changes in soil bacterial and fungal communities. An analysis of microbial and fungal communities confirmed that walnut–tea intercropped had a significant impact on the bacterial and fungal community diversity and composition as compared to the monoculture walnut and tea forests. In general, the intercropping practice significantly optimized bacterial and fungal community structure and harbor relatively enriched beneficial bacterial and fungal taxa that were mainly related to nutrient cycling and disease protection. Nevertheless, the functional potential of soil microbial communities using shotgun metagenomics and metatranscriptomics is needed to further study.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

Y-CB and Y-JX conceived the idea. Y-CB, C-YX, B-XL, QW, Q-ZW, and Y-NF collected and prepared the samples for soil, enzyme, and microbiome sequencing analysis. Y-CB, MR, J-YH, AI, and MH performed the bioinformatics analysis and prepared the figures. Y-CB, MH, and Y-JX wrote the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.

J-YH is employed by Shenyang Sinochem Agrochemicals R&D Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.