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Article

Leguminous Green Manure Intercropping Promotes Soil Health in a Citrus (Citrus reticulata) Orchard

by
Yuxin Xie
1,
Yulin Jing
2,
Yajie Wang
1,
Rongchun Zheng
1,
Qiurui Xu
1,
Zhenyu Sun
3,* and
Tingyu Duan
1,*
1
State Key Laboratory of Herbage Improvement and Grassland AgroEcosystems, Lanzhou University, Lanzhou 730020, China
2
Institute of Soil Fertilisation and Resource Environment, Nanchong Academy of Agricultural Sciences, Nanchong 637000, China
3
Institute of Plant Protection, Gansu Academy of Agricultural Sciences, Lanzhou 730070, China
*
Authors to whom correspondence should be addressed.
Agriculture 2024, 14(11), 1897; https://doi.org/10.3390/agriculture14111897
Submission received: 11 September 2024 / Revised: 17 October 2024 / Accepted: 24 October 2024 / Published: 26 October 2024
(This article belongs to the Special Issue Organic Management Approaches and Practices to Support Sustainable Horticultural and Fruit Plants Production)
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Abstract

:
The intercropping of green manure is an important and sustainable production method in citrus orchards (Citrus reticulata). However, few studies focus on the impact of annual and perennial green manure on soil health, particularly soil microbiome and properties in acid soil. Our research objective was to explore the potential effects on soil health by intercropping with annual and perennial leguminous green manures in acid soil citrus orchards of southwestern China. The leguminous green manures used were alfalfa (Medicago sativa) and hairy vetch (Vicia villosa). The results showed that intercropping with green manure increased the total nitrogen, nitrate nitrogen, and available phosphorous in the soil by 48.67~74.67%, 50.00~96.67%, and 44.48~45.04%, respectively. Intercropping with alfalfa significantly increased the activity of soil sucrase 63.75%, and intercropping with hairy vetch increased the activity of β-1,4-glucosidase 44.38% in the soil compared to the monoculture treatment. Intercropping hairy vetch and alfalfa altered the diversity and composition of the soil microbial community and enriched the soil with beneficial fungi and bacteria, including Mortierella and Streptomyces. The richness increased by 58.72% and 17.90% in alfalfa intercropping treatment. In conclusion, intercropping leguminous green manure improved the nutrients and activity of the enzymes in the soil and enriched the antagonistic microbiome in the soil, promoting soil health in the citrus orchard.

1. Introduction

Citrus (Citrus reticulata Blanco) is the most widely cultivated type of fruits in world. However, most of the citrus orchards are grown as a monoculture and lack cover crops, leading to soil erosion, soil degradation, and environmental issues [1,2,3]. Monoculture farming often relies on intensive inputs, such as synthetic fertilizers, to meet the demands of large-scale production. Numerous studies have indicated that excessive use of fertilizers over a long period can lead to soil nutrient imbalances, soil acidification, and reduced soil microbial activity, ultimately degrading soil quality [4,5]. Root exudates released under long-term monoculture increase pathogen accumulation and decrease plant beneficial microorganisms in the rhizosphere, playing a crucial role in soil health [6,7]. Moreover, intensive cultivation with the excessive use of chemical fertilizers has led to deleterious issues, such as a decline in fruit quality, altered fruit flavor, and a decrease in yields [8,9].
Incorporating green manure is a vital aspect of organic and sustainable agricultural practices. Green manure can enhance the ecosystem services in orchards, including the control of soil erosion, a reduction in nutrient leaching, improved sequestration of soil carbon (C) and nutrient availability, increased soil microbial diversity, and the suppression of pests and weeds [10,11,12]. In walnut (Juglans spp.) orchards, intercropping with Chinese violet (Orychophragmus violaceus), hairy vetch (Vicia villosa), and rattail fescue (Vulpia myuros) resulted in an increase of 12.7~24.6% in the content of total organic carbon (TOC) in the soil and increased the levels of β-1,4-glucosidase in the soil by 32.7–88.5% compared to the monoculture [13]. In perennial tree crop production systems, intercropping with green manure has a positive impact on the cycles of C, nitrogen (N), and phosphorous (P), which is an effective strategy that can increase the abundance of the genes related to the soil organic matter (SOM), promote the degradation of plants, and ultimately increase the content of SOM [14].
Soil, as an important ecosystem, contains large numbers of microorganisms, including fungi, protists, viruses, bacteria, and archaea [3,15]. Intercropping with green manure and tilling the intercropping green manure into the soil can enhance the soil nutrients, improve the activities of soil enzymes, and stimulate microbial activity, which leads to both short-term and long-term responses from the microbial population [16,17].
Soil microorganisms are not only involved in nutrient cycling and the transformation of organic matter but also modify the soil habitats through various biochemical and biophysical mechanisms [18]. Soil microbes also help in bioremediation by breaking down pollutants and promoting disease resistance in plants through beneficial microbial interactions [19,20]. Previous studies have found that intercropping changes the composition and function of microbial communities. Such microbially mediated changes to soil properties can have local effects on the composition and distribution of the microbiome, with obvious ecological consequences [21,22]. In the intercropping system of peanut (Arachis hypogaea) and cassava (Manihot esculenta), the ethylene released by cassava increased the abundance of Actinobacteria in the peanut rhizosphere, thus promoting the reassembly of the microbial symbiotic network in peanuts. In turn, this provides more available nutrients for the peanut root system, thus increasing the yields of peanut [23].
Intercropping has long been recognized for its ability to improve the biodiversity and health of soil [24,25,26]. Intercropping with green legume manures can reduce the damage caused by pests and pathogens by enriching the soil with microorganisms that accelerate nutrient cycling and with bacteria and fungi that have disease-resistant functions [27,28]. In the tea (Camellia sinensis)–soybean (Glycine max) intercropping system, the secretion of isoflavones by the soybean roots attracts beneficial bacteria. Soybean intercropping significantly increased the richness of soil bacterial communities relative to monoculture and promoted the growth of Rhodanobacter [28], which is antagonistic to the fungal pathogen Fusarium solani that causes root rot [29]. Compared with the maize (Zea mays) monoculture treatment, in the maize-edible legume intercropping systems, enriched beneficial microbiology species, including Epicoccum dendrobii, Bacillus megaterium and B. pseudofirmus, enhanced the ability of plant defense against pathogens [30]. Under such stressful conditions, legume-based intercropping has been reported to positively increase productivity compared to monoculture cropping systems [31].
Legumes are known for their tripartite symbiosis (arbuscular mycorrhizae (AM)–legume–Rhizobium), and the flavonoids that they secrete promote the colonization of AM in low-input systems [32]. Therefore, legumes with biological functions of N fixation are considered to be the ideal choice for intercropping with other crops [33,34]. Intercropping of green manure crops Medicago sativa and Trifolium repens had varying influence to the soil nutrients and soil water content in an apple orchard in the Loess Plateau. Intercropping of green manure increased the abundance of symbiotic fungi of soil and decreased the abundance of pathogenic fungi and is beneficial to the growth of apple trees [35]. However, in citrus orchards across various regions, the diversification of leguminous manure and tillage practices exert distinct influences on soil characteristics [36]. In citrus orchards of red soil, the response of relative abundance of beneficial microorganisms and pathogens to intercropping with different legumes remains inconclusive [37,38]. Additionally, the soil in southern China is generally acidic and has a profound impact on the cultivation of citrus fruits, for example, nutrient loss, particularly deficiencies of important elements such as calcium and magnesium. In such an environment, the growth of citrus trees is inhibited, making it difficult to improve both yield and quality [39,40]. To address this issue, our research aims to assess the impact of intercropping with annual and perennial leguminous green manures on soil nutrients and soil microbe in comparison to monoculture, providing an evaluation of the potential of leguminous green manures in sustainable citrus management. We hypothesized that intercropping with two leguminous green manures contribute to enhancing soil quality, decreasing pathogenic microorganisms, and recruiting beneficial microorganisms, ultimately fostering improved soil health. We examined the soils from citrus orchards under three different management practices, including citrus monoculture, citrus intercropped with hairy vetch, and citrus intercropping with alfalfa.

2. Materials and Methods

2.1. Experimental Site and Soil Sampling

The experimental site of citrus orchard was in Nanchong City (31°34′ N, 105°58′ E), Sichuan Province, China. In this region, the annual average temperature is 17.5 °C, the average altitude is 483 m, and the average annual precipitation is 800~1000 mm. The orchard was established in 2014 and planted with Citrus reticulata ‘Ai Yuan 38’, with the citrus rows spaced by 3.0 m × 3.0 m. This citrus orchard was a monoculture without any intercropping of green manure before 2021. The dominant soil type is red soil, and the basic soil properties were pH 5.56, SOC of 27.85 g/kg, soil total nitrogen (TN) of 1.50 g/kg, soil-available phosphorous (AP) of 23.18 mg/kg, soil-available potassium (AK) of 7.79 mg/kg, soil ammonia nitrogen (AN) of 2.17 mg/kg, and soil nitrate nitrogen (NN) of 7.79 mg/kg. Three treatments were established in 2021, including citrus monoculture without the intercropping of green manure (Control), citrus and hairy vetch (Vicia villosa R.) intercropping, and citrus and alfalfa (Medicago sativa L.) intercropping. Green manures were sown in a three-meter strip between rows of citrus trees. Vicia villosa was sown at a rate of 45 kg/ha, and Medicago sativa was sown at a rate of 30 kg/ha. Each treatment consisted of four replicate plots, with 12 citrus trees per plot. The two types of green manure were planted in September 2021, intercropping with citrus and covered around the year.

2.2. Soil Samples

The soil samples were collected during the intercropping of green manure in May 2023. In each plot, five soil samples were randomly collected from the inter-row area, 50 cm away from the citrus trees. The sampling depth was 0–15 cm; before sampling, the surface debris was removed, and then the soil was sampled. The five sub-samples were mixed together as one soil sample per plot. The soil samples were transported to the laboratory in a cooler and passed through a 2 mm sieve. The soil was then divided into two subsamples. One portion was stored at −80 °C for microbiological analyses, and the other was stored at 4 °C to analyze the extracellular enzymes within 1 week and determine the soil properties. The soil was left to air dry before being used for the determination of the chemical properties.

2.3. Characterization of the Properties of Chemicals in the Soil

The soil pH was determined based on air-dried samples using a 1:2.5 soil: water ratio (w/v). The contents of TN and TP of the soil were determined by the Kjeldahl and Mo-Sb antispetrophotography methods (DSH-UV755BUV-Vis Spectrophotometer, Guangzhou SH Biological Technology Co., Ltd., Guangzhou, China), respectively [41]. The ammonia nitrogen (NH4+) and nitrate nitrogen (NO3−) in the soil were extracted with 2 M KCl and then measured using a continuous flow analytical system (Seal Auto Analyzer AA3, Norderstedt, Germany). The content of soil-available potassium (AK) was measured using ammonium acetate extraction–flame photometry, and the AK extracts were titrated with a 1:5 ratio of 1 mol L−1 ammonium acetate (NH4OAc) (w/v) [42]. The SOC was determined using the potassium dichromate oil bath digestion method [43]. The soil-available phosphorus (AP) was extracted by 0.03 M ammonium fluoride (NH4F)-0.025 M HCl and then measured by molybdenum-blue colorimetry [44].

2.4. Enzyme Activity Assays

Soil enzyme activity is closely related to soil nutrients and reflects the strength of soil nutrient transformation (especially C, N, P), which characterize soil quality [45]. The activities of β-glucosidase (BG), acid phosphatase (AP) and catalase (CAT) were determined using the fluorometric microplate enzyme assay [46,47]. Soil homogenates were prepared with fresh soil (1 g) mixed with 125 mL of sodium acetate buffer. Then, 200 μL of each homogenate was incubated with 50 μL of the specific substrate at 25 °C. The reaction was terminated by the addition of 5 μL of NaOH, and the enzyme activity was determined by microplate fluorescence (SpectraMax i3x, Molecular Devices, San Jose, CA, USA). Urease (UE) activity was determined using the colorimetric method involving sodium phenol–sodium hypochlorite [48]. Sucrase (SR) activity was determined using the 3,5-dinitrosalicylic acid colorimetric method [49] (for details of the function of each enzyme and the corresponding reaction substrate, see Table S1).

2.5. Microbial DNA Extraction, PCR Amplification, and Illumina NovaSeq Sequencing

Approximately 2 g of soil samples were used for each treatment for the extraction of DNA. The total soil microbial DNA was extracted from the soil samples (0.3 g) using a TGuide S96 Kit (TianGen Biochemical Technology, Beijing, China). Based on the concentration and amplification range, detection and amplification were carried out on the Brilliant Laboratory 1000 (Revvity, Shanghai, China). The bacterial 16 S rRNA gene targeting primer pairs were 515 F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 926R (5′-CCGTCAATTCMTTTRAGTTT-3′), while the fungal internal transcribed spacer (ITS)-targeting primer pairs were ITS2F (5′-GCATCGATGAAGAACGCAGC-3’) and ITS2R (5′-TCCTCCGCTTATTGATATGC-3′). The PCR amplification was performed with a reaction mixture that contained 4 ng of DNA template, 2 μL of dNTPs (2 mM), 5 μL of KOD FX Neo Buffer, 0.3 μL of each primer, and 0.2 μL of KOD FX Neo (Beijing Biolink Biotechnology, Beijing, China), and the reaction mixture was brought to a final volume of 10 µL using ddH2O. PCR cycling was performed for the reaction as follows: initial denaturing at 95 °C for 5 min; annealing during 25 cycles at 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 40 s; and extension at 72 °C for 7 min. The PCR products were checked by 1.8% agarose gel electrophoresis, and the target band was close to 500 bp. The quality-checked DNA were used for subsequent paired-end sequencing using an Illumina No-vaSeq platform (San Diego, CA, USA), and Trimmomatic (version 0.33) was used for quality control and processing of the original sequence. The USEARCH (version 10) was used to assemble paired-end reads and eliminate chimeras (UCHIME, version 8.1). The final dataset for the 16S rRNA gene and ITS region consisted of an average of 73,342 and 66,915 sequences per sample, respectively. USEARCH (version 10.0) was employed to cluster sequences at a 97% similarity level, utilizing 0.005% of the total number of sequences sequenced as the threshold to filter OTUs. Based on Naive Bayes classifiers, the OTUs were then assigned to taxonomic groupings based on comparisons with Unite database version 8.0 (only fungi remained) and Silva database version 138 (only bacteria and archaea remained). The confidence level of the classifiers was 0.7.

2.6. Statistical Analysis

A one-way analysis of variance (ANOVA) was used to compare the significant differences between green manure intercropping on the soil properties, the abundance of microbial functional genus, the functional Shannon diversity, the enzyme activities, and the multifunctionality resistance, which were implemented in SPSS 26.0 (IBM, Inc., Armonk, NY, USA). A principal coordinate analysis (PCoA) of the functional genes abundance was conducted based on the Bray–Curtis distance, and the results were visualized using the “vegan” package in R 4.4.1. The pivotal predictors of soil multifunctionality resistance among the different factors were evaluated using a random forest analysis, which was performed using the “random Forest” package in R 4.4.1. A structural equation model (SEM) was constructed to evaluate the direct and indirect effects of a range of soil factors on multifunctionality resistance using AMOS 21.0 (IBM, Inc., Armonk, NY, USA). The following parameters were used to assess the model fitness: the root mean squared error of approximation (RMSEA) < 0.05, a lower chi-square value (χ2), a Fisher’s p value of 0.05 < p ≤ 1, and a lower Akaike information criterion (AIC).

3. Results

3.1. Soil Nutrients and Properties

Intercropping green manure in the citrus orchards had significant effects on the soil pH, TN, AN, and AP after 3 years of cultivation (p < 0.05), while the contents of soil TP, NN, and AK were similar between the intercropping green manure and the control. There were significantly higher contents of soil pH, TN, AN, and AP (p < 0.05) in the intercropped hairy vetch and alfalfa. The soil pH and the contents of TN, and AP of the intercropped hairy vetch were higher than those of the other treatments, and the soil pH, TN, and AP increased by 16.00%, 74.67%, and 45.04%, respectively, (p < 0.05) compared with the control. There were higher contents of AN in the soil of intercropped alfalfa than those in the other treatments, and its contents increased by 90.32% (p < 0.05) compared to the control (Table 1).

3.2. Activities of Soil Enzymes

The effects of different green manure treatments on the activities of the soil enzymes during the experimental period are summarized in Figure 1. Intercropping with green manure had no significant effect on the activities of urease, catalase and AcP. However, intercropping with hairy vetch significantly increased the activity of β-1,4-glucosidase by 44.38% compared to the control (p < 0.05). In contrast, intercropping with alfalfa significantly reduced the activity of β-1,4-glucosidase by 86.99% compared to the control (p < 0.05). Compared to the control, intercropping with alfalfa significantly increased the activity of soil sucrase 63.75% (p < 0.05). There was no significant difference in the activities of urease, catalase and AcP between the soil from the green manure intercropping and the control.

3.3. Soil Microbial Diversity and Communities Under Long-Term Intercropping of Different Green Manures

The operational taxonomic units (OTUs) used to classify the species in each sample after clustering were distributed between 5141 and 5748. There were 2663, 3330, and 2706 specific OTUs contained in the control, hairy vetch intercropping, and alfalfa intercropping, respectively. A total of 1247 OTUs were common to the three treatments. Among the three treatments, the hairy vetch intercropping treatment contained the highest number of OTUs at 5748. In contrast, the alfalfa intercropping treatment contained the fewest OTUs at 5141 (Figure 2).
The rarefaction curve of each sample tended to flatten out, and the rarefaction curve analysis exhibited a high gene sequencing depth and a substantial possibility of observing the community diversity (Figure S1). The application of green manure intercropping did not result in a significant impact on the abundance of soil fungi as indicated by the Chao1, ACE, and Simpson indices in comparison to the control. The intercropping of hairy vetch and alfalfa was found to result in a reduction of 10.48% and 13.60% in the richness of soil fungi, respectively, compared to the control. The intercropping of alfalfa significantly reduced the soil fungal Shannon index, whereas the intercropping of hairy vetch had no significant effect on this index (Table 2).
The results of the study indicated that intercropping with hairy vetch increased the soil bacterial richness and the Chao1, ACE, and Shannon indices. In contrast, intercropping with alfalfa decreased the soil bacterial richness and the Chao1, ACE, and Shannon indices compared to the control. Green manure intercropping had no significant effect on the soil bacterial richness and the Chao1, ACE, Shannon, and Simpson indices (p > 0.05).
The variation in the soil bacterial and fungal community structure from the different treatments was investigated using a PCoA (Figure 3). The results of the PCoA of the fungal communities indicated a significant separation of fungal communities on the PC1 axis under the three treatments. PERMANOVA also confirmed that the different types of green manure intercropping had a significant effect on the soil fungal communities (R2 = 0.4905, p = 0.001). The PERMANOVA results for the bacteria demonstrated significant differences in their communities under the three treatments (R2 = 0.2773, p = 0.006). The PCoA analysis revealed that the alfalfa intercropping was highly clustered in comparison to the control, whereas the hairy vetch intercropping was significantly segregated from the other two treatments on the PC1 axis.

3.4. Soil Microbial Community Composition Under the Long-Term Intercropping of Different Types of Green Manures

The impact of different orchard management techniques on the composition of soil microbial communities is shown in Figure 4. A total of 13 phyla, 49 classes, 484 genera, and 612 species of soil fungi were identified. The most prevalent phyla across all three treatments were Ascomycota (62.4%), Basidiomycota (11.4%), Chytridiomycota (1.7%), and Mortierellomycota (1.6%) (Figure 4A). The results of the ANOVA indicated that there were significant differences in the relative abundances of the Basidiomycota (p < 0.001), Mortierellomycota (p = 0.020), Rozellomycota (p = 0.015), and Glomeromycota (p = 0.039) under the three treatments (Table S2). There was a significantly higher relative abundance of the Basidiomycota, Rozellomycota, and Glomeromycota under the hairy vetch intercropping treatment compared to the control (p < 0.05); there was also a significantly higher relative abundance of Mortierellomycota under the alfalfa intercropping treatment compared to the control (Figure S2).
At the fungal genus level, the control and alfalfa intercropping treatments were grouped together and separated from the hairy vetch intercropping. This suggests that the community compositions of the dominant fungal genera were more similar between the control and alfalfa intercropping treatments than between these and the hairy vetch intercropping (Figure 4B). The results of the ANOVA indicated that the relative abundance of the three dominant fungal genera Cladosporium (p < 0.001), Fusarium (p = 0.043), and Alternaria (p = 0.001) differed significantly among the three treatments (Table S3).
Intercropping of the two green manures significantly decreased the relative abundance of Alternaria in comparison to the control. The alfalfa intercropping treatment was observed to significantly decrease the relative abundance of Cladosporium, while the hairy vetch intercropping treatment was found to significantly decrease the relative abundance of Fusarium in comparison to the alfalfa intercropping treatment.
Additionally, the hairy vetch intercropping treatment resulted in a reduction in the relative abundance of Fusarium in comparison to the control. Both green manure intercropping treatments demonstrated a reduction in the relative abundance of Alternaria in comparison to the control (Figure S3).
A total of 42 phyla, 89 classes, 879 genera, and 1063 species of soil bacteria were identified. In the three treatments, the most prevalent bacterial phyla were Proteobacteria (24.8%), Acidobacteriota (13.5%), Actinobacteriota (10.6%), and Bacteroidota (9.7%) (Figure 4C). The results of the ANOVA indicated that among the nine most abundant bacterial phyla, there were significant differences in the relative abundances of Chloroflexi (p = 0.043) and Gemmatimonadota (p = 0.017) among the three different treatments (Table S4).
There was a significantly lower relative abundance of Gemmatimonadota under hairy vetch intercropping than in the other two treatments (Figure S4). At the genus level, we summarized the response of the top 20 most abundant bacterial genera to the cropping systems, and the ANOVA showed that UTCFX1, Pirellula, Dongia, and Terrimonas differed significantly among the varying management practices. There were significantly higher relative abundances of UTCFX1 (p = 0.039), Pirellula (p = 0.021), Dongia (p = 0.040), and Terrimonas (p = 0.046) under the hairy vetch intercropping than in the other two treatments (Table S5). The results of the cluster analysis indicated that there was a similar composition of the major bacterial genus between the alfalfa intercrop and the control (Figure 4D). The relative abundance of Pirellula was found to be significantly higher than the control, while the relative abundances of UTCFX1, Dongia and Terrimonas were significantly higher than the alfalfa intercropping under the hairy vetch intercropping (Figure S5).

3.5. Comprehensive Analysis of the Soil Properties and Microbial Diversity

A correlation analysis revealed that there was a significant positive correlation between the soil TN and soil pH (p < 0.05), between soil TP and soil SOC (p < 0.05), and between soil AN and soil sucrase (p < 0.01). The Mantel test indicated that the soil AN, NN, AK, AP, and β-1,4-glucosidase all had a positive effect on the diversity of soil fungi and bacteria. The content of soil AN and activity of β-1,4-glucosidase significantly positively affected the soil fungal diversity (p < 0.05), while the soil NN significantly positively affected the soil bacterial diversity (p < 0.05) (Figure 5).

3.6. Difference Analysis of the Soil Microbial Communities Under the Different Treatments

Based on the linear discriminant analysis effect size (LEfSe), the linear discriminant analysis (LDA) score histogram (with a cutoff of ±3.5) was used to display the microbial taxonomic units with significantly different abundances between the groups. The results of the cladogram analysis of LEfSe showed that there were significant differences in the abundances of multiple taxonomic units between the two green manures intercropping and the control (Figure S6).
At the family level, there were differences in the abundances of 12 types of fungi, and at the genus level, there were differences in the abundance of 11 types of fungi (Figure 6A). Further analysis found that at the genus level, Mortierella, Gibberella, Ramularia, and Fusarium were highly enriched under the intercropping treatment of alfalfa, while Sarocladium, Botrytis, Filobasidium, Sporidiobolus, Plectosphaerella, Gibellulopsis, and Cladosporium were highly enriched under the intercropping treatment of hairy vetch (Figure 6B).
The results of the cladogram analysis of LEfSe showed that there were significant differences in the abundances of multiple bacterial taxonomic units between the two types of green manure intercropping and the control (Figure S7). At the family level, there were differences in the abundances of 12 types of bacteria, and at the genus level, there were differences in the abundances of 11 genera of bacteria (Figure 6C). An additional analysis revealed that at the genus level, Truepera, Pedobacter, and Lysobacter were highly enriched under the intercropping treatment of alfalfa, while UTCFX1 and Streptomyces were highly enriched under the intercropping treatment of hairy vetch. In particular, compared with the other two treatments, the monoculture treatment (control) significantly increased the abundance of Ulvibacter (Figure 6D).

3.7. RDA of the Beneficial Microbial and the Spearman’s Correlation Analysis

A redundancy analysis (RDA) was used to test the correlation between the eight selected environmental variables, five soil enzymes, and the beneficial microbes under the different types of green manure intercropping. The RDA was performed on the genus data, and the two effective fungal genera in the samples were Mortierella and Sarocladium. The three effective bacterial genera in the samples were UTCFX1, Lysobacter, and Streptomyces. The soil biochemical properties and microbial analysis showed that the first two RDA components explained 76.08% of the total variation, and the soil NN, pH, and AK were the most effective soil indicators (Figure 7A). The soil enzymes and microbial analysis showed that the first two RDA components explained 65.28% of the total variation, and the soil BG and AcP were the most effective indicators of soil enzymes (Figure 7B).
As shown in Table S6, the relative abundance of Mortierella positively correlated with most of the soil properties, including the soil SOC, TP, AN, NN, AK, and AP. However, it significantly negatively correlated with the activity of BG (p = 0.007). Sarocladium significantly positively correlated with the soil TN (p = 0.024). The relative abundance of Lysobacter negatively correlated with most of the soil properties and soil enzymes, and Lysobacter significantly negatively correlated with the soil pH (p = 0.014) and soil AcP (p = 0.039). Streptomyces significantly negatively correlated with the NN (p = 0.007). The soil BG had a significantly positive effect on the abundances of UTCFX1 and Streptomyces (p < 0.05).

4. Discussion

4.1. Responses of the Soil Nutrients, Properties and Enzyme Activities to Green Manure Intercropping

Legumes are widely distributed in natural ecosystems and are considered to be the key species that promote the efficiency of ecosystems [50,51,52]. In intercropping systems, when the soil N is limited, owing to low availability or increased N competition due to intercropping, legumes have been proven to be forced to fix the atmospheric N instead of competing with the crop for inorganic N the soil [53]. This study showed that the soil TN and AN were significantly higher in the citrus–legume intercropping system. In fact, legume intercropping and soil tillage can provide a wider range of additional services, including increased soil SOC and mineral nutrients [54,55]. In this study, the soil AP was significantly higher under legume intercropping. The ability of legume intercropping systems to enhance the acquisition of P is well known [56].
Plants have been shown to be able to receive environmental stimuli and modify the distribution of their roots depending on the availability of nutrient and competition with the root systems of other species [57]. The increase in root length density and surface area under intercropping systems directly improves exploration of the soil, which thereby enhances the acquisition of P in the soil [58,59]. In addition, it has been generally accepted that intercropping, as a form of species diversity, can increase the activities of soil enzymes, which is a critical function in ecosystem productivity. In this study, the improvements in the activity of acid phosphatase and sucrase were observed under the intercropping treatment. This finding is consistent with previous studies [5,60]. However, the activities of urease, catalase, and β-1,4-glucosidase were decreased under the intercropping patterns, which is inconsistent with the findings of previous studies, possibly owing to the differences in the intercropping system, sampling stage, and the history of cultivation. A correlation analysis also showed that the surcease significantly positively correlated with the soil AN, which indicated that the higher activities of sucrase in the soil corresponded to an increase in the accumulation of available N, which was likely owing to their positive feedback [61].

4.2. Responses of the Microbial Community Diversity to Green Manure Intercropping

The application of green manure to the soil is considered a valuable practice of orchard management in organic agriculture. Past findings have generally suggested positive effects of cover crops on the microbial properties in the soil [62,63], This study showed that the fungal alpha-diversity decreased and bacteria alpha-diversity increased under the intercropping with hairy vetch. These findings suggest that the intercropping with hairy vetch induces alterations in the structure of soil microbial communities. One possible reason for this result could be that significant increase in soil available N and P content and soil bacteria are more sensitive than fungi in response to N and P enrichment [18,64].
In addition, we found that the hairy vetch intercropping increased the soil bacterial richness and that the alfalfa intercropping decreased the soil bacterial richness. One possible reason for this finding could be explained by the stronger root systems of alfalfa compared to hairy vetch, which may enhance the root selection process and cause fewer bacteria to survive in the rhizosphere [35]. Moreover, significant positive effects of AN and BG were found on the fungal alpha-diversity, and significant positive effects of NN were found on the bacterial alpha-diversity. This indicates that the improvements in the utilization of N sources and metabolic activity help to increase the microbial diversity for intercropping systems. In the legume intercropping patterns, N is transferred from the atmospheric N to the soil by the rhizosphere, which ultimately drives symbiotic N fixation and differences in the microbial community [65].

4.3. Responses of the Soil Fungal Community Composition to Green Manure Intercropping

The composition of soil fungi is shaped by various factors, including growth status and environmental conditions [66,67]. In this study, among the top 10 phyla in terms of relative abundance, Ascomycota was the most abundant in all the cropping systems studied (Figure 4A), which is consistent with the results of previous studies [68]. We found that the Basidiomycota, Mortierellomycota, Rozellomycota, and Glomeromycota significantly increased under intercropping treatments. The relative abundance of the Basidiomycota significantly increased under hairy vetch intercropping, and members of the Basidiomycota have been shown to play a role in the degradation of lignocellulose throughout composting, and their high abundance is beneficial for the degradation of organic matter [69]. Mortierellomycota comprised the core microbes that consumed organic fragments and degraded lignocellulose and mineral P [60,70]. Glomeromycota can colonize the roots of most terrestrial plants, including citrus, and establish a symbiotic relationship [70]. It has been proven that mycorrhizal symbiosis can promote the growth of plants and their uptake of nutrients, as well as enhance their tolerance to stress [71].
Among the top 20 fungal genera in terms of relative abundance, the relative abundance of Fusarium decreased under hairy vetch intercropping, and the relative abundance of Alternaria significantly decreased under green manure intercropping. Alternaria and Fusarium are cosmopolitan fungal genera, and species of Fusarium are important plant pathogens [72]. Contamination with species of Alternaria and Fusarium are responsible for some of the world’s most devastating plant diseases, including citrus, and they can seriously reduce crop yields and cause considerable economic losses [72,73]. This study showed that the intercropping of different types of leguminous green manures displayed the microbial taxonomic units with significantly different abundances between groups. In particular, intercropping with alfalfa increased the abundances of Mortierella, Gibberella, and Penicillium compared to the two other treatments. Mortierella positively correlated with most of the soil properties. This suggests that the responses of the microbial compositions to intercropping crops depend on the soil properties. Intercropping with hairy vetch increased the abundances of Sarocladium, Filobasidium, and Sporidiobolus compared with the other two treatments. Previous studies showed that Mortierella was found widely in various environments, and some strains of this genus were considered to be plant-growth-promoting fungi [74]. The genus Sarocladium has been reported to be an antagonist of Fusarium pathogens [75]. Sarocladium significantly positively correlated with the soil TN. The abundances of the Sarocladium were influenced by the soil N dynamics as a result of the degradation of hairy vetch residues. Cunninghamella elegans efficiently biotransformed fluorotelomer alcohols and has potential for the remediation of environmental pollutants [76,77]. These results may be owing to the differences in changes in the soil properties by the intercropping of green manure, as our results showed that the relative abundance of Mortierella positively correlated with most of the soil properties, including the soil SOC, TP, AN, NN, AK, and AP, while it significantly negatively correlated with the activity of BG.

4.4. Responses of the Soil Bacterial Community Composition to Green Manure Intercropping

The dominant phyla under patterns of intercropping were basically composed of Proteobacteria, Acidobacteriota, Actinobacteriota, and Bacteroidota, which is consistent with the findings of the global citrus rhizosphere microbiome [78]. In contrast to the fungi, these dominant phyla were not particularly responsive to environmentally induced changes in intercropping conditions. An additional analysis revealed that at the genus level, Truepera, Pedobacter, and Lysobacter were highly enriched under the intercropping treatment of alfalfa, while UTCFX1 and Streptomyces were highly enriched under the intercropping treatment of hairy vetch. The genus Lysobacter plays an important role in the rhizosphere microbial community, including the improvement of soil physical and chemical properties and its antifungal effects. Lysobacter can promote plant growth and improve the soil properties by producing compounds, including indole-3-acetic acid and chitinase [76]. In addition, Lysobacter is a promising source of biocontrol agents of plant diseases. It can primarily control plant pathogenic bacteria, fungi, nematodes, oomycetes, and protists by relying on multiple mechanisms, such as the competition for space, predation, induction of plant resistance mechanisms, and the production of antibiotics, lytic enzymes and toxic compounds [79,80,81]. Lysobacter can produce various extracellular enzymes, such as proteases, chitinases, and β-1,3-glucanases, and it also produces various secondary metabolites, such as cyclic lipopeptides (CLP) and polycyclic tetramate macrolactams (PTMs), which have antibiotic activity [76].
Soil texture and pH are some environmental factors that determine the abundance of species of Lysobacter in agricultural soils [76]. In this study, Lysobacter significantly negatively correlated with the soil pH (p = 0.014) and soil AcP (p = 0.039). This is not consistent with Postma et al., who found that Lysobacter members were commonly isolated from soils with high pH values ranging from 6 to 8.8 [82]. The higher relative abundances of UTCFX1, Pirellula, Dongia, and Terrimonas were observed under green manure intercropping. The abundance of UTCFX1 increases under conditions of low dissolved oxygen, and it also plays a crucial role in N metabolism. They encode and transcribe genes related to N metabolism and are involved in nitrate and nitrite respiration [83,84]. In our study, intercropping with hairy vetch increased the relative abundance of Streptomyces, and it significantly negatively correlated with the NN. This may have been due to the soil properties shaped by hairy vetch, which are suitable for the survival of Streptomyces [85,86,87,88]. The genus Streptomyces, an actinomycete, is a natural source of diverse metabolites that have numerous clinical, agricultural, and biotechnological applications. Streptomyces have multiple functions, and some include producing antibiotics and phytohormones, promoting plant growth, and enhancing the resistance of plants to biotic and abiotic stresses [86]. For example, it significantly improved the growth of the tomato plants and decreased diseases caused by Fusarium oxysporum in tomatoes (Solanum lycopersicum) [86]. The mechanism including Streptomyces’s capacity of intensive production of antibiotics, the biosynthesis of plant growth regulators [87], and other metabolites and competition for nutrients help in its role as an antagonist of pathogens [88]. The metabolites of Streptomyces can be antibacterial, antifungal, antitumor, anthelmintic, insecticidal, and cytotoxic [85]. Streptomyces JD211 has been shown to induce systemic resistance in rice (Oryza sativa), and its activities of catalase, phenylalanine ammonia-lyase (PAL), and β-1,3-glucanase significantly increased along with pathogenesis-related protein 1 [89]. In this study, the soil BG significantly positively correlated with Streptomyces. The variation in the properties of Streptomyces was stimulated by higher enzyme activities and well adapted to the soil, owing to its physiological metabolism [90]. In particular, the differential responses of the composition of the beneficial soil community to the intercropping patterns were largely dependent on environmental changes, soil nutrient parameters and soil enzyme activities. Therefore, these results suggest that the legume-based intercropping systems can also promote diversity of the rhizobacterial community, soil health, and agroecosystem functions by enhancing symbiotic and non-symbiotic beneficial populations [91].
Previous studies have indicated that organic fertilization supports a disease-suppressive and, consequently, plant-health-increasing soil microbiome [92,93]. A commonality in orchard management practices is the need to mitigate the threat of soil-borne pathogens that can significantly impact citrus growth [92,94]. Sustainable orchard management measures focus on enhancing the soil nutrients and improve beneficial microbes in the soil and their ecological interactions, thereby avoiding the adverse side effects on the environment and human health caused by monoculture [95,96]. Our study demonstrated intercropping with legume green manure has potential for sustainable management of citrus orchard in acidic soils.

5. Conclusions

In this study, citrus intercropping with the green manure crops hairy vetch and alfalfa significantly increased the total nitrogen, nitrate nitrogen, and available phosphorous in the soil. Intercropping with alfalfa and hairy vetch significantly increased the activity of soil sucrose and β-1,4-glucosidase, respectively. Intercropping green manure changed the diversity and composition of the soil microbial community, for example, and enriched the soil with beneficial fungi and bacteria, including Mortierella and Streptomyces. Green manure significantly decreased the relative abundance of fungal pathogens such as Alternaria, Cladosporium, and Fusarium, while it increased the bacteria of Pirellula. We also found that soil AN, NN, AK, AP, and β-1,4-glucosidase all had a positive effect on the diversity of soil fungi and bacteria. Our findings highlight the distinct responses of the bacterial and fungal communities to green manure intercropping and further illuminate the potential of use green manure for sustainable citrus management.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture14111897/s1, Table S1. Detailed description of soil extracellular enzymes. Table S2. Analysis of variance (ANOVA) of the top 10 fungi at the fungal phylums level under three treatments. Table S3. Analysis of variance (ANOVA) of the top 20 fungi at the fungal genus level under three treatments. Table S4. Analysis of variance (ANOVA) of the top 10 Bacteria at the bacterial phylums level under three treatments. Table S5. Analysis of variance (ANOVA) of the top 20 Bacteria at the bacterial genus level under three treatments. Table S6. Spearman correlation analysis of five beneficial microorganisms in relation to soil physico-chemical and soil enzymes. Figure S1. Rarefaction curves of all samples. Fungi (A) and bacteria (B). Figure S2. Relative abundance of fungal phyla that differed significantly under the three treatments. * and ** indicate the significance level at p < 0.05 and p < 0.01 p < 0.001. Figure S3. Relative abundance of fungal genera that differed significantly under the three treatments. * and ** indicate the significance level at p < 0.05 and p < 0.01. Figure S4. Relative abundance of bacterial phyla that differed significantly under the three treatments. * and ** indicate the significance level at p < 0.05 and p < 0.01. Figure S5. Relative abundance of bacterial genera that differed significantly under the three treatments. * and ** indicate the significance level at p < 0.05 and p < 0.01. Figure S6. Plot cladogram at different fungal taxonomic levels among different groups. Figure S7. Plot cladogram at different fungal taxonomic levels among different groups.

Author Contributions

Conceptualization, methodology, writing—reviewing and editing, Y.X.; writing—original draft, Y.X.; software and formal analysis, Y.X. and Y.W.; funding acquisition, writing—reviewing and editing, T.D. and Y.X.; investigation and data curation, Y.J., R.Z. and Q.X.; project administration, Z.S. and T.D.; supervision, T.D.; visualization, Y.X., Y.W. and R.Z.; resources and software, Y.W. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the China Modern Agriculture Research System (CARS-22 Green Manure).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Lei, J.C.; Wang, S.; Wang, Y.D.; Luo, W. Determining the planting year of navel orange trees in mountainous and hilly areas of southern China: A remote sensing based method. J. Mt. Sci. 2024, 21, 1–13. [Google Scholar] [CrossRef]
  2. Guan, G.; Zhang, S.; He, T.Y.; Guo, F.P.; Zhu, J. Sod culture treatments positively affect soil fungal diversity, soil enzyme activities, and nutrient uptake in navel orange orchards. Soil Sci. Plant Nutr. 2024, 24, 5130–5143. [Google Scholar] [CrossRef]
  3. Castellano-Hinojosa, A.; Kanissery, R.; Strauss, S.L. Cover crops in citrus orchards impact soil nutrient cycling and the soil microbiome after three years but effects are site-specific. Biol. Fert. Soils 2023, 59, 659–678. [Google Scholar] [CrossRef]
  4. Zhang, Y.J.; Ye, C.; Su, Y.W.; Peng, W.C.; Lu, R.; Liu, Y.X.; Huang, H.C.; He, X.H.; Yang, M.; Zhu, S.S. Soil acidification caused by excessive application of nitrogen fertilizer aggravates soil-borne diseases: Evidence from literature review and field trials. Agric. Ecosyst. Environ. 2022, 340, 108176. [Google Scholar] [CrossRef]
  5. Liu, B.; Xia, H.; Jiang, C.C.; Riaz, M.; Yang, L.; Chen, Y.F.; Fan, X.P.; Xia, X.E. 14 years applications of chemical fertilizers and crop straw effects on soil labile organic carbon fractions, enzyme activities and microbial community in rice-wheat rotation of middle China. Sci. Total Environ. 2022, 841, 156608. [Google Scholar] [CrossRef]
  6. An, S.H.; Wei, Y.L.; Li, H.M.; Zhao, Z.J.; Hu, J.D.; Philp, J.; Ryder, M.; Toh, R.; Li, J.S.; Zhou, Y.; et al. Long-term monocultures of american ginseng change the rhizosphere microbiome by reducing phenolic acids in soil. Agriculture 2022, 12, 640. [Google Scholar] [CrossRef]
  7. Zhao, X.; Liu, Y.; Yue, L.; Tian, Y.; Zhou, Q.; Wang, Y.; Liu, S.F.; Guo, Z.H.; Zhang, Y.B.; Wang, R.Y. Continuous monoculture alters the fungal community and accumulates potential pathogenic strains in the rhizosphere of Codonopsis pilosula. Phytobiomes J. 2024, 8, 340–349. [Google Scholar] [CrossRef]
  8. Liu, L.Y.; Zheng, X.Q.; Wei, X.C.; Kai, Z.; Xu, Y. Excessive application of chemical fertilizer and organophosphorus pesticides induced total phosphorus loss from planting causing surface water eutrophication. Sci. Rep. 2021, 11, 23015. [Google Scholar] [CrossRef]
  9. Zheng, X.Q.; Wei, L.; Lv, W.G.; Zhang, H.Q.; Zhang, Y.; Zhang, H.Y.; Zhang, H.L.; Zhu, Z.K.; Ge, T.D.; Zhang, W.J. Long-term bioorganic and organic fertilization improved soil quality and multifunctionality under continuous cropping in watermelon. Agric. Ecosyst. Environ. 2024, 359, 108721. [Google Scholar] [CrossRef]
  10. Adetunji, A.T.; Ncube, B.; Mulidzi, R.; Lewu, F.B. Management impact and benefit of cover crops on soil quality: A review. Soil Tillage Res. 2020, 204, 104717. [Google Scholar] [CrossRef]
  11. Kim, N.; Zabaloy, M.C.; Guan, K.Y.; Villamil, M.B. Do cover crops benefit soil microbiome? a meta-analysis of current research. Soil Biol. Biochem. 2020, 142, 107701. [Google Scholar] [CrossRef]
  12. Wang, T.; Duan, Y.; Liu, G.D.; Shang, X.W.; Liu, L.F.; Zhang, K.X.; Li, J.Q.; Zou, Z.W.; Zhu, X.J.; Fang, W.P. Tea plantation intercropping green manure enhances soil functional microbial abundance and multifunctionality resistance to drying-rewetting cycles. Sci. Total Environ. 2022, 810, 151282. [Google Scholar] [CrossRef] [PubMed]
  13. Dong, N.G.; Hu, G.L.; Zhang, Y.Q.; Qi, J.X.; Chen, Y.H.; Hao, Y.B. Effects of green-manure and tillage management on soil microbial community composition, nutrients and tree growth in a walnut orchard. Sci. Rep. 2021, 11, 16882. [Google Scholar] [CrossRef] [PubMed]
  14. Castellano-Hinojosa, A.; Strauss, S.L. Impact of cover crops on the soil microbiome of tree crops. Microorganisms 2020, 8, 328. [Google Scholar] [CrossRef]
  15. Fierer, N. Embracing the unknown: Disentangling the complexities of the soil microbiome. Nat. Rev. Microbiol. 2017, 15, 579–590. [Google Scholar] [CrossRef]
  16. Mbuthia, L.W.; Acosta-Martínez, V.; DeBruyn, J.; Schaeffer, S.; Tyler, D.; Odoi, E.; Mpheshea, M.; Walker, F.; Eash, N. Long term tillage, cover crop, and fertilization effects on microbial community structure, activity: Implications for soil quality. Soil Biol. Biochem. 2015, 89, 24–34. [Google Scholar] [CrossRef]
  17. Rao, G.D.; Sui, J.K.; Zhang, J.G. Metabolomics reveals significant variations in metabolites and correlations regarding the maturation of walnuts (Juglans regia L.). Biol. Open 2016, 5, 829–836. [Google Scholar] [CrossRef]
  18. Zhou, Z.H.; Wang, C.K.; Luo, Y.Q. Meta-analysis of the impacts of global change factors on soil microbial diversity and functionality. Nat. Commun. 2020, 11, 3072. [Google Scholar] [CrossRef]
  19. Lopes, M.J.D.; Dias, M.B.; Gurgel, E.S.C. Successful plant growth-promoting microbes: Inoculation methods and abiotic factors. Front. Sustain. Food Syst. 2021, 5, 606454. [Google Scholar] [CrossRef]
  20. Khan, M.F.; Chowdhary, S.; Koksch, B.; Murphy, C.D. Biodegradation of amphipathic fluorinated peptides reveals a new bacterial defluorinating activity and a new source of natural organofluorine compounds. Environ. Sci. Technol. 2023, 57, 9762–9772. [Google Scholar] [CrossRef]
  21. Banerjee, S.; van der Heijden, M.G.A. Soil microbiomes and one health. Nat. Rev. Microbiol. 2022, 21, 6–20. [Google Scholar] [CrossRef] [PubMed]
  22. Philippot, L.; Chenu, C.; Kappler, A.; Rillig, M.C.; Fierer, N. The interplay between microbial communities and soil properties. Nat. Rev. Microbiol. 2024, 22, 226–239. [Google Scholar] [CrossRef] [PubMed]
  23. Chen, Y.; Bonkowski, M.; Shen, Y.; Griffiths, B.S.; Jiang, Y.J..; Wang, X.Y.; Sun, B. Root ethylene mediates rhizosphere microbial community reconstruction when chemically detecting cyanide produced by neighbouring plants. Microbiome 2020, 8, 4. [Google Scholar] [CrossRef] [PubMed]
  24. Li, X.F.; Wang, Z.G.; Bao, X.G.; Sun, J.H.; Yang, S.C.; Wang, P.; Wang, C.B.; Wu, J.P.; Liu, X.R.; Tian, X.L.; et al. Long-term increased grain yield and soil fertility from intercropping. Nat. Sustain. 2021, 4, 943–950. [Google Scholar] [CrossRef]
  25. Tilman, D. Benefits of intensive agricultural intercropping. Nat. Plants 2020, 6, 604–605. [Google Scholar] [CrossRef]
  26. Zhu, Y.Y.; Chen, H.R.; Fan, J.H.; Wang, Y.Y.; Li, Y.; Chen, J.B.; Fan, J.X.; Yang, S.S.; Hu, L.P.; Leung, H.; et al. Genetic diversity and disease control in rice. Nature 2000, 406, 718–722. [Google Scholar] [CrossRef]
  27. Wang, C.X.; Liang, Q.; Liu, J.N.; Zhou, R.; Lang, X.Y.; Xu, S.Y.; Li, X.C.; Gong, A.D.; Mu, Y.T.; Fang, H.C.; et al. Impact of intercropping grass on the soil rhizosphere microbial community and soil ecosystem function in a walnut orchard. Front. Microbiol. 2023, 14, 1137590. [Google Scholar] [CrossRef]
  28. Zhong, Y.J.; Liang, L.N.; Xu, R.N.; Xu, H.Y.; Sun, L.L.; Liao, H. Intercropping tea plantations with soybean and rapeseed enhances nitrogen fixation through shifts in soil microbial communities. Front. Agric. Sci. Eng. 2022, 9, 344–355. [Google Scholar] [CrossRef]
  29. Huo, Y.; Kang, J.P.; Park, J.K.; Li, J.; Chen, L.; Yang, D.C. Rhodanobacter ginsengiterrae sp. nov., an antagonistic bacterium against root rot fungal pathogen Fusarium solani, isolated from ginseng rhizospheric soil. Arch. Microbiol. 2018, 200, 1457–1463. [Google Scholar] [CrossRef]
  30. Jalloh, A.A.; Mutyambai, D.M.; Yusuf, A.A.; Subramanian, S.; Khamis, F. Maize edible-legumes intercropping systems for enhancing agrobiodiversity and belowground ecosystem services. Sci. Rep. 2024, 14, 14355. [Google Scholar] [CrossRef]
  31. Bargaz, A.; Isaac, M.E.; Jensen, E.S.; Carlsson, G. Nodulation and root growth increase in lower soil layers of water-limited faba bean intercropped with wheat. J. Plant Nutr. Soil Sci. 2016, 179, 537–546. [Google Scholar] [CrossRef]
  32. Siqueira, J.O.; Safir, G.R.; Nair, M.G. Stimulation of vesicular-arbuscular mycorrhiza formation and growth of white clover by flavonoid compounds. New Phytol. 1991, 118, 87–93. [Google Scholar] [CrossRef]
  33. Alam, M.Z.; Lynch, D.H.; Sharifi, M.; Burton, D.L.; Hammermeister, A.M. The effect of green manure and organic amendments on potato yield, nitrogen uptake and soil mineral nitrogen. Biol. Agric. Hortic. 2016, 32, 221–236. [Google Scholar] [CrossRef]
  34. Murugan, R.; Kumar, S. Influence of long-term fertilization and crop rotation on changes in fungal and bacterial residues in a tropical rice-field soil. Biol. Fertil. Soils 2013, 49, 847–856. [Google Scholar] [CrossRef]
  35. Ding, T.T.; Yan, Z.C.; Zhang, W.Z.; Duan, T.Y. Green manure crops affected soil chemical properties and fungal diversity and community of apple orchard in the loess plateau of China. J. Soil Sci. Plant Nutr. 2021, 21, 1089–1102. [Google Scholar] [CrossRef]
  36. Zhang, G.L.; Bai, J.H.; Zhai, Y.J.; Jia, J.; Zhao, Q.Q.; Wang, W.; Hu, X.Y. Microbial diversity and functions in saline soils: A review from a biogeochemical perspective. J. Adv. Res. 2024, 59, 129–140. [Google Scholar] [CrossRef]
  37. Castellano-Hinojosa, A.; Maltais-Landry, G.; Martens-Habbena, W.; Strauss, S.L. Depth-dependent effects of cover crops in citrus orchards on soil carbon and nitrogen cycling, greenhouse gas emissions, and soil microbial communities. Appl. Soil Ecol. 2023, 192, 105071. [Google Scholar] [CrossRef]
  38. Bazany, K.E.; Delgado-Baquerizo, M.; Thompson, A.; Wang, J.T.; Otto, K.; Adair, R.C.J.; Borch, T.; Leach, J.E.; Trivedi, P. Management-induced shifts in rhizosphere bacterial communities contribute to the control of pathogen causing citrus greening disease. J. Sustain. Agric. Environ. 2022, 1, 275–286. [Google Scholar] [CrossRef]
  39. Gao, Z.C.; Xu, Q.X.; Si, Q.; Zhang, S.P.; Fu, Z.Y.; Chen, H.S. Effects of different straw mulch rates on the runoff and sediment yield of young citrus orchards with lime soil and red soil under simulated rainfall conditions in southwest China. Water 2022, 14, 1119. [Google Scholar] [CrossRef]
  40. Wang, Y.H.; Long, Q.; Li, Y.Y.; Kang, F.R.; Fan, Z.H.; Xiong, H.Y.; Zhao, H.Y.; Luo, Y.Y.; Guo, R.; He, X.H.; et al. Mitigating magnesium deficiency for sustainable citrus production: A case study in southwest China. Sci. Hortic. 2022, 295, 110832. [Google Scholar] [CrossRef]
  41. Lu, R.K. Assay on Agro-Chemical Properties of Soil, 3rd ed.; China Agricultural Science and Technology Press: Beijing, China, 2000; pp. 146–170. [Google Scholar]
  42. Bao, S.D. Soil and Agricultural Chemistry Analysis, 3rd ed.; China Agricultural Press: Beijing, China, 1981; pp. 103–106. [Google Scholar]
  43. Nelson, D.W.; Sommers, L.E. Total carbon, organic carbon, and organic matter. In Methods of Soil Analysis; Sparks, D.L., Page, A.L., Helmke, P.A., Loeppert, R.H., Soltanpour, P.N., Tabatabai, M.A., Johnston, C.T., Sumner, M.E., Eds.; American Society of Agronomy: Madison, WI, USA, 1982; pp. 961–1010. [Google Scholar] [CrossRef]
  44. Bray, R.H.; Kurtz, L.T. Determination of total organic and available forms of phosphorus in soils. Soil Sci. 1945, 59, 39–45. [Google Scholar] [CrossRef]
  45. Adetunji, A.T.; Lewu, F.B.; Mulidzi, R.; Ncube, B. The biological activities of β-glucosidase, phosphatase and urease as soil quality indicators: A review. J. Soil Sci. Plant Nutr. 2017, 17, 794–807. [Google Scholar] [CrossRef]
  46. German, D.P.; Weintraub, M.N.; Grandy, A.S.; Lauber, C.L.; Rinkes, Z.L.; Allison, S.D. Optimization of hydrolytic and oxidative enzyme methods for ecosystem studies. Soil Biol. Biochem. 2011, 43, 1387–1397. [Google Scholar] [CrossRef]
  47. Saiya-Cork, K.R.; Sinsabaugh, R.L.; Zak, D.R. The effects of long term nitrogen deposition on extracellular enzyme activity in an acer saccharum forest soil. Soil Biol. Biochem. 2002, 34, 1309–1315. [Google Scholar] [CrossRef]
  48. Hoffmann, G.; Teicher, K. Ein kolorimetrisches verfahren zur bestimmung der ureaseaktivität in böden. J. Plant Nutr. 1961, 95, 55–63. [Google Scholar] [CrossRef]
  49. Ge, Y.Y.; Wang, Q.L.; Wang, L.; Liu, W.X.; Liu, X.Y.; Huang, Y.J.; Christie, P. Response of soil enzymes and microbial communities to root extracts of the alien Alternanthera Philoxeroides. Arch. Agron. Soil Sci. 2018, 64, 708–717. [Google Scholar] [CrossRef]
  50. Altieri, M.A. The ecological role of biodiversity in agroecosystems. Agric. Ecosyst. Environ. 1999, 74, 19–31. [Google Scholar] [CrossRef]
  51. Malézieux, E.; Crozat, Y.; Dupraz, C.; Laurans, M.; Makowski, D.; Ozier-Lafontaine, H.; Rapidel, B.; de Tourdonnet, S.; Valantin-Morison, M. Mixing plant species in cropping systems: Concepts, tools and models. A review. Agron. Sustain. Dev. 2009, 29, 43–62. [Google Scholar] [CrossRef]
  52. Vandermeer, J.; van Noordwijk, M.; Anderson, J.; Ong, C.; Perfecto, I. Global change and multi-species agroecosystems: Concepts and issues. Agric. Ecosyst. Environ. 1998, 67, 1–22. [Google Scholar] [CrossRef]
  53. Jensen, E.S. Grain yield, symbiotic N2 fixation and interspecific competition for inorganic N in pea-barley intercrops. Plant Soil 1996, 182, 25–38. [Google Scholar] [CrossRef]
  54. Jayaraman, S.; Sinha, N.K.; Mohanty, M.; Hati, K.M.; Chaudhary, R.S.; Shukla, A.K.; Shirale, A.O.; Neenu, S.; Naorem, A.K.; Rashmi, I.; et al. Conservation tillage, residue management, and crop rotation effects on soil major and micro-nutrients in semi-arid vertisols of India. J. Soil Sci. Plant Nutr. 2021, 21, 523–535. [Google Scholar] [CrossRef]
  55. Vance, C.P.; Graham, P.H.; Allan, D.L. Biological Nitrogen Fixation: Phosphorus—A Critical Future Need? In Current Plant Science and Biotechnology in Agriculture; Pedrosa, F.O., Hungria, M., Yates, G., Newton, W.E., Eds.; Springer: Dordrecht, The Netherlands; New York, NY, USA, 2000; pp. 509–514. [Google Scholar] [CrossRef]
  56. Hinsinger, P.; Betencourt, E.; Bernard, L.; Brauman, A.; Plassard, C.; Shen, J.B.; Tang, X.Y.; Zhang, F.S. P for two, sharing a scarce resource: Soil phosphorus acquisition in the rhizosphere of intercropped species. Plant Physiol. 2011, 156, 1078–1086. [Google Scholar] [CrossRef] [PubMed]
  57. Cahill, J.F.; McNickle, G.G.; Haag, J.J.; Lamb, E.G.; Nyanumba, S.M.; Clair, C.C.S. Plants integrate information about nutrients and neighbors. Science 2010, 328, 1657. [Google Scholar] [CrossRef]
  58. Bargaz, A.; Noyce, G.L.; Fulthorpe, R.; Carlsson, G.; Furze, J.R.; Jensen, E.S.; Dhiba, D.; Isaac, M.E. Species interactions enhance root allocation, microbial diversity and P acquisition in intercropped wheat and soybean under P deficiency. Appl. Soil Ecol. 2017, 120, 179–188. [Google Scholar] [CrossRef]
  59. Sun, Y.B.; Bi, H.X.; Xu, H.S.; Duan, H.Q.; Peng, R.D.; Wang, J.J. Variation of Fine Roots Distribution in Apple (Malus pumila M.)-Crop Intercropping Systems on the Loess Plateau of China. Agronomy 2018, 8, 280. [Google Scholar] [CrossRef]
  60. Gong, X.W.; Liu, C.J.; Li, J.; Luo, Y.; Yang, Q.H.; Zhang, W.L.; Yang, P.; Feng, B.L. Responses of rhizosphere soil properties, enzyme activities and microbial diversity to intercropping patterns on the Loess Plateau of China. Soil Tillage Res. 2019, 195, 104355. [Google Scholar] [CrossRef]
  61. Peng, X.H.; Zhu, Q.H.; Zhang, Z.B.; Hallett, P.D. Combined turnover of carbon and soil aggregates using rare earth oxides and isotopically labelled carbon as tracers. Soil Biol. Biochem. 2017, 109, 81–94. [Google Scholar] [CrossRef]
  62. Daryanto, S.; Fu, B.J.; Wang, L.X.; Jacinthe, P.A.; Zhao, W.W. Quantitative synthesis on the ecosystem services of cover crops. Earth Sci. Rev. 2018, 185, 357–373. [Google Scholar] [CrossRef]
  63. Venter, Z.S.; Jacobs, K.; Hawkins, H.J. The impact of crop rotation on soil microbial diversity: A meta-analysis. Pedobiologia 2016, 59, 215–223. [Google Scholar] [CrossRef]
  64. Chen, Y.C.; Yin, S.W.; Shao, Y.; Zhang, K.R. Soil bacteria are more sensitive than fungi in response to nitrogen and phosphorus enrichment. Front. Microbiol. 2022, 13, 999385. [Google Scholar] [CrossRef]
  65. Chamkhi, I.; Cheto, S.; Geistlinger, J.; Zeroual, Y.; Kouisni, L.; Bargaz, A.; Ghoulam, C. Legume-based intercropping systems promote beneficial rhizobacterial community and crop yield under stressing conditions. Ind. Crops Prod. 2022, 183, 114958. [Google Scholar] [CrossRef]
  66. Cao, T.T.; Luo, Y.C.; Shi, M.; Tian, X.J.; Kuzyakov, Y. Microbial interactions for nutrient acquisition in soil: Miners, scavengers, and carriers. Soil Biol. Biochem. 2024, 188, 109215. [Google Scholar] [CrossRef]
  67. Yu, Y.; Zhou, Y.; Janssens, I.A.; Deng, Y.; He, X.J.; Liu, L.L.; Yi, Y.; Xiao, N.W.; Wang, X.D.; Li, C.; et al. Divergent rhizosphere and non-rhizosphere soil microbial structure and function in long-term warmed steppe due to altered root exudation. Glob. Chang. Biol. 2024, 30, e17111. [Google Scholar] [CrossRef] [PubMed]
  68. Liu, H.; Pan, F.J.; Han, X.Z.; Song, F.B.; Zhang, Z.M.; Yan, J.; Xu, Y.L. A comprehensive analysis of the response of the fungal community structure to long-term continuous cropping in three typical upland crops. J. Integr. Agric. 2020, 19, 866–880. [Google Scholar] [CrossRef]
  69. Abdellah, Y.A.Y.; Li, T.Z.; Chen, X.; Cheng, Y.; Sun, S.S.; Wang, Y.; Jiang, C.; Zang, H.L.; Li, C.Y. Role of psychrotrophic fungal strains in accelerating and enhancing the maturity of pig manure composting under low-temperature conditions. Bioresour. Technol. 2021, 320, 124402. [Google Scholar] [CrossRef]
  70. Wang, G.W.; Jin, Z.X.; George, T.S.; Feng, G.; Zhang, L. Arbuscular mycorrhizal fungi enhance plant phosphorus uptake through stimulating hyphosphere soil microbiome functional profiles for phosphorus turnover. New Phytol. 2023, 238, 2578–2593. [Google Scholar] [CrossRef]
  71. Zhang, F.; Zou, Y.N.; Wu, Q.S.; Kuca, K. Arbuscular mycorrhizas modulate root polyamine metabolism to enhance drought tolerance of trifoliate orange. Environ. Exp. Bot. 2020, 171, 103962. [Google Scholar] [CrossRef]
  72. Arie, T. Fusarium diseases of cultivated plants, control, diagnosis, and molecular and genetic studies. J. Pestic. Sci. 2019, 44, 275–281. [Google Scholar] [CrossRef]
  73. Fernandes, C.; Casadevall, A.; Gonçalves, T. Mechanisms of Alternaria pathogenesis in animals and plants. FEMS Microbiol. Rev. 2023, 47, fuad061. [Google Scholar] [CrossRef]
  74. Ozimek, E.; Hanaka, A. Mortierella species as the plant growth-promoting fungi present in the agricultural soils. Agriculture 2021, 11, 7. [Google Scholar] [CrossRef]
  75. Tian, Y.; Tan, Y.L.; Liu, N.; Yan, Z.; Liao, Y.C.; Chen, J.; de Saeger, S.; Yang, H.; Zhang, Q.Y.; Wu, A.B. Detoxification of deoxynivalenol via glycosylation represents novel insights on antagonistic activities of Trichoderma when confronted with Fusarium graminearum. Toxins 2016, 8, 335. [Google Scholar] [CrossRef] [PubMed]
  76. Panthee, S.; Hamamoto, H.; Paudel, A.; Sekimizu, K. Lysobacter species: A potential source of novel antibiotics. Arch. Microbiol. 2016, 198, 839–845. [Google Scholar] [CrossRef] [PubMed]
  77. Fulano, A.M.; Shen, D.Y.; Kinoshita, M.; Chou, S.H.; Qian, G.L. The homologous components of flagellar type iii protein apparatus have acquired a novel function to control twitching motility in a non-flagellated biocontrol bacterium. Biomolecules 2020, 10, 733. [Google Scholar] [CrossRef]
  78. Xu, J.; Zhang, Y.; Zhang, P.F.; Trivedi, P.; Riera, N.; Wang, Y.Y.; Liu, X.; Fan, G.Y.; Tang, J.L.; Coletta, H.D.; et al. The structure and function of the global citrus rhizosphere microbiome. Nat. Commun. 2018, 9, 4894. [Google Scholar] [CrossRef]
  79. Brescia, F.; Pertot, I.; Puopolo, G. Chapter 16—Lysobacter. In Beneficial Microbes in Agro-Ecology: Bacteria and Fungi; Amaresan, N., Kumar, M.S., Annapurna, K., Kumar, K., Sankaranarayanan, A., Eds.; Academic Press: Amsterdam, The Netherlands, 2020; pp. 313–338. [Google Scholar] [CrossRef]
  80. Lin, L.; Zhou, M.M.; Shen, D.Y.; Han, S.; Fulano, A.M.; Chou, S.H.; Qian, G.L. A non-flagellated biocontrol bacterium employs a PilZ-PilB complex to provoke twitching motility associated with its predation behavior. Phytopathol. Res. 2020, 2, 12. [Google Scholar] [CrossRef]
  81. Semenov, M.V.; Krasnov, G.S.; Semenov, V.M.; Ksenofontova, N.; Zinyakova, N.B.; van Bruggen, A.H.C. Does fresh farmyard manure introduce surviving microbes into soil or activate soil-borne microbiota? J. Environ. Manag. 2021, 294, 113018. [Google Scholar] [CrossRef] [PubMed]
  82. Postma, J.; Schilder, M.T.; Bloem, J.; van Leeumen-Haagsma, W.K. Soil suppressiveness and functional diversity of the soil microflora in organic farming systems. Soil Biol. Biochem. 2008, 40, 2394–2406. [Google Scholar] [CrossRef]
  83. De Paepe, J.; De Paepe, K.; Gòdia, F.; Rabaey, K.; Vlaeminck, S.E.; Clauwaert, P. Bio-electrochemical COD removal for energy-efficient, maximum and robust nitrogen recovery from urine through membrane aerated nitrification. Water Res. 2020, 185, 116223. [Google Scholar] [CrossRef]
  84. Wang, Y.L.; Niu, Q.G.; Zhang, X.; Liu, L.; Wang, Y.B.; Chen, Y.Q.; Negi, M.; Figeys, D.; Li, Y.Y.; Zhang, T. Exploring the effects of operational mode and microbial interactions on bacterial community assembly in a one-stage partial-nitritation anammox reactor using integrated multi-omics. Microbiome 2019, 7, 122. [Google Scholar] [CrossRef]
  85. Elabyad, M.S.; Elsayed, M.A.; Elshanshoury, A.R.; Elsabbagh, S.M. Towards the biological control of fungal and bacterial diseases of tomato using antagonistic Streptomyces spp. Plant Soil 1993, 149, 185–195. [Google Scholar] [CrossRef]
  86. Lee, J.; Kim, S.; Jung, H.; Koo, B.K.; Han, J.A.; Lee, H.S. Exploiting bacterial genera as biocontrol agents: Mechanisms, interactions and applications in sustainable agriculture. J. Plant Biol. 2023, 66, 485–498. [Google Scholar] [CrossRef]
  87. Goudjal, Y.; Toumatia, O.; Sabaou, N.; Barakate, M.; Mathieu, F.; Zitouni, A. Endophytic actinomycetes from spontaneous plants of Algerian Sahara: Indole-3-acetic acid production and tomato plants growth promoting activity. World J. Microbiol. Biotechnol. 2013, 29, 1821–1829. [Google Scholar] [CrossRef] [PubMed]
  88. Som, N.F.; Heine, D.; Holmes, N.; Knowles, F.; Chandra, G.; Seipke, R.F.; Hoskisson, P.A.; Wilkinson, B.; Hutchings, M.I. The MtrAB two-component system controls antibiotic production in Streptomyces coelicolor A3 (2). Microbiology 2017, 163, 1415–1419. [Google Scholar] [CrossRef] [PubMed]
  89. Shao, Z.Y.; Li, Z.; Fu, Y.H.; Wen, Y.P.; Wei, S.J. Induction of defense responses against Magnaporthe oryzae in rice seedling by a new potential biocontrol agent Streptomyces JD211. J. Basic. Microbiol. 2018, 58, 686–697. [Google Scholar] [CrossRef]
  90. Salwan, R.; Sharma, V. Chapter 15—Bioactive compounds of Streptomyces: Biosynthesis to applications. In Studies in Natural Products Chemistry; Rahman, A.U., Ed.; Academic Press: Amsterdam, The Netherlands, 2020; Volume 64, pp. 467–491. [Google Scholar] [CrossRef]
  91. Zhang, X.X.; Zhang, R.J.; Gao, J.S.; Wang, X.C.; Fan, F.L.; Ma, X.T.; Yin, H.Q.; Zhang, C.W.; Feng, K.; Deng, Y. Thirty-one years of rice-rice-green manure rotations shape the rhizosphere microbial community and enrich beneficial bacteria. Soil Biol. Biochem. 2017, 104, 208–217. [Google Scholar] [CrossRef]
  92. Lin, D.; Xu, J.Y.; Wang, L.; Du, S.; Zhu, D. Long-term application of organic fertilizer prompting the dispersal of antibiotic resistance genes and their health risks in the soil plastisphere. Environ. Int. 2024, 183, 108431. [Google Scholar] [CrossRef]
  93. Chen, D.L.; Wang, X.X.; Zhang, W.; Zhou, Z.G.; Ding, C.F.; Liao, Y.W.K.; Li, X.G. Persistent organic fertilization reinforces soil-borne disease suppressiveness of rhizosphere bacterial community. Plant Soil. 2020, 452, 313–328. [Google Scholar] [CrossRef]
  94. Bassanezi, R.B.; Lopes, S.A.; de Miranda, M.P.; Wulff, N.A.; Volpe, H.X.L.; Ayres, A.J. Overview of citrus huanglongbing spread and management strategies in Brazil. Trop. Plant Pathol. 2020, 45, 251–264. [Google Scholar] [CrossRef]
  95. Morugán-Coronado, A.; Linares, C.; Gómez-López, M.D.; Faz, A.; Zornoza, R. The impact of intercropping, tillage and fertilizer type on soil and crop yield in fruit orchards under mediterranean conditions: A meta-analysis of field studies. Agric. Syst. 2020, 178, 102736. [Google Scholar] [CrossRef]
  96. Xiang, Y.Z.; Chang, S.X.; Shen, Y.Y.; Chen, G.; Liu, Y.; Yao, B.; Xue, J.M.; Li, Y. Grass cover increases soil microbial abundance and diversity and extracellular enzyme activities in orchards: A synthesis across China. Appl. Soil Ecol. 2023, 182, 104720. [Google Scholar] [CrossRef]
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Figure 1. Soil enzyme activities in response to the green manure–citrus intercropping. Solid lines in the figure indicate median. The box boundaries indicate upper and lower quartiles, the whisker caps indicate 90th and 10th percentiles, and the circles indicate the distribution of soil enzyme data. (A) Urease. (B) Catalase. (C) Acid phosphatase. (D) β-1,4-glucosidase. (E) Sucrase. Different lowercase letters indicate significant differences in soil physicochemical properties for the three treatments at p < 0.05 level. Control: monoculture of citrus; V. villosa: hairy vetch intercropping with citrus; and M. sativa: alfalfa intercropping with citrus.
Figure 1. Soil enzyme activities in response to the green manure–citrus intercropping. Solid lines in the figure indicate median. The box boundaries indicate upper and lower quartiles, the whisker caps indicate 90th and 10th percentiles, and the circles indicate the distribution of soil enzyme data. (A) Urease. (B) Catalase. (C) Acid phosphatase. (D) β-1,4-glucosidase. (E) Sucrase. Different lowercase letters indicate significant differences in soil physicochemical properties for the three treatments at p < 0.05 level. Control: monoculture of citrus; V. villosa: hairy vetch intercropping with citrus; and M. sativa: alfalfa intercropping with citrus.
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Figure 2. OTUs number of soil fungal (A) and bacterium (B) in monoculture of citrus (control), V. villosa intercropping with citrus and M. sativa intercropping with citrus. Red: alfalfa intercropping with citrus; green: monoculture of citrus; blue: hairy vetch intercropping with citrus.
Figure 2. OTUs number of soil fungal (A) and bacterium (B) in monoculture of citrus (control), V. villosa intercropping with citrus and M. sativa intercropping with citrus. Red: alfalfa intercropping with citrus; green: monoculture of citrus; blue: hairy vetch intercropping with citrus.
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Figure 3. Principal coordinate plots showing the effects of different practices on the composition of the overall soil fungal (A) and bacterial (B) communities based on Bray–Curtis distances. (OTUs with relative abundance more than 0.01%). Control = monoculture of citrus; V. villosa = hairy vetch intercropping with citrus; and M. sativa = alfalfa intercropping with citrus. Different lowercase letters indicate significant differences in soil physicochemical properties for the three treatments (p < 0.05).
Figure 3. Principal coordinate plots showing the effects of different practices on the composition of the overall soil fungal (A) and bacterial (B) communities based on Bray–Curtis distances. (OTUs with relative abundance more than 0.01%). Control = monoculture of citrus; V. villosa = hairy vetch intercropping with citrus; and M. sativa = alfalfa intercropping with citrus. Different lowercase letters indicate significant differences in soil physicochemical properties for the three treatments (p < 0.05).
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Figure 4. Relative abundances of the dominant fungi ((A) at phylum level; (B) at genus level) and bacteria ((C) at phylum level; (D) at genus level) in the monoculture of citrus, V. villosa intercropping with citrus, and M. sativa intercropping with citrus. The top 10 dominant phyla and the top 20 genera of bacteria and fungi were used in the analysis, respectively. The relative abundance of each phylum or genus was calculated by the average relative abundance of this phylum or genus across all soils divided by the average total relative abundance of all phyla in each cropping sequences.
Figure 4. Relative abundances of the dominant fungi ((A) at phylum level; (B) at genus level) and bacteria ((C) at phylum level; (D) at genus level) in the monoculture of citrus, V. villosa intercropping with citrus, and M. sativa intercropping with citrus. The top 10 dominant phyla and the top 20 genera of bacteria and fungi were used in the analysis, respectively. The relative abundance of each phylum or genus was calculated by the average relative abundance of this phylum or genus across all soils divided by the average total relative abundance of all phyla in each cropping sequences.
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Figure 5. Correlation analyses of soil properties and Mantel test analyses of soil microbial (fungal, bacterial) diversity and soil properties in soils under different orchard management techniques. pH, soil pH; SOC, soil organic carbon; TN, total nitrogen, soil; TP, total phosphorus, soil; AN, soil ammonia nitrogen (NH4+); NN, soil nitrate nitrogen (NO3−); AP, soil available phosphorus; AK, soil available potassium; CAT, soil catalase; AcP, acid phosphatase; UE, urease; SR, sucrase; BG, β-1,4-glucosidase. The R2 value represents the explained variation. * and ** indicate the significance level at p < 0.05 and p < 0.01.
Figure 5. Correlation analyses of soil properties and Mantel test analyses of soil microbial (fungal, bacterial) diversity and soil properties in soils under different orchard management techniques. pH, soil pH; SOC, soil organic carbon; TN, total nitrogen, soil; TP, total phosphorus, soil; AN, soil ammonia nitrogen (NH4+); NN, soil nitrate nitrogen (NO3−); AP, soil available phosphorus; AK, soil available potassium; CAT, soil catalase; AcP, acid phosphatase; UE, urease; SR, sucrase; BG, β-1,4-glucosidase. The R2 value represents the explained variation. * and ** indicate the significance level at p < 0.05 and p < 0.01.
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Figure 6. LDA distribution histogram and representative differential genera among the control, hairy vetch intercropping, and alfalfa intercropping groups. (A,C) LDA distribution histogram based on a LDA score cutoff of ±3.5. (B) Fungal representative differential genera, including Sarocladium, Filobasidium, Sporidiobolus, Mortierella, Gibberella, and penicillium. (D) Bacterial differential genera, including Truepera, Pedobacter Lysobacter, UTCFX1, Streptomyces, and Ulvibacter. Abbreviation: LDA, linear discriminant analysis.
Figure 6. LDA distribution histogram and representative differential genera among the control, hairy vetch intercropping, and alfalfa intercropping groups. (A,C) LDA distribution histogram based on a LDA score cutoff of ±3.5. (B) Fungal representative differential genera, including Sarocladium, Filobasidium, Sporidiobolus, Mortierella, Gibberella, and penicillium. (D) Bacterial differential genera, including Truepera, Pedobacter Lysobacter, UTCFX1, Streptomyces, and Ulvibacter. Abbreviation: LDA, linear discriminant analysis.
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Figure 7. RDA ordination plot based on the relationship (A) between the beneficial microbial community and the environmental variables and (B) between the beneficial microbial community and the soil enzymes under the different treatments. The soil properties include the pH, soil organic carbon (SOC), total nitrogen content (TN), total phosphorus content (TP), soil ammonia nitrogen (AN), soil nitrate nitrogen (NN), available phosphorus (AP), and available potassium (AK). The soil enzymes include catalase (CAT), acid phosphatase (AcP), urease (UE), surcease (SR), and BG (β-1,4-glucosidase). Samples of the control are shown as green circles. The hairy vetch intercropping treatments and alfalfa intercropping treatments are shown as blue square and pink triangle symbols, respectively. The soil properties and soil enzymes are indicated by blue arrows, and soil beneficial microbial are indicated by red arrow symbols, respectively.
Figure 7. RDA ordination plot based on the relationship (A) between the beneficial microbial community and the environmental variables and (B) between the beneficial microbial community and the soil enzymes under the different treatments. The soil properties include the pH, soil organic carbon (SOC), total nitrogen content (TN), total phosphorus content (TP), soil ammonia nitrogen (AN), soil nitrate nitrogen (NN), available phosphorus (AP), and available potassium (AK). The soil enzymes include catalase (CAT), acid phosphatase (AcP), urease (UE), surcease (SR), and BG (β-1,4-glucosidase). Samples of the control are shown as green circles. The hairy vetch intercropping treatments and alfalfa intercropping treatments are shown as blue square and pink triangle symbols, respectively. The soil properties and soil enzymes are indicated by blue arrows, and soil beneficial microbial are indicated by red arrow symbols, respectively.
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Table 1. Effect of intercropping different green manures on soil nutrients and properties.
Table 1. Effect of intercropping different green manures on soil nutrients and properties.
TreatmentpHSOC
(g kg−1)		TN
(g/kg)		TP
(g/kg)		AN
(mg/kg)		NN
(mg/kg)		AK
(mg/kg)		AP
(mg/kg)
Monoculture5.56 ± 0.23 b27.85 ± 6.41 a1.50 ± 0.24 b0.62 ± 0.08 a2.17 ± 0.09 c9.54 ± 1.31 a7.79 ± 0.90 a23.18 ± 2.18 b
V. villosa6.45 ± 0.13 a36.24 ± 2.26 a2.62 ± 0.05 a0.68 ± 0.05 a3.15 ± 0.17 b12.36 ± 1.78 a13.31 ± 1.96 a33.62 ± 1.94 a
M. sativa6.27 ± 0.11 a26.80 ± 9.40 a2.23 ± 0.22 a0.66 ± 0.05 a4.13 ± 0.22 a11.67 ± 1.11 a11.72 ± 2.59 a33.49 ± 3.52 a
p-value0.0090.5710.0070.526<0.0010.3860.1740.032
Abbreviations: SOC, soil organic carbon; TN, total nitrogen; TP, total phosphorus; AN, soil ammonia nitrogen (NH4+); NN, soil nitrate nitrogen (NO3−); AP, available phosphorus; AK, available potassium. The data represent the mean ± S.D.; n = 3. Different letters within a column indicate significant differences among treatments at the p < 0.05 level (one-way ANOVA). Different lowercase letters indicate significant differences in soil physicochemical properties for the three treatments (p < 0.05). Control: monoculture of citrus; V. villosa: hairy vetch intercropping with citrus; and M. sativa: alfalfa intercropping with citrus.
Table 2. Effect of different green manure treatment applications on soil microbial alpha diversity.
Table 2. Effect of different green manure treatment applications on soil microbial alpha diversity.
FungiControlV. villosaM. sativap-Value
richness815 ± 44 a737 ± 41 a717 ± 83 a0.494
Chao1821.64 ± 46.32 a739.35 ± 41.46 a723.99 ± 85.57 a0.504
ACE823.43 ± 46.08 a739.95 ± 41.45 a725.83 ± 85.17 a0.499
Shannon4.76 ± 0.09 a4.46 ± 0.12 ab4.22 ± 0.2 b0.075
Simpson0.97 ± 0 a0.94 ± 0.01 a0.94 ± 0.01 a0.093
BacteriaControlV. villosaM. sativap-Value
Richness1927 ± 78.86 a2110 ± 77.73 a1787 ± 154.39 a0.169
Chao11931.41 ± 80.89 a2120.3 ± 78.46 a1792.33 ± 154.96 a0.165
ACE1934.31 ± 81.34 a2121.76 ± 79.66 a1795.92 ± 154.95 a0.170
Shannon6.9235 ± 0.05 a7.0616 ± 0.06 a6.6355 ± 0.25 a0.180
Simpson1 ± 0.00 a1 ± 0.00 a0.99 ± 0.00 a0.09
Data are expressed as mean ± SD. Treatments with similar lowercase letters within a row are not significantly different at p < 0.05.
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Xie, Y.; Jing, Y.; Wang, Y.; Zheng, R.; Xu, Q.; Sun, Z.; Duan, T. Leguminous Green Manure Intercropping Promotes Soil Health in a Citrus (Citrus reticulata) Orchard. Agriculture 2024, 14, 1897. https://doi.org/10.3390/agriculture14111897

AMA Style

Xie Y, Jing Y, Wang Y, Zheng R, Xu Q, Sun Z, Duan T. Leguminous Green Manure Intercropping Promotes Soil Health in a Citrus (Citrus reticulata) Orchard. Agriculture. 2024; 14(11):1897. https://doi.org/10.3390/agriculture14111897

Chicago/Turabian Style

Xie, Yuxin, Yulin Jing, Yajie Wang, Rongchun Zheng, Qiurui Xu, Zhenyu Sun, and Tingyu Duan. 2024. "Leguminous Green Manure Intercropping Promotes Soil Health in a Citrus (Citrus reticulata) Orchard" Agriculture 14, no. 11: 1897. https://doi.org/10.3390/agriculture14111897

APA Style

Xie, Y., Jing, Y., Wang, Y., Zheng, R., Xu, Q., Sun, Z., & Duan, T. (2024). Leguminous Green Manure Intercropping Promotes Soil Health in a Citrus (Citrus reticulata) Orchard. Agriculture, 14(11), 1897. https://doi.org/10.3390/agriculture14111897

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