Organic Mulching Alters the Composition, but not the Diversity, of Rhizosphere Bacterial and Fungal communities

Background: Organic mulching is an effective forest management technique that provides carbon and nutrient sources to soil ecosystems, thereby improving the soil environment and promoting plant growth. Although the importance of rhizosphere microbiomes in plant and soil ecosystem functions has been widely recognised, the effect of organic mulching on rhizosphere microorganisms and the underlying mechanisms are unclear. Methods: We performed a eld experiment in a 15-year-old Ligustrum lucidum forest of urban green space. The diversity and composition of the rhizosphere bacterial and fungal communities following organic mulching were assessed by combining 16S ribosomal RNA and internal transcribed spacer amplicon sequencing. The correlations between microbial diversity, composition, and ne-root traits, as well as rhizosphere soil properties, were also analysed. Results: Organic mulching did not signicantly affect the diversity of the rhizosphere bacterial or fungal communities. Additionally, organic mulching increased the bacterial diversity after 6 months, with a 20-cm-thick mulch layer showing a greater effect than 5- or 10-cm layers. Organic mulching signicantly altered the rhizosphere bacterial and fungal community composition; after 6 months of mulching, the community compositions were signicantly associated with ne-root traits (specic root length, nitrogen, and phosphorus concentration) and enzyme (urease and dehydrogenase) activity. Moreover, alterations in the bacterial and fungal communities occurred at the order level within each mulching stage. Bacterial diversity is affected by fungal diversity and rhizosphere soil properties (water content and organic carbon) in time-dependent manners. Hence, organic mulching appears to directly affect the fungal composition while indirectly affecting the bacterial composition via inuencing rhizosphere soil properties (dissolved organic carbon and peroxidase activity). Conclusions: Organic mulching affects the rhizosphere bacterial and fungal community composition through different pathways; however, the underlying mechanisms, including the effects of time and soil layers, require further exploration combined with multi-index measurements and long-term dynamic monitoring. Our results suggest that the changes in the rhizosphere microbial community composition after organic mulching result from changes in their work or functions rather than in their lack of diversity. In particular, the altered bacterial and fungal communities (at the order level) differed during the various mulching stages. We demonstrated that ne-root traits and enzymatic activity play important roles in the rhizosphere microbial community composition during the early stage of organic mulching. Our study further reveals the regulatory mechanism by which organic mulching affects the rhizosphere bacterial and fungal communities. Multi-index measurements and long-term dynamic monitoring should be performed to continuously explore the mechanism of nutrient exchange and energy ow between soil and plants to provide a foundation for soil improvement and forest productivity.


Background
The understanding of soil microorganisms is limited because of the large number and variety of soil microorganisms. The causes of the vast microbial variation in rhizosphere soil are complex because of the complicated rhizosphere environment, which is directly in uenced by living roots. In fact, the nutrient content of rhizosphere soil, as well as its soluble organic matter, enzyme activity, and microbial diversity, are higher than those of bulk soil (Turpault et al. 2007;De Feudis et al. 2017). Thus, the microbial responses in bulk and rhizosphere soils to different soil management practices are distinct (Maarastawi et al. 2018). Although the essential roles of rhizosphere microbiomes (rhizobiomes) have been demonstrated in soil and plants, few studies have considered rhizosphere-related traits, particularly ne-root architecture and characteristics (Kuzyakov and Razavi 2019).
Rhizosphere microorganisms are in uenced by ne roots and the rhizosphere soil environment. Plant roots provide effective C and N sources for rhizobiomes by producing exudates and metabolites (Phillips and Fahey 2008). Roots speci cally structure their environment to optimise their functions (water and nutrient uptake) while also establishing habitats for microorganisms and their activities (Kuzyakov and Razavi 2019). Thus, root physiological features shape rhizobiomes and exudation (Sasse et al. 2018). For example, root morphology (root surface structure, number, and length) represents a major index for explaining the mechanism by which plants are thought to modulate microbial interactions (Sasse et al. 2018). The C, N, and P contents in roots de ne the root exudate composition and nutrient availability to some extent (Dotaniya and Meena 2015), as well as dramatically affect the rhizosphere environment and microorganisms. Fine roots (diameter ≤ 2 mm) are highly dynamic and vital components of forests that are more sensitive than other roots to environmental change (Yuan and Chen 2010). Further, ne root abrasion serves as one of the main lipids in leachates from the root zone (Jandl et al. 2013). However, little is known regarding ne-root biology and the relationships between ne-root traits and the rhizosphere environment, including rhizobiomes, limiting the ability to predict the responses of soil microorganisms to environmental changes (Zak et al. 2000).
Although soil physicochemical properties are known to signi cantly affect soil microorganisms, the predominant factors involved remain controversial. For example, some studies showed that soil pH and permanganate oxidisable C/soil organic C (SOC) represent the major drivers of the microbial community structure (Liu et al. 2017; Ramírez et al. 2020). However, another study indicated that light fractions of organic C and inorganic N are the key factors responsible for regulating the microbial community structure, whereas SOC controls microbial residue accumulation (Jing et al. 2019). External disturbances, such as climate change, precipitation, and fertilisation, can alter the soil environment, including its temperature, moisture, and nutrient elements, while also affecting plant growth and, consequently, soil properties and microorganisms (Hopkins et al. 2014;Wei et al. 2017). This is a reciprocal process in which soil microorganisms also affect plant diversity and productivity while signi cantly affecting soil fertility (Van Der Heijden et al. 2008; Leff et al. 2015).
However, considering the various interactions occurring within this intricate relationship, the changes in soil microorganism communities, particularly within the rhizosphere environment, are complex; therefore, detailed research efforts are needed to resolve these issues.
Organic mulching is an important practice in agricultural soil conservation. Indeed, organic mulching has recently become widely used for urban greening and plantation for soil remediation and plant growth. It not only alters the physical characteristics of soil, including the temperature and bulk density, but also provides C and nutrients to the soil, which in turn in uence nutrient uptake by plants (Kader et al. 2017). Changes in the soil environment caused by mulching affect the rhizosphere, as microorganisms are sensitive to various factors such as soil temperature and water content (Dotaniya and Meena 2015). Furthermore, the decomposition of organic mulch and exudate from the roots, occurring after plant growth, provides different nutrients for microorganisms. The thickness of the mulch and decomposed available nutrients decrease over time, thereby weakening the effects on soil physical conditions and nutrient content. Hence, the rhizobiomes and rhizosphere environment are dynamic. The rhizosphere environment also changes according to the natural conditions, including high seasonal variation (Calvaruso et al. 2014). The speci c response of rhizobiomes to organic mulching is complicated and not well understood, particularly in the context of forest ecosystems.
Accordingly, the aim of this study was to improve the understanding of microbial diversity and composition after organic mulching of a Ligustrum lucidum forest in an urban green space, as well as the relationship between the rhizosphere environment ( ne-root traits and rhizosphere soil properties) and rhizobiomes. We aimed to determine how the bacterial and fungal community diversity and composition in rhizosphere soil respond to organic mulching, which biotic and/or abiotic factors in the rhizosphere environment ( ne-root traits and rhizosphere soil properties) affect the diversity and composition of rhizobiomes, and how these factors in uence the diversity and composition of rhizobiomes.
According to the historical records of Zhongshan Cemetery in Nanjing, the area previously contained buildings that were demolished, after which the region was covered with plantation forestry in 50-60 cm of soil. We analysed 15-year-old pure stands of Ligustrum lucidum W.T. Aiton (broad-leaf privet; family: Oleaceae) with tree spacing > 2 m and canopy density approximately 85%. The average tree height was 7.5 m, average crown was 2.5 m, and average diameter at breast height was 10.9 cm. The basic physical and chemical properties of the soil are shown in Table 1.

Experimental design
Four adjacent trees were randomly selected as an experimental plot with 32 experimental plots (128 trees) established.
According to the 'Technical speci cation for the application of organic mulch on urban and rural greening' of Shanghai,  Table 2.

Field sampling
The soil was sampled twice, after 6 and 12 months of organic mulching. During each sampling, soil was recovered from three randomly selected experimental plots (i.e., n = 3 per treatment, 12 trees) with each experimental plot used only once. The soil pro les were sampled 50 cm away from the tree trunk, and each pro le was divided into two layers (0-20 cm and 20-40 cm below the mulch layer). Soil blocks of 20 × 20 × 20 cm 3 were recovered. The ne roots were removed by hand.
Rhizosphere soil was collected by gently shaking off the soil adhered to the roots. All ne roots and soil samples were placed in self-sealing bags and immediately transported to the laboratory for analysis. The soil samples were sieved (2 mm) and stored at 4°C until physicochemical analysis. In addition, 5-10 g of rhizosphere soil was collected from each sample, and after removing impurities such as plant roots and animal remains, the soil was placed in sterile centrifuge tubes, which were then placed in an ice box and transported to the laboratory where they were stored at -80°C for subsequent microbial sequencing.

Laboratory analysis
The physiochemical properties and enzyme activities of the rhizosphere soil were determined as described in our previous study (Sun et al. 2021a, b). These properties included the water content, pH, SOC, dissolved organic C (DOC), total N (TN), dissolved N (DN), ammonium, nitrate, microbial biomass C (MBC), microbial biomass N (MBN), total P (TP), enzyme (invertase, urease, peroxidase, and peroxidase) activity, and ne-root traits, namely, speci c root length (SRL), speci c surface area (SSA), root tissue density (RTD), ne root biomass (FRB), and ne root C and N concentrations (FRC and FRN, respectively). Additionally, the total ne root P (FRP) was detected calorimetrically following digestion (Campbell et al. 1991).
Genomic DNA was extracted from rhizosphere samples using the FastDNA Spin Kit for Soil (MP Biomedicals, Santa Ana, CA, USA). The DNA purity and concentration were detected and monitored using a Nanodrop ND-2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Next, 1% agarose gel electrophoresis was performed to assess the DNA quality, and the quali ed DNA was stored at − 80°C for subsequent polymerase chain reaction (PCR) analysis. The V4-V5 hypervariable region fragments of the bacterial 16S ribosomal RNA gene were ampli ed with primers 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) using a thermocycler PCR system (GeneAmp

Statistical analysis
Obtained raw FASTQ les were processed using Trimmomatic software for sequence quality control and ltering. FLASH software was used for stitching according to the overlap relation. After the samples were differentiated, UPARSE software (Edgar 2013) was used for operational taxonomic unit (OTU) clustering according to a 97% similarity level. The species classi cation annotation determined using the Silva database was compared with the RDP classi er (Pruesse et al. 2007).
The diversity index was calculated using Mothur software (Schloss et al. 2009).
All statistical analyses were performed using R v.3.5.3 software (core Team 2018), and the corresponding gures were created using the 'ggplot2' software package in R. Linear mixed effects models were calculated using the R package 'lme4' to evaluate differences in rhizosphere soil bacterial and fungal diversity (Shannon index), rhizosphere soil properties, and ne-root traits among treatments, soil layer, time after organic mulching, and their interactions (Bates et al. 2015). Treatment it was improved by removing meaningless direct or indirect paths. The model was evaluated and reduced based on the goodness of t, whereas the AIC was applied to ensure optimal selection among different models. That is, the model with the lowest AIC value was selected as the nal model. We implemented SEM using the piecewiseSEM package with 'plot' as the random effect to account for autocorrelation among split plots (Lefcheck 2016). All variables were initially tested for a normal distribution. P < 0.05 was considered as statistically signi cant.

Results
Changes in bacterial and fungal diversity after organic mulching  (Table 3). After 6 months of mulching, the Shannon index for the bacterial community was signi cantly affected, and bacterial diversity increased with increased mulching amounts (Fig. 1). Moreover, the Shannon index for the bacterial community under OM5 and OM10 after 6 months of mulching, as well as that under OM0 after 12 months of mulching differed signi cantly between the topsoil and subsoil.

Changes in microbial community composition after organic mulching
Organic mulching signi cantly affected the bacterial and fungal community composition; the interaction of the treatment × soil layer signi cantly affected the bacterial community composition except for at 6 months ( Fig. 2 and Table 4). FRP signi cantly affected the bacterial community composition after 6 months of mulching, accounting for 33% of the observed changes, whereas the fungal community composition was signi cantly in uenced by SRL, FRN, and urease and dehydrogenase activity, accounting for 26%, 28%, 33%, and 30% of the changes, respectively ( Fig. 2 and Table S1). Note: * P < 0.05; ** P < 0.01; *** P < 0.001.
Relationship between microbial community diversity, composition, and rhizosphere ne-root traits SEM analysis revealed that the water content, SOC, and FRB signi cantly affected the bacterial and fungal community diversity (Fig. 4a). Bacterial community diversity was signi cantly affected by fungal community diversity and rhizosphere soil properties (water content and SOC). Rhizosphere soil properties were signi cantly affected by time. All factors in SEM that affected bacterial community diversity accounted for 44% of the changes. In addition, FRB was signi cantly affected by time, accounting for 69% of the variation in FRB.
Peroxidase activity, DOC, and FRN were shown by SEM analysis to signi cantly affect the bacterial and fungal community composition (Fig. 4b). Organic mulching directly affected the fungal community composition, accounting for 9% of its variation, and indirectly affected the bacterial community composition through rhizosphere soil properties (peroxidase activity and DOC). All factors identi ed by SEM to affect the bacterial community explained 27% of its changes. Soil layers also signi cantly affected rhizosphere soil properties, accounting for 20% of its changes, together with mulching. FRN was positively affected by time and negatively affected by soil layers.

Discussion
Effect of organic mulching on rhizosphere bacterial and fungal community diversity Although, in general, organic mulching did not signi cantly affect the diversity of the rhizosphere bacterial and fungal communities, it increased bacterial community diversity after 6 months. Soil environmental and microphysical factors signi cantly affect the bacterial community, whereas none of these parameters showed a signi cant correlation with the fungal community (Pudasaini et al. 2017;Tan et al. 2019). Similarly, fungal diversity and rhizosphere soil properties, which include the water content and SOC, were determined to be critical factors affecting bacterial diversity according to the SEM results (Fig. 4a); however, no index signi cantly affected fungal diversity among our measured variables. Compared to fungi, which control the decomposition of organic matter, rhizosphere bacteria appear to have a greater advantage in terms of organic matter transformation and assimilation because of their interaction with the roots, which leads to greater changes In addition, soil depth signi cantly affects soil properties. In the current study, soil layers indirectly affected the bacterial composition but not the fungal composition. Hence, to some extent, the composition of the bacterial community had a stronger spatial dependence compared to the fungal community (Yang et al. 2018). As previously mentioned, bacteria may play important roles in transforming and assimilating organic matter in the rhizosphere, whereas altered C sources at various soil depths affect the microbial community structure (Preusser et al. 2019).

Conclusions
Our results suggest that the changes in the rhizosphere microbial community composition after organic mulching result from changes in their work or functions rather than in their lack of diversity. In particular, the altered bacterial and fungal communities (at the order level) differed during the various mulching stages. We demonstrated that ne-root traits and enzymatic activity play important roles in the rhizosphere microbial community composition during the early stage of organic mulching. Our study further reveals the regulatory mechanism by which organic mulching affects the rhizosphere bacterial and fungal communities. Multi-index measurements and long-term dynamic monitoring should be performed to continuously explore the mechanism of nutrient exchange and energy ow between soil and plants to provide a foundation for soil improvement and forest productivity.    Rhizosphere bacterial and fungal communities with signi cant differences after organic mulching at the order level. a: Topsoil, b: subsoil. Bacterial and fungal classes containing at least 1% of total OTUs (x axis) for each sampling time are shown in the gure. Figure 4