| Open Peer Review | Environmental Microbiology | Research Article

Grass-legume mixtures maintain forage biomass under microbial

diversity loss via gathering Pseudomonas in root zone soil
Yu Liu,1 Wei Yan,2 Tongyao Yang,1 Yining An,1 Xiaomeng Li,1 Hang Gao,1 Ziheng Peng,1 Gehong Wei,1 Shuo Jiao1

AUTHOR AFFILIATIONS See affiliation list on p. 14.

ABSTRACT As the most common approach for the restoration of degraded grasslands,
the use of grass-legume mixtures has long been recognized for its role in increas­
ing aboveground biomass and resisting grassland degradation. However, whether the
legumes in these mixtures can help neighboring plants resist the decline in biomass
caused by the loss of soil microbial diversity remains a question worthy of investigation.
To address this, we employed a dilution method to create a gradient of decreasing
microbial diversity in soil and utilized full-factorial combinations of legumes and two
grasses to investigate the crucial role of legumes in the mixture. The results showed that
compared to monoculture, the mixture of Medicago sativa L. and Elymus dahuricus Turcz.
enhanced the biomass of grass species under conditions of soil microbial diversity loss.
We then discovered that a significantly enriched Pseudomonas (ASV53), in the grass-
legume mixtures under conditions of microbial diversity loss, was positively correlated
with plant biomass and nitrogen-fixing (nifH) gene abundance, implying that it could
be a keystone species. In addition, the grass-legume mixture increased the deterministic
processes of microbial community enrichment in the root zone soil by enhancing the
process of homogeneous selection. Functional predictions revealed that grass-legume
mixtures increased the potential abundance of N-related and phototrophy-related

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microbial communities in the root zone soil. This study provides an important insight
into the mechanism underlying the role of legumes in increasing and maintaining grass
biomass despite soil microbial diversity loss.

IMPORTANCE Grass-legume mixtures are a common practice for establishing artificial

grasslands, directly or indirectly contributing to the improvement of yield. In addition,
this method helps maintain soil and plant health by reducing the use of chemical
fertilizers. The impact of grass-legume mixtures on yield and its underlying microbial
mechanisms have been a focus of scientific investigation. However, the benefits of
mixtures in the context of soil microbial diversity loss remain a problem worthy Editor Nick Bouskill, E. O. Lawrence Berkeley National
of exploration. In this study, we examined different aboveground and belowground Laboratory, Berkeley, California, USA
diversity combinations to elucidate the mechanisms by which grass-legume mixtures
Address correspondence to Gehong Wei,
help maintain stable yields in the face of diversity loss. We identified the significantly weigehong@nwsuaf.edu.cn, or Shuo Jiao,
enriched Pseudomonas genus microbial ASV53, which was gathered through homogene­ shuojiao@nwsuaf.edu.cn.
ous selection and served as a keystone in the co-occurrence network. ASV53 showed a Yu Liu and Wei Yan contributed equally to this article.
strong positive correlation with biomass and the abundance of nitrogen-fixing genes. Author order was determined alphabetically.
These findings provide a new theoretical foundation for utilizing grass-legume mixtures The authors declare no conflict of interest.
to enhance grass yields and address the challenges posed by diversity loss.
See the funding table on p. 15.

KEYWORDS microbial diversity loss, grass-legume mixtures, root zone, microbial Received 18 July 2023
community, nitrogen fixation bacteria Accepted 22 September 2023
Published 30 October 2023

G rasslands are one of the most important biomes on Earth, covering nearly one- Copyright © 2023 Liu et al. This is an open-access
article distributed under the terms of the Creative
third of the planet’s land surface (1). They provide a wide range of ecological,
Commons Attribution 4.0 International license.

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economic, and social benefits, including carbon sequestration, livestock grazing, and
biodiversity conservation (2). However, grasslands are facing numerous threats,
with degradation being one of the most significant challenges. Grassland degradation
is the loss of grassland productivity and biodiversity due to various factors such as
overgrazing, land-use change, climate change, and invasive species (3). This phenom­
enon has become a global concern, with significant impacts on the environment and
human societies; consequently, the restoration of degraded grasslands has emerged
as a pressing concern among scientists and local governments (4). Restoration manage­
ment of degraded grasslands typically involves a range of techniques, including fencing
to exclude large herbivores, raking to remove dead vegetation and reseeding with
appropriate plants, fertilization to enhance soil fertility and productivity, turf transplan­
tation to establish new vegetation patches, as well as controlling rodent and weed
populations to minimize competition for resources (5). Among these practices, the
establishment of artificial or semi-artificial grasslands is currently the most widely used
method, as it can select suitable grass species, improve grassland productivity, and
relieve the pressure on natural grassland (6).
Currently, grass-legume mixtures are the most commonly used planting pattern for
artificial grasslands compared with traditional monoculture (7, 8). The key reason for
selecting legumes as the main crop for a mixture in agriculture is their ability to fix
nitrogen, which is highly valued for enhancing soil fertility and reducing dependence
on synthetic nitrogen fertilizers (9). However, not all the nitrogen fixed by legumes
necessarily flows to neighboring plants. Studies have found that when the soil environ­
ment changes, legumes will prioritize their use of fixed nitrogen, rather than exhibit­
ing the “good neighbor” role as previously believed (10). Although there have been
numerous studies exploring the benefits and mechanisms of grass-legume mixtures on
grass yield, most of these studies have primarily focused on investigating the impact
of different aboveground plant combinations (11, 12). The enrichment of underground
microbial communities in response to these combinations and their potential role in
helping plants resist the adverse effects of diversity loss has been largely overlooked (13).
Plants and microbial communities have a long co-evolutionary history, and their
interactions play crucial roles in shaping soil microbial structures and ecosystem

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functioning (14). One important way that plants influence soil microbial communities
is via the release of root exudates, which can attract or repel different microbial taxa
(15). As a result, plants can selectively enrich specific microbial groups in the root
zone, leading to a plant-specific microbial community structure. Due to their beneficial
effects on plant growth and health, plant growth-promoting bacteria (PGPR) have been
extensively studied as potential biofertilizers and biocontrol agents for various crops (16–
18). Within the aforementioned PGPR, the most notable are the nitrogen-fixing bacteria
that assist plants in acquiring nitrogen from the environment. Apart from the symbiotic
nitrogen-fixing bacteria that trigger differentiated structures on the host plant (root
nodules of legumes and actinorhizal plants), there is also a diverse range of free-living
nitrogen-fixing bacteria that can associate with the root system of graminaceous plants,
such as Klebsiella pneumoniae, Azotobacter vinelandii, and Azospirillum brasilense (19).
Pseudomonas stutzeri A1501 can assist with nitrogen fixation in the Poaceae family,
further expanding the range of nitrogen-fixing bacteria. P. stutzeri A1501 can colonize
the root surface and invade the superficial layers of the root cortex (20). However,
it is not clear whether the root zone of grasses will become enriched with specific
microbial communities due to the influence of adjacent legumes within mixed grass-
legume plantings. Nonetheless, plant species and cultivation methods do affect the
community structure of root zone microbes, which has theoretical guidance significance
in agricultural production (21), as such, it is important to clarify the role of legumes in
these mixed plantings.
Members of Poaceae and Fabaceae are the representative species types in grasslands,
with Medicago sativa L. (legume), Elymus dahuricus Turcz. (grass), and Festuca elata Keng.
(grass) being three commonly used forage species for the establishment of artificial

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pastures (22–24). In this study, we used these three plants for pot cultivation under
different plant combinations and soil microbial diversities. The use of pot experiments
enables the identification of microbial communities that have a significant impact on
plant growth, as they provide a more sensitive reflection of microbial changes within
the root zone (25). We systematically investigated the enriched microbial communi­
ties of the root zone soil in the grass-legume mixture. Our first objective was to
determine whether the grass-legume mixture could help enhance the yield of grass
in the context of soil microbial diversity loss. Subsequently, we intended to decipher
the underlying mechanisms behind this phenomenon by focusing on the significantly
enriched microbial taxa. Our findings revealed that the mixture exhibited deterministic
enrichment of the Pseudomonas genus member ASV53 in the face of diversity loss.
This organism showed a significant positive correlation with biomass and N-related
functions. Through the assistance of these communities, the grass was able to mitigate
the negative impacts of soil microbial diversity loss and maintain biomass stability.

RESULT
The construction of a gradient for soil microbial diversity and the impact of
grass-legume mixtures on biomass
We successfully created a loss gradient of soil microbial diversity and found that the
microbial α-diversity in the root zone decreased with increasing dilution (Fig. S1a),
and significant differences in β-diversity were observed across soil dilution gradients
(Fig. S1b). When comparing the biomass of grass under monoculture and grass-legume
mixtures conditions, we found that grass-legume mixtures (MP) resulted in a significant
(P < 0.05) increase in the total biomass compared to monoculture (P) (Fig. 1a). To
further ascertain the factors contributing to the total biomass increase, we compared
the individual plant weights of grasses and legumes under monoculture and in mixture.
The results revealed that the increase in individual plant biomass was more pronounced
in the grass than in legumes (Fig. 1b). This was confirmed even under conditions of
soil microbial diversity loss, as the individual weights of both grass (M) and legume
(P) decreased with the decline in microbial diversity in monoculture, the grass-legume

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mixtures (MP) increased the biomass of grass and maintained its biomass stability
despite the reduction in soil microbial diversity (Fig. 1c). These findings suggest that
grass-legume mixtures can be an effective approach for improving productivity in the
establishment of artificial grasslands. In addition, due to the involvement of legumes, we
also compared the content of ammonium and nitrate nitrogen in the soil under different
plant combinations and found no significant difference (P > 0.05) between monocultures
and the mixtures (Fig. S3). This suggests that the increase in grass biomass under the
mixture was not achieved by changing the physicochemical properties of the soil.

Microbial community diversity, structure, and composition of grass-legume

mixtures
To investigate the impact of different plant combinations on the composition and
structure of root zone soil microbial communities, we first compared the differences in
microbial α-diversity between monoculture and grass-legume mixtures. The observed
richness, Shannon, and inverse Simpson indices showed no significant difference (P >
0.05) between monocultures and grass-legume mixtures, indicating that microbial
diversity may not be the main driver of yield benefits from the mixture (Table S2). We
then used the significance test methods of multi-response permutation procedure
(MRPP), analysis of similarities (ANOSIM), and permutational multivariate analysis of
variance (PERMANOVA) to explore the β-diversity of root zone soil microbial communi­
ties under different plant combinations based on Bray-Curtis distance (Table S3). The
results showed that the microbial communities in monocultures and the mixtures were
not significantly different (P > 0.05) (Fig. S4).
To summarize, there were no significant differences in microbial α- and β- diversity of
the root zone between monocultures and grass-legume mixtures. However, there was a

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FIG 1 The variation in biomass under different plant combinations. Lowercase letters indicate significant differences. M_mono, Medicago sativa L. monoculture;
G_mono, Festuca elata Keng. Monoculture; P_mono, Elymus dahuricus Turcz. monoculture; GP_mix, Festuca elata Keng. and Elymus dahuricus Turcz. mixture;
MG_mix, Medicago sativa L. and Festuca elata Keng. mixture; MP_mix, Medicago sativa L. and Elymus dahuricus Turcz. mixture; MGP_mix, a mixture of Medicago
sativa L., Festuca elata Keng. and Elymus dahuricus Turcz.. (a) The overall changes in biomass within the seven plant cultivation patterns. (b) Comparison of the
individual plant weight of Medicago sativa L. and Elymus dahuricus Turcz. when grown separately versus in a mixture. Analyses of variances (ANOVAs) were
conducted to test for significant differences, with F values and their significance given in the top left-hand corner (P < 0.05). (c) The changes in individual plant
weight of Medicago sativa L. and Elymus dahuricus Turcz. under the condition of soil microbiome diversity loss. The values in the figure legend named 100,
102, 104, and 106, represent the dilution gradients of the soil suspension. Specifically, 100 signifies the undiluted original mother solution, 102 corresponds to a
100-fold dilution, 104 denotes a 10,000-fold dilution, and 106 signifies a 100,000-fold dilution.

significant increase in biomass for the grasses in the mixture; thus, we believe that there
must be some soil microbial community members that play a crucial role despite their
insignificant diversity changes. Therefore, a classifier program was used to annotate the
root zone soil microbial communities, and comparisons were made at the phylum and
genus levels under different plant combinations. All ASVs were classified into 525 genera
and 42 phyla. We found that the microbial composition in the root zone of different plant
combinations was relatively similar. Proteobacteria (59.75%) were dominant (Fig. S2a) in
P monoculture compared with the MP mixture. The t-test results showed that Bacteroi­
dota were significantly less abundant in the mixtures (11.28%) than in monocultures
(16.35%) (Fig. S2b). At the genus level, Pseudomonas (23.28%) was dominant among
different plant combinations (Fig. S2c), and we found the abundance of Pseudomonas
was significantly higher in MP mixture (26.35%) than in P monoculture (14.38%) (Fig.

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S2d). Acinetobacter, a genus known to contain several pathogens, was ubiquitous in the
root zone soils and its relative abundance in MP mixture was 0.46%, while in P monocul­
ture it was 4.13% (Fig. S2d). To further determine the role of Pseudomonas in the mixture
plantings, we also used the method of linear discriminant analysis effect size (LEfSe) to
identify microbial taxa that showed significant changes across different soil dilution
gradients. The results indicated significant enrichment of Pseudomonas taxa by plants at
a dilution of 106 (Fig. 2a). Finally, we utilized the tree map to integrate and present the
results of both direct comparison of community relative abundance and LEfSe analysis
results (Fig. 2b). The above results implied that the grass-legume mixtures increased the
relative abundance of potential PGPR, such as Pseudomonas, and reduced the relative
abundance of pathogenic bacteria (e.g., Acinetobacter).

The enriched Pseudomonas genus member ASV53 in the grass-legume

mixtures and its functions
To ascertain which specific bacteria within the Pseudomonas genus played an important
role, we conducted a differential analysis comparing the ASVs that showed significant
changes among M and P monocultures and the MP mixture. Our analysis showed
that ASV53 was remarkably enriched in the mixture (Fig. 3a). We then conducted the
correlation between the abundance of ASV53, the copy number of nifH genes, and the
individual plant biomass of grass under the MP mixture. The results revealed a significant
positive correlation between the abundance of ASV53 and the copy number of nifH
genes (R2 = 0.9764, P < 0.05), as well as the biomass (R2 = 0.9543, P < 0.05) of grass (Fig.
3b). A previous study showed that P. stutzeri A1501 could help fix nitrogen in the Poaceae
family, expanding the understanding of nitrogen-fixing microbes (20). Given the strong
correlation between ASV53 and nitrogen fixation genes in our study, we constructed a
phylogenetic tree of all annotated Pseudomonas sequences and the downloaded 16S
rRNA gene sequences of the A1501 strain from NCBI and found that ASV53 and A1501

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FIG 2 We compared microbial taxa that exhibited significant changes across soil dilution gradients and identified which taxa were enriched in monoculture
and mixtures. (a) LEfSe plots showing bacterial abundance enriched in monoculture and mixtures. Histograms of different colors indicate taxa (ranging from the
genus level to the class level) that were abundant in the corresponding soil dilution gradient (P < 0.05); (b) The circular packing chart of taxonomic differences
between monoculture and mixture soils under different soil dilution gradients. The outermost to innermost circles represent phylum, class, order, and family
levels, respectively. The red font lists the genera that were the focus of this study. Solid circles represent genera. Bacterial taxa that were significantly enriched are
indicated in dark blue for monoculture and yellow for mixtures.

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FIG 3 Identifying the key microbes under different plant combinations and exploring their functions. (a) ASVs exhibiting significant differential abundance
were identified through differential analysis among monocultures of M_mono and P_mono, and the MP_mix. (b) Investigation of the relationship between the
abundance of ASV53 and the individual plant biomass of Elymus dahuricus Turcz., as well as the abundance of the nitrogen fixation gene nifH in the nitrogen
cycling. (c) Construction of an evolutionary tree to determine the phylogenetic relationship between all obtained Pseudomonas ASVs in our study and P. stutzeri
A1501, a known nitrogen-fixing Pseudomonas species.

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had a very close relationship (Fig. 3c). As the sequence information for ASV53 is V4-V5
region derived from high-throughput 16S rRNA sequencing data, we selected a segment
of the 16S rRNA from the A1501 whole-genome sequence and aligned it with ASV53. The
similarity between ASV53 and A1501 was 98.12%. The Pearson correlation heatmap also
indicated that ASV53 had a strong correlation with the nifH gene in all plant combina­
tions, but only in the MP mixture did ASV53 show a significant positive correlation with
plant biomass (Fig. S5a through c).
We then delved into the role of ASV53 in the entire microbial co-occurrence network.
In general, the MP mixture increased the number of nodes and edges in the network,
resulting in more positive correlations between nodes and promoting network stability.
In addition, different plant combinations significantly altered the modules within the
co-occurrence network (Fig. 4). More specifically, the MP mixture transformed the
network’s dominance from modules 1 and 5 to module 4, and Pseudomonas ASV53
became a crucial key node in the network, with the highest degree (Fig. 4a). To examine
the role of ASV53 further, we introduced functional gene data into the network and
scrutinized the correlation between nitrogen cycling-related genes and network nodes
in the context of the MP mixture. The outcomes revealed a significant positive relation­
ship between ASV53 and the nitrogen fixation gene nifH (Fig. S6b). Furthermore, while
assessing the changes in nitrogen cycling-related genes in the root zone soil across
various plant combinations, we observed that the MP mixture considerably boosted
the soil’s nitrogen fixation gene nifH content (Fig. S5). All the results mentioned above
indicated that the MP mixture can help enrich the Pseudomonas ASV53 in the root zone
soil of grass and that it has nitrogen-fixing ability, contributing to the biomass of grass.

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FIG 4 The changes in microbial co-occurrence networks and the role of key microbe ASV53 within different plant combinations. (a) The variations in
co-occurrence networks between monocultures of M and P, and MP mixture, as well as the changes in the abundance of ASV53 under the condition of soil
microbiome diversity loss. (b) The changes in the basic properties of the network structure in three plant combinations.

Bacterial community assembly process under monoculture and grass-legume

mixtures
We used null models to investigate whether the mixtures could bring about changes in
community assembly processes and further confirmed the effects of the mixture on the
root zone soil microbial communities. Undominated processes contributed the largest
fraction to the assembly of all plant combinations, in our focus on M and P monocultures
and their mixture, the proportions were 61.05%, 57.78%, and 53.03%, respectively (Fig.
5). Furthermore, the MP mixture resulted in a more deterministic community assembly
process (Homogeneous Selection) of root zone soil microbes, increasing from 20.53%,
13.33% under monoculture to 33.33%, indicating the enrichment of specific bacteria
by plants. Furthermore, we also predicted the potential microbial functions in the root
zone soil under M and P monocultures and MP mixture. The results were consistent
with the previous results, showing enrichment of nitrogen-fixing-related functions, and

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FIG 5 Assembly of the monocultures of M_mono, P_mono, and MP_mix. The inner circle represents the contribution of stochastic and deterministic processes to
bacterial assembly. The outer circle represents the percentage of detailed ecological processes belonging to stochastic or deterministic processes.

a decrease in potential disease risk (Fig. 6). Based on all the results above, we concluded
that the MP mixture could help to more deterministically enrich microbial communities
with nitrogen-fixing functions in root zone soil, particularly for grass (Fig. 7).

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FIG 6 Prediction of soil microbial functions under the scenarios of monocultures of M and P, and MP mixture. The respective
functionalities, from top to bottom, are as follows: carbon cycling-related functions, nitrogen cycling-related functions,
disease-related functions, and light-related functions.

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FIG 7 The model diagram depicts the phenomenon of grass-legume mixture aiding in biomass enhancement and enriching
Pseudomonas of root zone soil under conditions of microbial diversity loss. When Elymus dahuricus Turcz. (P) and Medicago
sativa L. (M) were grown individually, both species experienced a decrease in biomass under conditions of soil microbial
diversity loss. However, in the grass-legume mixture, homogeneous selection led to a significant enrichment of Pseudomonas
in the root zone. These Pseudomonas exhibited a strong positive correlation with biomass and the nitrogen-fixing gene nifH,
served as the keystone in the microbial co-occurrence network, and contributed to biomass increase.

DISCUSSION

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In this study, we first compared the biomass trends of grass under different plant
combinations and found that the MP mixture significantly increased the biomass of
grass, and could maintain its production stability under our simulated soil microbial
diversity loss. However, this increase in yield was not significantly correlated with
soil physicochemical properties, so, we then focused on the diversity and structural
differences of root zone microbial communities among the different plant combinations.
The results showed no significant differences in α- and β-diversity between monocul­
tures and mixture, but we found that the MP mixture significantly enriched Pseudomonas
under soil diversity loss. We then identified ASV53 which had a very close phylogenetic
relationship with the previously reported nitrogen-fixing P. stutzeri A1501. ASV53 not
only showed an important role in the microbial co-occurrence network but also had
a positive correlation with biomass and nitrogen fixation gene abundance in the MP
mixture. Moreover, the root zone soil of grass enriched ASV53 through homogeneous
selection, and functional prediction results showed that the mixture soil exhibited a
stronger N-cycling potential while reducing the risk of pathogenic bacteria occurrence.

The yield increase from the grass-legume mixture

The use of grass-legume mixtures is a common practice in both agricultural production
and the restoration of degraded grasslands (11, 26). Besides the nitrogen fixation ability
of legumes that can increase nitrogen input to neighboring plants, the mechanism
underlying the yield increase in grass species through mixture has long been a focus
of attention for ecologists and agricultural experts (27). Combining previous long-term
field experiments (28) with the conclusions of our study, we found that there were no

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significant changes in soil nitrate nitrogen, ammonium nitrogen content, total nitrogen,
total carbon, moisture content, and soil conductivity among different plant combina­
tions. This indicates that the grass-legume mixture may not have a direct impact on the
physical and chemical properties of soil, and the main reason for the increase in biomass
of grass species under mixed planting is not related to soil properties. This could be
because the nitrogen fixed by legumes is directed to meet their own needs before being
provided to surrounding plants, as confirmed by related research in tropical forests (29).
The “good neighbor” role of legumes requires a certain level of soil nitrogen content, and
only under conditions of high soil available nitrogen, can legumes reduce competition
with neighboring plants, ultimately achieving mutualism and harmonious coexistence
(10).
In addition, the loss of soil microbial diversity is also a significant problem faced
by grassland degradation (30). Many studies have shown that there is an important
relationship between microbial diversity and productivity in the soil (31, 32). Different
types of microbial communities play distinct roles, some can decompose organic matter
and release nutrients (33), while others may convert these nutrients into forms that
can easily be taken up by plants (34). Thus, a more diverse microbial community may
promote better nutrient cycling and increase plant nutrient use efficiency, ultimately
leading to higher crop yields. Some fungi can form symbiotic relationships with plants,
such as mycorrhizal fungi that associate with plant roots and help them absorb nutrients
(35, 36). We first compared the variations in biomass across different plant combinations.
We observed that the M and P mixture increased the biomass of P, while mixtures had
no significant effect on the biomass of G. Similar observations were made with plant
combinations GP and MGP. This is the reason why we focused our subsequent analysis
exclusively on the individual growth of M and P and their mixed cultivation as MP. The
occurrence of these patterns is not only attributed to the crucial role of the soil microbial
community, which we have been investigating but also to the inherent characteristics
of the plants themselves (37). As commonly used grass species in establishing artificial
grasslands, G and P exhibit preferences for specific soil environments and tolerances.
Empirical knowledge from livestock farming indicates that P thrives in nutrient-rich
soils (38). When P is mixed with M, the nitrogen-fixing capacity of M provides a more

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abundant nitrogen source, creating a nutrient-rich environment that favors the growth
of P. On the contrary, G responds sensitively to soil fertilization, and excessively high
nitrogen content in the soil can be detrimental to its growth (39). Thus, a mixture
with M does not increase G biomass. In addition, P demonstrates strong adaptability
to temperature, while G prefers cooler environments (40). The controlled temperature
conditions in our study may not have reached the optimal growth temperature for G.
Consequently, there were no significant differences in biomass, whether in monoculture
or mixture. In our artificially simulated soil microbial diversity loss experiment, both M
and P monocultures showed a decrease in biomass. However, the MP mixtures helped to
maintain the stable biomass of the grass, indicating that the mixtures must have formed
a protective mechanism to help the root zone microbes resist the adverse effects of
microbial diversity loss.

Grass-legume mixtures changed the composition of the root zone bacterial

community from grass
In the restoration processes of degraded grasslands, the selection of plant species and
planting methods can vary depending on local soil properties or climatic conditions,
and both can be important factors influencing soil microbial communities (41). In our
study, after a 2-month pot experiment, we found no significant differences in the
α- and β-diversity of soil microbial communities among different plant combinations.
This phenomenon could be attributed to multiple factors. First, functional redundancy
among soil microbial communities (42) can result in a similar overall functional diversity
regardless of changes in plant combinations. Second, the experimental scale, such as
pot experiments, may capture changes in microbial taxa with more sensitivity, but the

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short duration of the experiment may not have reached the threshold to significantly
impact overall α- and β-diversity. Lastly, other environmental factors, including soil type,
moisture, temperature, and nutrient availability, apart from plant combinations, can also
influence soil microbial communities and their diversity. If these environmental factors
are similar across different plant combinations, they may mask any potential differences
in the α- and β-diversity of soil microbial communities.
Bacteria are key components of important functions in soil and can have beneficial
effects on plant growth through direct or indirect interactions (43). Among them,
genera such as Pseudomonas, which are known to promote plant growth, can help
plants through mechanisms such as nodulation (44), antagonism (45), production of
IAA (indole-3-acetic acid) (46), and siderophore (47). We found that the MP mixture
significantly increased the abundance of Pseudomonas in the root zone soil. In addition,
the abundance of Acinetobacter, a group of potential pathogens found in a variety
of environments, including fresh and marine water, soil, and marine sediments (48),
was decreased in the mixture, compared to P monoculture. Altogether, the significant
changes in abundance of these two key microbial genera resulted in the increased yield
of the grass-legume mixture.
After identifying significant changes at the genus level, we aimed to further
investigate which ASV played a dominant role in the process of the MP mixture. We
employed microbial co-occurrence network analysis to identify key nodes in different
plant combinations and found that ASV53 had the most connections with other nodes
in the network for this mixture. As the ASV53 sequence was annotated as belonging
to the Pseudomonas genus in the database, we constructed a phylogenetic tree using
the 16S rRNA sequences of all Pseudomonas ASVs in our study and the 16S rRNA
sequence of P. stutzeri A1501, a nitrogen-fixing Pseudomonas strain reported in the
literature (20). The results showed that ASV53 was closely related to A1501. P. stutzeri
A1501 is a nitrogen-fixing bacterium isolated from paddy fields, which exhibits significant
nitrogen-fixing activity under microaerobic conditions, and its nitrogen fixation products
can be rapidly absorbed and utilized by rice plants (49). To investigate whether ASV53
possessed nitrogen fixation and plant growth-promoting abilities similar to A1501, we
incorporated nitrogen cycling-related functional gene data into the network analysis

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and found a strong positive correlation between ASV53 and the nitrogenase gene (nifH).
Moreover, we found that only in the MP mixture was the abundance of ASV53 signifi-
cantly positively correlated with the copy number of nifH and biomass. We also found
that homogeneous selection was an important process through which grass-enriched
ASV53 in the root zone soil. Although our study did not investigate the specific mech­
anisms of ASV53’s interactions with plants in depth, the analysis results suggest that
ASV53, similar to A1501, plays an important role in nitrogen fixation, helping grasses
and leading to increased biomass of grasses and resistance to the adverse effects of
decreased biodiversity during mixture planting patterns. This finding could be further
validated in subsequent experiments by isolating ASV53 from the soil of grass-legume
mixtures and investigating its genetic and phenotypic characteristics.

Conclusion
In conclusion, there was a significant increase in the biomass of grass under grass-
legume mixtures, and the legume maintained biomass stability when microbial diversity
in the soil was reduced. However, grass-legume mixtures showed no significant impact
on soil physicochemical properties. To elucidate the underlying mechanism of inter­
cropping on grasses, we conducted differential analysis and network construction and
identified that the pseudomonad ASV53 showed a significant increase in the mixture
over the monoculture. We found that ASV53 was closely related to the nitrogen-fixing P.
stutzeri A1501 and its abundance was significantly correlated with nitrogen-fixing gene
copy number and grass biomass under a mixture planting pattern, and it was enriched
in grass root zone soil by homogeneous selection. This study reveals new support for the

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restoration of artificially degraded grasslands, providing a new perspective on the soil

microbial interactions in the root zone during grass-legume mixtures.

MATERIALS AND METHODS

Building of model soil
Due to the physical properties of the local farmland soils, soil compaction often occurs
in pot experiments. To avoid compaction and repair the physical structure of the soil,
making the gaps between soil particles looser for the flow of water and nutrients, we
created a model soil by mixing fresh farmland soil (108°09′E, 34°31′N) passed through
a 2-mm sieve with an equal amount of fine sand. The model soil was then placed in
a double-layered sealed bag, leaving an air outlet. The soil samples in the bag were
sterilized in a sterilization pot at 121°C for 90 min and immediately sealed tightly with the
hope to prevent airborne bacteria from entering.

Construction of soil microbial and plant community diversity

The gradient of the diversity of soil microbial communities was constructed using the
dilution gradient method of bacterial suspension. After collecting and sieving the soil
samples, 100 g of the sample was weighed and placed in 1 L of sterile water, then
stirred to fully disperse the soil and obtain a mother liquor with a concentration of 100,
which was then sealed and reserved. The mother liquor was then diluted separately with
sterile water to achieve dilution factors of 102, 104, and 106. An amount of 300 mL of
the different bacterial suspensions was added to each bag of the sterilized model soil,
resulting in different gradients of soil microbial diversity (50). To achieve plant diversity,
we conducted treatments in each pot with a single plant species, two plant species
mixtures, and a three-plant species mixture (51). Each pot contained a total of 24 plants,
with 24 plants of each single species under monoculture (5 pots per soil microbial
diversity gradient), and 12 plants of each species under two plant mixtures (4 pots per

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soil microbial diversity gradient), for pots with the three plant species mixture, there
were 8 plants of each species (4 pots per soil microbial diversity gradient). For the sake
of simplicity, we will use the abbreviations M, P, and G to represent Medicago sativa L.
(legume), Elymus dahuricus Turcz. (grass), and Festuca elata Keng. (grass), respectively, in
the following text.

Establishment of a sterilized environment

The planting pots (21.5 cm in diameter and 14.8 cm in height) were covered with a
plastic bag with a height of 30 cm, and the interface between the plastic bag and
the pot was taped. Two holes were cut on each side of the plastic bag, and a 16 ×
16 cm sealing film was attached to the holes with tape to allow for air circulation.
First, a layer of non-woven fabric with a diameter of 20 cm was laid in the pot,
followed by a 1 cm thick layer of quartz sand on top of the fabric, and another
layer of non-woven fabric on top of the sand. After that, the different gradients of
microbial diversity soil were added. To avoid contamination from external sources,
we used sterilized forceps and gently planted the seeds that had reached the
planting conditions into the soil, with a sowing depth of about 2 cm. To ensure
consistency and minimize external factors, we maintained a temperature of 25°C,
relative humidity of 60% relative humidity (RH), CO2 concentration of 400 PPM, and
12 h of light each day throughout the experiment. We weighed the pot as a whole
to determine the amount of water needed and a sterile syringe to spray sterilized
water from the top of the bag and evenly watered the plants every 48–72 h. After
watering, the small hole was sealed with tape. All equipment used above had been
pre-sterilized, and all operations were carried out in a laminar flow hood.

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Sampling and measurement

The root zone soil samples and plant samples were collected after 2 months of planting.
A complete root system was dug out to collect root zone soil. Aboveground structures
were harvested from each pot, then dried at 70°C for 72 h, and their dry weight was
recorded after cooling. Our experiment included different gradients of soil microbial
diversity and combinations of different plant species. Under four soil microbial diversity
gradients (100, 10−2, 10−4, and 10−6), seven plant combinations (M, G, P, GP, MG, MP, and
MGP) were set, each combination had four replicates. In total, we collected a total of 112
root zone soil samples.

DNA extraction and amplicon sequencing

DNA was extracted from root zone soil using the FastDNA SPIN Kit for Soil (MP Biochemi­
cals, Solon, OH, USA), following the manufacturer’s instructions, and stored at −80°C until
use. We performed 16S rDNA amplicon sequencing of the root zone soil samples. For
prokaryotic amplicon preparation, the V4-V5 region of the 16S rDNA gene was amplified
by PCR using 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 907R (5′-CCGTCAATTCCTTTG­
AGTTT-3′) primers. PCRs were performed in triplicate in a 15-µL reaction mixture which
contained 7.5 µL of Phusion High-Fidelity PCR Master Mix (New England Biolabs, Ipswich,
MA, USA), 1 µL of forward and reverse primers (3 mM), 2.5 µL of template DNA (5 ng
µL−1), and 4 µL of ddH2O. PCR conditions were as follows: 1 cycle × 98°C for 1 min;
30 cycles × 98°C for 10 s, 50°C for 30 s, and 72°C for 30 s; 1 cycle ×72°C for 5 min. The
quality of amplicons was detected through electrophoresis on 2% agarose gel, and the
purity of amplicons was ensured using the Qiagen Gel Extraction Kit (Qiagen, Hilden,
Germany). The triplicate PCRs for each sample were combined and quantified on a
QuantiFluor-ST Fluorometer (Promega, Madison, WI, USA) following the manufacturer’s
protocol. 16S rDNA sequencing was performed on the Illumina HiSeq 2500 platform
(Illumina Inc., San Diego, CA, USA) at Novogene (Beijing, China) using high-output mode
with the paired-end method after library construction (NEB Next Ultra DNA Library Prep
Kit). DADA2 was used to process raw sequencing reads for each sample (clean data),
infer the unique amplicon variant (ASV) through error-corrected reads further quality

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control through the error model, and filter chimeras using the DADA2 pipeline (52).
Subsequently, the sequences were filtered, trimmed, and truncated at 210 bp of forward.
Then, based on the Bayesian algorithm, we used the SILVA reference database (v.12.8)
to classify representative sequences from each ASV (53). Non-bacterial ASVs (chloroplast,
mitochondria, unknown, Archaea, and plants) and ASVs with fewer than two reads were
also removed.

Quantitative microbial nitrogen cycling

Due to the presence of legumes in the experiment, we focused on the changes in
nitrogen-related functional genes of microbes in the root zone soil (54). We selected
five functional genes that represented the processes of nitrogen fixation, nitrification,
and denitrification, including nifH, AOA, AOB, nosZ, nirS, and nirK (55). Quantification was
carried out using the Thermo QuantStudio 6 Flex Real-Time PCR System. The primer
sequences and amplification programs used for each nitrogen cycling-related gene are
shown in Table S1. The reaction volume of 20 µL contained 10 µL of Maxima SYBR Green
qPCR Master Mix (Thermo), 0.5 µL of forward primer (10 µmol), 0.5 µL of reverse primer
(10 µmol), 10–20 ng of DNA template, and 8 µL of sterile ultrapure water. Each primer set
was conducted in triplicate qPCRs at a threshold cycle (CT) of 31 as the detection limit.
Finally, we averaged the three values, and each functional gene was standardized [0,1] by
the min-max approach (56).

Statistical analysis
Statistical analyses were implemented in R 4.1.2 unless otherwise stated. α-diversity was
calculated for each sample using richness, Shannon, and Inverse Simpson indices based

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on the normalized ASV abundance table using the rarefied method (57). Significance
testing between the two groups was conducted using the Wilcoxon test, with an
adjustment of the P-value for false discovery rate (FDR) (58). Bacterial α-diversity was
evaluated by calculating the Bray-Curtis distance matrices and visualized by principal
coordinate analysis (PCoA) or nonmetric multidimensional scaling analysis (NMDS).
These analyses were then subjected to MRPP, one-way analysis of variance (ANOSIM),
and PERMANOVA using the Adonis function of the “vegan” package in R to compare
community structure under monoculture and grass-legume mixtures (59). To clarify the
differences in composition between monoculture and mixed culture in classification
groups, and evaluate whether certain key bacterial taxa exhibit differential abundance
across different soil dilution gradients, the following analyses were conducted. First,
“LEfSe” (60) was employed to compare taxa that displayed significant changes under
various dilution gradients. Next, changes in bacterial abundance at the phylum and
genus levels among different plant combinations were directly compared. Finally,
enriched microbial taxa under different soil dilution gradients in both monoculture
and mixture were visualized via a tree map using the “ggraph” package (61). To further
investigate whether changes in productivity resulting from grass-legume mixtures were
attributable to specific microbial taxa, we utilized the DESeq2 package (62) in R to
identify ASVs that exhibited significant differences between mixtures and monoculture
conditions. Co-occurrence networks of root zone bacteria in grass-legume mixtures and
monocultures were constructed, with a focus on significantly different ASVs. Spearman
correlation scores were computed, retaining only correlations that were both statistically
significant (P < 0.01) and robust (Spearman’s r > 0.7 or r < −0.7). Network visualization
and analysis were performed using Gephi, where each node represented an ASV, and
each edge indicated a strong and significant correlation between the two nodes (63).
Phylogenetic trees were annotated and visualized in iTOL (Interactive Tree Of Life, an
online tool to display and operation of evolutionary trees) to determine whether ASVs
exhibiting significant differences are closely related to P. stutzeri A1501 (64). Subse­
quently, the correlation between the identified ASVs and yield as well as nitrogen fixation
genes was assessed. The Pearson correlation coefficient was calculated using the cor.test
function in R (65), and the resulting correlation matrix was visualized as a heatmap using

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the “corrplot” package (66). To predict the geochemical material cycle processes of the
bacteria, the prokaryotic taxonomic group (FAPROTAX) functional annotation database
was employed (67). When assessing the potential contribution of neutral processes to
microbial community assembly, we used neutral assembly (dominance test) models to
predict the relationship between the occurrence frequency of ASVs and their relative
abundance (68, 69). The model evaluated whether the microbial assembly process of the
bacterial community conformed to a niche-based process (prediction outside the model)
or neutral model (prediction inside the model).

ACKNOWLEDGMENTS

This work was supported by the National Key Research and Development Program
of China (Grant No.: 2021YFD1900500), the National Science Foundation for Excellent
Young Scholars of China (Grant No.: 42122050), and the National Science Foundation of
China (Grant Nos.: 42077222, 41830755, and 41807030).
All authors contributed intellectual input and assistance to this study and the
manuscript preparation. S.J. developed the original framework. Y.L. performed the
experiments with the help of Y.Y., Y.A., X.L., H.G., and Z.P.; Y.L. carried out the data analysis
and wrote the paper with the help of S.J. and G.W.

AUTHOR AFFILIATIONS
1
National Key Laboratory of Crop Improvement for Stress Tolerance and Production,
Shaanxi Key Laboratory of Agricultural and Environmental Microbiology, College of Life
Sciences, Northwest A&F University, Yangling, Shaanxi, China

Month XXXX Volume 0 Issue 0 10.1128/msystems.00755-23 14

Research Article mSystems

2
Gansu Vocational College of Agriculture, Lanzhou, China

AUTHOR ORCIDs

Yu Liu http://orcid.org/0000-0002-9362-6306
Shuo Jiao http://orcid.org/0000-0002-1367-6769

FUNDING

Funder Grant(s) Author(s)

MOST | National Key Research and Development 2021YFD1900500 Shuo Jiao
Program of China (NKPs)
National Science Foundation of Excellent Young 42122050 Shuo Jiao
Scholars of China
National Science Foundation of China 42077222, 41830755, Shuo Jiao
41807030

AUTHOR CONTRIBUTIONS

Yu Liu, Data curation, Investigation, Methodology, Project administration, Writing –

original draft | Wei Yan, Investigation, Methodology | Tongyao Yang, Investigation,
Methodology, Project administration | Yining An, Investigation, Methodology | Xiao­
meng Li, Investigation, Methodology | Hang Gao, Investigation, Methodology | Ziheng
Peng, Conceptualization, Investigation, Methodology | Gehong Wei, Funding acquisi­
tion, Resources, Validation | Shuo Jiao, Conceptualization, Funding acquisition, Project
administration, Resources, Supervision, Validation

DATA AVAILABILITY

The raw sequence data reported in this paper have been deposited in the Genome
Sequence Archive (70) in the National Genomics Data Center (71), China National
Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences,

Downloaded from https://journals.asm.org/journal/msystems on 04 December 2023 by 147.96.60.115.

under BioProject accession no. PRJCA018459 and are publicly accessible at https://
ngdc.cncb.ac.cn/gsa.

ADDITIONAL FILES

The following material is available online.

Supplemental Material

Supplemental material (mSystems00755-23-s0001.docx). Supplemental figures and

tables.

Open Peer Review

PEER REVIEW HISTORY (review-history.pdf). An accounting of the reviewer comments

and feedback.

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