Responses of soil microbial communities

and nutrient dynamics under continuous

alfalfa (Medicago sativa L.) cultivation
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panelPeiyan Qin , Long Hu , Yidan Liu , Xiao Hu , Xike Zhang , Al
a 1 a 1 a b a

exandre Soares Rosado , Gehong Wei , Chun Chen

c d a a

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Highlights
 •
Bacterial and fungal richness increased initially during the early
stages of continuous alfalfa cultivation but declined after a
decade.
 •
Bacterial and fungal communities showed significant changes in
structure and composition along continuous alfalfa cultivation.
 •
Bacterial genus Subgroup 10 and fungal
genera Cordyceps and Phaeomycocentrospora were linked to
soil carbon and nitrogen turnover.
 •
During 10-year continuous alfalfa cultivation, SOM, TN, NO -N 3
−

accumulated while NH -N and AP decreased.

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Abstract
Alfalfa (Medicago sativa L.) cultivation is a widespread soil erosion
prevention measure in the Loess Plateau of China. However, long-
term continuous alfalfa cultivation can adversely affect soil physical,
chemical, and biological properties. Previous studies have primarily
assessed the effects of continuous alfalfa cultivation on soil physical
and chemical properties, whereas the dynamics of microbial
communities and their influence on soil nutrient cycling require
further exploration. To address this research gap, we investigated the
responses in bacterial and fungal communities and nutrients of fields
continuously cultivated with alfalfa for 3, 5, and 10 years, in
comparison with those of the adjacent abandoned land. Soil sampling
was conducted in the Loess Plateau (Guyuan, China) in July 2019. Our
findings demonstrated that continuous alfalfa cultivation significantly
affected soil nutrient availability by augmenting SOM, TN, NO -N, TP,
3
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TK, and AK content while reducing NH -N and AP content with the

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prolongation of cultivation. At the microbial level, continuous alfalfa

cultivation altered microbial community diversity, structure, and
composition. The bacterial and fungal richness was enriched for
approximately 5 years, followed by deterioration after a decade.
Differences in alpha diversity among duration groups were limited,
whereas the clear variations in beta diversity indicated clear shifts in
community composition. Several genus-level microbial biomarkers,
such as the bacterial genus Subgroup 10 and the fungal
genera Cordyceps and Phaeomycocentrospora, were identified as
important predictors of SOM, TN, and NO -N content through
3
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random forest analyses, indicating their potential roles in soil C and N

cycling. These key taxa may participate in soil C and N turnover
through organic matter degradation, nitrification, denitrification, or
other soil processes. These findings underscore the potential effects of
continuous alfalfa cultivation on soil microbial communities and their
functional contributions to nutrient cycling in the Loess Plateau.
Consequently, this study underscores the importance of limiting the
cultivation duration in continuous alfalfa cultivation systems and the
crucial roles of microbial communities in regulating soil nutrients.
More attention should be paid to microbe–soil interactions in
continuous cultivation systems to achieve long-term soil ecological
integrity and productivity.
Introduction
Soil erosion represents a pervasive global concern with significant
implications for both the environment and socioeconomic progress,
warranting widespread attention from scientists and governments
(Borrelli et al., 2020). Among the regions heavily impacted by soil
erosion, the Loess Plateau in northwestern China is among the largest
affected areas, with erosion imposing substantial on-site and off-site
challenges on the national environment and society (Wen and Deng,
2020). The consequences of soil erosion in this region manifest as
land degradation, soil nutrient depletion, water scarcity, loss of
biodiversity, and diminished agricultural productivity (Zhao et al.,
2013). Consequently, various strategies have been employed to combat
soil erosion, including mulching techniques (Prosdocimi et al., 2016),
optimized drainage systems (Anache et al., 2017), contour farming
practices (Ricci et al., 2020), cessation of overgrazing (Nicu, 2018),
reforestation initiatives (Veldkamp et al., 2020), and vegetation
planting (Hou et al., 2016). Among these, vegetation planting has
emerged as a widely adopted and effective approach to address soil
erosion, with alfalfa (Medicago sativa L.) representing an herbaceous
legume possessing several advantageous traits for erosion control (Li
et al., 2017), such as water retention and C sequestration. Alfalfa helps
with water retention in the soil by increasing water infiltration
through its widespread roots and acting as mulches that protect
against evaporation of water from the soil surface (Daane et al., 2018).
In addition, alfalfa increases the water storage capacity of soil by
promoting soil aggregate stability through organic matter input via
dead root decomposition and root secretion (Blanco-Canqui and Jasa,
2019). Additionally, alfalfa offers notable economic benefits and is
highly recognized for its ability to fix atmospheric N through symbiotic
association with rhizobia, rendering it a vital N source for ecosystems
and the agricultural industry (Wang et al., 2018). Consequently, the
multifaceted advantages of alfalfa have led to its extensive cultivation
as a soil conservation strategy. For instance, Hunt et al. (2019)
demonstrated that incorporating crops with extensive and deep root
systems, including alfalfa, contributed to soil retention and erosion
prevention within arable fields. Moreover, Song et al. (2021)
highlighted the potential of alfalfa root growth in fostering SOM
accumulation and nutrient mineralization, thereby supporting
sustainable land use practices in the semiarid region of the Loess
Plateau.
Alfalfa cultivation plays a vital role in maintaining a balanced
terrestrial ecosystem in the Loess Plateau (Li and Huang, 2008).
However, prolonged and uninterrupted alfalfa cultivation can have
detrimental effects on soil properties (Jiang et al., 2006; Huang et al.,
2018; Wang et al., 2021). Previous studies have identified various
negative consequences of long-term alfalfa cultivation on soil
characteristics, including compaction of the soil structure and
decreased nutrient levels. For instance, She et al. (2009) observed an
increase in soil bulk density and a significant reduction in saturated
soil hydraulic conductivity in the upper 40-cm soil layer due to
continuous alfalfa cultivation. Additionally, Wang et al. (2021)
documented a progressive decline in AP content with an increasing
duration of alfalfa cultivation. Although numerous investigations have
explored the physical and chemical aspects of soil under continuous
alfalfa cultivation, the impact on soil microorganisms has received
comparatively less attention. The soil microbial community plays a
crucial role in nutrient cycling processes, encompassing N fixation
(Kuypers et al., 2018), C sequestration (Zhu et al., 2020), and P
mineralization (Dai et al., 2020). Therefore, it is essential to
investigate the influence of continuous alfalfa cultivation on the
diversity, structure, and composition of the soil microbial community.
Furthermore, deeper investigation into the links of these changes in
the microbial community to soil nutrient dynamics is warranted.
Existing research has several limitations regarding the investigation of
microbial communities and their associations with soil nutrient
dynamics under continuous alfalfa cultivation. First, a few studies
reported changes in soil microbial biomass associated with continuous
alfalfa cultivation (Sun et al., 2009; Wang et al., 2009; Raiesi and
Beheshti, 2014; Jiang et al., 2022). However, the ecological traits of
the microbial community, such as diversity and composition, were not
explored. Second, although other alfalfa cultivation-related research
investigated microbial ecology, these studies did not address the
effects of continuous cultivation or the role of the fungal community.
For instance, Tong et al. (2023) focused on the different effects of
alfalfa–corn rotation and alfalfa monoculture systems on soil bacterial
communities. Similarly, Zhang et al. (2018) investigated the bacterial
community in a mulberry (Morus alba L.)/alfalfa (Medicago
sativa L.) intercropping system. Finally, the contribution of the
microbial community to soil nutrient dynamics in continuous alfalfa
cultivation systems has not been adequately studied.
Therefore, further research is necessary to elucidate the effects of
continuous alfalfa cultivation on the soil microbial community,
particularly the fungal community. Moreover, the contributions of
changes in the microbial community on soil nutrient dynamics await
further investigation. The diversity of soil microbial communities is a
key factor influencing various basic ecological processes, such as
organic matter decomposition and nutrient cycling (Bastida et al.,
2019; Crowther et al., 2019). Changes in microbial composition also
reflect the impact of environmental changes, and they have further
implications for nutrient dynamics through the enrichment or decline
of functional taxa (Hermans et al., 2020). Soil chemical properties
including SOM, TN, NH -N, NO -N, TP, AP, TK, and AK content,
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3
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reflect the soil nutrient condition, and they are crucial for determining
the microbial functions related to nutrient turnover and investigating
the relationship between the microbial community and soil nutrient
cycling (Jiao et al., 2018).
The primary objectives of this research were to assess the temporal
dynamics of the ecological characteristics of microbial communities
under continuous alfalfa cultivation and evaluate changes in soil
nutrient parameters and the potential contributions of key bacterial
and fungal taxa to soil nutrient dynamics in the context of continuous
alfalfa cultivation. We hypothesized that the effects of continuous
alfalfa cultivation on microbial communities and soil nutrients are
duration-dependent and that some key microbial taxa act as crucial
contributors to the dynamics of soil nutrients. To test our hypotheses,
we sampled soil from abandoned land and fields subjected to 3, 5, and
10 years of alfalfa cultivation in the Loess Plateau. We examined the
diversity, structure, composition, and potential functions associated
with the nutrient cycling of soil bacterial and fungal communities and
assessed various soil physicochemical properties and enzyme
activities.
Section snippets

Experimental design
The study sites were located in Guyuan, Ningxia Hui Autonomous
Region, China (35°14′19″N to 36°31′12″N, 105°19′15″E to
106°57′18″E). This region is characterized by the semiarid Loess
Plateau with an average annual precipitation of 499.7 mm and an
average annual temperature of 7.0 °C. The predominant soil type in
the area is Cambic Arenosol (WRB, 2022), which encompasses a vast
expanse of >120,000 ha of alfalfa planting fields
(Guyuan_People's_Government, 2022). Four types of fields were

Dynamics of soil physicochemical parameters and enzyme

activity under continuous alfalfa cultivation
In this study, we investigated the changes in pH, SOM, TN, NH -N,
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and NO -N levels; and urease activity in soil under different durations

3
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of continuous alfalfa cultivation (Fig. 1). Soil pH exhibited

considerable decreases after 3 (8.33) and 10 years (8.12) of continuous
alfalfa cultivation compared to the control (8.52, p < 0.05). In
particular, the lowest pH was observed in the 10-year soil,
distinguishing it from the other groups. The SOM and TN content
noticeably increased after

Discussion
This study revealed that continuous alfalfa cultivation increased SOM,
TN, NO -N, TP, TK, and AK levels and urease, ALP, and CAT activity
3
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in soil while simultaneously decreasing soil pH and NH -N and AP

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levels. Changes in soil nutrient levels could significantly affect crop

productivity and ecosystem functions (Fornara et al., 2013; Delgado-
Baquerizo et al., 2017; Seabloom et al., 2021). Evident contributions to
the dynamics of soil characteristics from microbial communities were
confirmed by

Conclusion
The results revealed that continuous alfalfa cultivation exerted a
double-edged influence on soil nutrient availability. Specifically, we
observed increases in SOM, TN, NO -N, TP, TK, and AK content with
3
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increasing cultivation time, whereas NH -N and AP content declined.

4
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In addition, the continuous cultivation of alfalfa enriched the soil

microbial diversity for approximately 5 years, followed by
deterioration after a decade of cultivation. The microbial structure and
composition also

CRediT authorship contribution statement

Peiyan Qin: Writing – review & editing, Writing – original draft,
Visualization, Formal analysis. Long Hu: Writing – original draft,
Visualization, Formal analysis. Yidan Liu: Investigation, Formal
analysis. Xiao Hu: Investigation, Formal analysis. Xike
Zhang: Investigation, Data curation. Alexandre Soares
Rosado: Writing – review & editing. Gehong Wei: Validation,
Supervision, Resources, Project administration. Chun Chen: Writing
– review & editing, Supervision, Project administration,

Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

Acknowledgements
This work was supported by the National Natural Science Foundation
of China (42277118) and the Talent Introduction Startup Fund
of Northwest A&F University of China. The authors appreciate and
express gratitude to Dr. Gupta Vadakattu at CSIRO, Australia for his
valuable comments and suggestions on the manuscript, and to Wen
Luo and Chengyao Ren at the College of Life Sciences, Northwest A&F
University, for their essential help with data analysis.
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