Agricultural Water Management 295 (2024) 108781

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Agricultural Water Management

journal homepage: www.elsevier.com/locate/agwat

Rehydration under extreme drought conditions affected rhizosphere

microorganisms more than bulk soil in broomcorn millet farmland
Lixin Tian a, *, Yu Yang a, Youhong Song a, Baili Feng b, *
a
School of Agronomy, Anhui Agricultural University, Hefei, Anhui 230036, China
b
College of Agronomy, Northwest A & F University, Yangling, Shaanxi 712100, China

A R T I C L E I N F O A B S T R A C T

Keywords: Rehydration is a primary adaptation strategy for alleviating the detrimental effects of drought on crop growth.
Broomcorn millet However, the effects of rehydration on microbial communities in various compartments under drought condi­
Water regimes tions remain poorly understood. Herein, we explored the response patterns of bacterial and fungal community
Rhizosphere-associated microbiome
composition, diversity, ecological network, and assembly process in the bulk soil and rhizosphere of broomcorn
Network analysis
Assembly process
millet farmland at 5, 10, 20, and 30 days after rehydration during drought conditions. Compared to drought,
rehydration significantly decreased the organic matter (OM), total nitrogen (TN), available potassium (AK),
available phosphorus (AP), nitrate nitrogen (NO-3-N), and ammonium nitrogen (NH+ 4 -N) contents in rhizosphere,
but had no obvious influences on the OM and TN contents in bulk soil on the four sampling days. In the
rhizosphere of broomcorn millet field, Firmicutes and Actinobacteria were enriched in drought condition, but
Proteobacteria and Bacteroidetes were enriched in rehydration regime. Compared to drought, the relative
abundances of Eurotiomycetes, Dothideomycetes, Mortierellomycetes, and Leotiomycetes were enhanced, but
Sordariomycetes was reduced on the 5th and 10th days after rehydration. Rehydration increased the bacterial
and fungal observed ASVs and Shannon index in the rhizosphere to varying degrees, but had little effect on the
bulk soil. Null-model analysis indicated that rehydration deceased the stochastic process of fungal communities
in the bulk soil, whereas had no influence on deterministic process of bacterial community in the bulk soil and
rhizosphere. More importantly, rhizosphere properties had a greater impact on the bacterial and fungal com­
munity composition, diversity, and assembly process than bulk soil. Network analysis revealed that rehydration
improved the interconnected taxa in the rhizosphere bacterial network, but reduced microbial interactions in the
rhizosphere fungal network compared to drought. This work provides a theoretical foundation for elucidating the
role of rehydration regime in governing the ecological services of microbiome of broomcorn millet farmland
under drought conditions.

1. Introduction
Dunhuang City of Gansu province is located at the westernmost end
of the Hexi Corridor in Northwest China. It is a typical extremely arid
region because of its large evaporation and very little precipitation (Qi
et al., 2018). In recent years, global climate change has led to the decline
of groundwater level, coupled with population growth and rapid
development of tourism in the region, further exacerbating the deteri­
oration of the originally fragile ecological environments of Dunhuang
Oasis (Liang et al., 2020; Zhang et al., 2014).Additionally, this region
mainly planted crops that consumed more water, including grapes (He
et al., 2023a) and cotton (Wang et al., 2008), which further exacerbated
the crisis of water scarcity (Zhang et al., 2003). Therefore, the region
needs to further optimize water resource allocation to achieve the goal
of improving water utilization efficiency and crop yield. A previous
study in this region showed that supplemental irrigation at the heading
stage can significantly improve the photosynthetic capacity of foxtail
millet leaves and grain yield compared to no irrigationand supplemental
irrigation at jointing stage and filling stage, respectively (Liu et al.,
2017).However, currently, no study has explored the dynamic changes
of soil microorganisms in broomcorn millet farmland after rehydration
in the extremely arid region.
Soil microorganisms have a vital role in the services and functions of
farmland ecosystem as they can serve as bio-fertilizers or biological

* Corresponding authors.
E-mail addresses: tianlixin@ahau.edu.cn (L. Tian), fengbaili@nwafu.edu.cn (B. Feng).

https://doi.org/10.1016/j.agwat.2024.108781
Received 28 November 2023; Received in revised form 8 March 2024; Accepted 16 March 2024
Available online 21 March 2024
0378-3774/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-
nc/4.0/).
L. Tian et al.
insecticides to improve the soil micro-environment and thus ensure crop
growth (Jiao et al., 2018; Kaur et al., 2021). Rhizosphere, acting as a
reservoir of seeds present in the bulk soil, represents the immediate
interface between the bulk soil and plant roots. This zone offers a suit­
able habitat for a diverse array of soil microorganisms (Fan et al., 2017;
Ling et al., 2022). Previous studies observed that water deficit reduced
the fungal alpha diversities of bulk soil and rhizosphere in grape fields
(Carbone et al., 2021) and also changed the fungal and bacterial com­
munity structures (Barnard et al., 2013), which was attributed to the
selection of low-humidity-tolerant taxa in this severe environment
(Naylor and Coleman-Derr, 2018). In addition, the variations of root
morphology and exudates under drought stress can also influence the
compositions and structures of root-associated microbial communities
(Henry et al., 2007; Naylor and Coleman-Derr, 2018; Santos-Medellín
et al., 2017). However, rehydration regime on the dynamic variations of
the diversities, compositions, and structures of rhizosphere-associated
microbial community under extreme drought conditions have rarely
been reported.
In various ecosystems, the interactions between taxa form a complex
microbial network, thereby maintaining the stability of the ecosystem
(Duffy et al., 2017). More importantly, the interactions between
different taxa can be understood by assessing several network topolog­
ical properties, which not only can reflect the community’s response to
environmental changes, including extreme weather, but also provide
more significant insights beyond microbial diversity and composition
(Guan et al., 2020). Several studies have confirmed that the stability of
soil fungal networks was higher than that of bacterial networks under
drought conditions (Chen et al., 2022; de Vries et al., 2018), and aridity
decreased the diazotrophic and protistan co-occurrence network
complexity (Chen et al., 2022; Lei et al., 2024). Meanwhile, the rhizo­
sphere bacterial network of drought-tolerant cultivar was more complex
than that of drought-sensitive cultivar (Dao et al., 2023; Liu et al.,
2021a). At present, however, the variations in microbial networks after
rehydration under extreme drought conditions, especially in distinct
compartments, are still rarely reported.
Clarifying the microbial assembly process is a key theme of concern
in microbial ecology nowadays (Ning et al., 2019). The assembly pro­
cesses are dynamically governed by stochastic process and deterministic
process, and the ratio of the two will undergo coordinated changes when
agricultural measures disturb the soil micro-environment (Li et al.,
2023; Tian et al., 2022a). Drought stress shaped the relative ratios of
deterministic and stochastic processes in the rhizoplane and rhizosphere
bacteria assembly, and the relative importance of assembly processes
was associated with community diversity and composition of rhizode­
posits (Munoz-Ucros et al., 2021; Zhang et al., 2016). Previous studies
reported that severe water deficit caused strong and continuous envi­
ronmental filtration, which in turn led to deterministic community as­
sembly (Li et al., 2023), and soil aridity was a key environmental factor
affecting the bacterial community assembly (Lei et al., 2024; Pan et al.,
2022). However, a comprehensive understanding of how the bacterial
and fungal assembly processes in different compartments respond to the
rehydration process under extreme drought conditions is still scant.
Broomcorn millet is considered the future smart minor grain crop
due to its barrenness tolerance, drought tolerance, and superior water-
use efficiency (Ma et al., 2023; Yang et al., 2023). In this work, we
explored the fungal and bacterial community composition, diversity,
ecological network, and assembly process in the bulk soil and rhizo­
sphere of broomcorn millet field after rehydration for different days
under extreme drought conditions. We aimed to (i) analyze the varia­
tions in the fungal and bacterial community composition and diversity
after rehydration under extreme drought conditions, as well as their
relationships with environmental variables; (ii) explore whether rehy­
dration under extreme drought conditions can improve the taxa in­
teractions in fungal and bacterial networks, thereby increasing network
stability and complexity; (iii) clarify how rehydration regulated the
bacterial and fungal assembly processes under extreme drought
2
Agricultural Water Management 295 (2024) 108781
conditions, as well as the critical environmental variables that influ­
enced the assembly processes. This study provides new insights for
providing implementable farmland management practices in extreme
drought regions.
2. Materials and methods
2.1. Experimental design
A field experiment was implemented at the Dunhuang experiment
station of Gansu Academy of Agricultural Sciences, Gansu Province,
China (40◦ 08′N, 94◦ 43′E). The region is a typical warm-temperate arid
climate, with an average altitude of 1187 m. The mean annual tem­
perature is 9.4◦ C, mean annual evaporation is 2486 mm, and dryness is
19.6. The mean annual precipitation is 9.34 mm, 17.34 mm, and
29.34 mm from 2020 to 2022, respectively. The precipitation during the
growing season of crops is 15.52 mm in 2022. The soil type is
anthropogenic-alluvial soil.
This experiment was implemented in 2022 with a randomized block
design with triplicate in each plot (20 m2 per plot) with both water re­
gimes: (i) No irrigation during the whole growth stage of broomcorn
millet (Drought); (ii) Irrigating 600 m3 ha− 1 water only conducted once
at the flowering stage of broomcorn millet (Rehydration). Broomcorn
millet variety “Shaanmi No. 2” was adopted in this study. The planting
density is 600,000 plants ha− 1. The sowing date was 20 April, and the
harvest date was 10 September. To ensure the emergence of crops
neatly, 600 m3 ha− 1 water was uniformly irrigated before sowing. When
the soil moisture content reached about 15%, a rototiller was employed
to incorporate fertilizers into the soil after applying base fertilizer,
including 750 kg ha− 1 organic fertilizer (organic matter ≥ 20%,
N+P2O5+K2O=15%, Gansu Luneng Agricultural Science and Technol­
ogy Co., Ltd, Gansu, China) and 600 kg ha− 1 compound fertilizer (N-
P2O5-K2O = 19–19–19, Jiashili Fertilizer Co., Ltd, Hubei, China). Other
farmland management measures are carried out following the standards
of local farmers.
2.2. Agronomic characteristics and yield compositions
At the maturity stage, three plants were randomly collected from
each plot to estimate the plant height, stem diameter, spike length, and
aboveground dry weight. Plant height was determined by measuring
from the base to the top of the main stem with a steel tape. Stem
diameter was estimated using vernier caliper at the base of the main
stem. The leaf, stem, and spike of broomcorn millet were dissected and
dried in an oven at 105 ◦ C for 30 minutes to eliminate moisture and then
at 80 ◦ C until a constant weight was achieved. Dry weight was estimated
using an electronic analytical balance.
At the maturity stage, the plants were manually harvested from
15 m2 area of each plot, and the broomcorn millet grains were naturally
dried to weigh the grain yield. Meanwhile, three main spikes were
randomly collected from each plot to calculate the spike length, 1000-
grain weight, and grain weight per spike of broomcorn millet.
2.3. Soil moisture content
Soil samples were collected at 20 cm intervals from a depth of
0–100 cm with a soil auger (diameter 38 mm) in each plot on the 5th (30
July), 10th (4 August), 20th (14 August), and 30th days (24 August)
after rehydration, respectively. Soil moisture content was determined by
subjecting the samples to oven drying at 105 ◦ C until a constant weight
was achieved.
2.4. Soil sampling
The bulk soil and rhizosphere were collected on the 5th, 10th, 20th,
and 30th days after rehydration, respectively. About 10 plants were
L. Tian et al.
selected from each plot with a clean spade and gently shaken to elimi­
nate large soil clumps. Collecting soil adhering to a distance of 2 mm
from the plant roots and mixed it into a rhizosphere sample (Tian et al.,
2022). The bulk soil and rhizosphere samples underwent sieving
through a 10 mesh, resulting in two portions. One portion was subjected
to DNA extraction at − 80 ◦ C, while the other portion was kept at − 20 ◦ C
for soil properties analysis. Soil properties, including soil pH, organic
matter (OM), total nitrogen (TN), nitrate nitrogen (NO-3-N), ammonium
nitrogen (NH+ 4 -N), available potassium (AK), and available phosphorus
(AP). Specific assays were described in the Supplementary Material.
2.5. DNA extraction and sequencing
We collected total DNA from 500 mg of fresh bulk soil and rhizo­
sphere samples employing the FastDNA® spin kit (MP Biomedicals Co.,
Ltd., Santa Ana, CA, USA) following the manufacturer’s protocols. The
concentration and purification of DNA were estimated using NanoDrop
UV-Vis 2000 spectrophotometer (Thermo Scientific, Wilmington, DE,
USA). The DNA quality was assessed through one percent agarose gel
electrophoresis. The extracted samples’ total DNA was sent to Majorbio
Bio-pharm Technology Co., Ltd. (Shanghai, China) for sequencing.
The primer pair consisting of 338 F (5’-ACTCCTACGGGAGGCAGCA-
3’) and 806 R (5’-GGACTACHVGGGTWTCTAAT-3’) was used, with
unique barcodes, to amplify the V3–V4 region of bacterial 16 S rRNA
genes. Additionally, the primer pair comprising ITS1F (5′-
CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-
GCTGCGTTCTTCATCGATGC-3′), with unique barcodes, was utilized to
amplify the internal transcribed spacer (ITS) region of fungal rRNA
genes. The preparation of the PCR reaction system was described in the
Supplementary Material. The PCR products were purified employing an
AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA,
USA) and quantified employing QuantiFluor™-ST (Promega, USA) as
per the manufacturer’s instructions. The purified amplicons were then
combined in equal proportions and subjected to pair-end 2 × 300 bp
sequencing on the Illlumina Miseq platform at Majorbio Bio-pharm
Technology Co., Ltd. (Shanghai, China). The ITS rRNA and 16 S rRNA
gene sequencing reads were initially demultiplexed and subjected to
quality control using Trimmomatic software. Subsequently, the reads
were merged together employing the FLASH software (http://ccb.jhu.
edu/software/FLASH/). A detailed account of the specific implementa­
tion standard can be observed in the Supplementary Material. The
sequence resulting from the application of DADA2 noise reduction
processing is commonly referred to as the amplicon sequence variant
(ASV). The fungal and bacterial ASV sequences were aligned with UNITE
databases (http://unite.ut.ee/index.php, v8.2) (Nilsso et al., 2019) and
Silva data (https://www.arb-silva.de/, v138) (Quast et al., 2013),
respectively. Raw sequences of bacteria and fungi are released in the
NCBI Sequence Read Archive under PRJNA1028264 and
PRJNA1028307, respectively.
2.6. Statistical analysis
Fungal classes and bacterial phyla that exhibited a relative abun­
dance higher than one percent were retained for analysis. Linear
Discriminant Analysis Effect Sizes (LEfSe) was utilized to explore po­
tential biomarkers in the microbial community of the bulk soil and
rhizosphere under both water regimes. This analysis covered six taxo­
nomic levels, spanning from the kingdom to genus level for fungal and
bacterial taxa. The LEfSe approach relied on LDA scores to discern sta­
tistically significant differences (Segata et al., 2011). The observed ASVs
and Shannon index were assessed adopting picante package. Principal
coordinate analysis (PCoA) based on Bray–Curtis distances was imple­
mented to exhibit the clustering of rehydration regimes and distinct
compartments using ape package. Analysis of similarities (ANOSIM) and
permutational multivariate analysis of variance (PERMANOVA) were
employed to evaluate the impacts of water regimes, compartments, and
3
Agricultural Water Management 295 (2024) 108781
their interactions on the dissimilarity of bacterial and fungal commu­
nities. The anosim function in the vegan package was utilized for ANO­
SIM, while the adonis function was used for PERMANOVA. To enhance
the linearity and normality of the data, environmental variables (no
including pH) were subjected to a log10(x+1) transformation. The se­
lection of environmental variables was carried out using the forward
selection procedure, which utilized the ordiR2step function in the vegan
package. The forward selection process was terminated when no further
increment in the model selection criteria (R2) was observed upon adding
additional environmental variables, or when the model’s significance
level (p < 0.05) was attained. The important environmental variables
identified were subsequently adopted for constructing distance-based
redundancy ordinations (db-RDA) using the capscale function in the
vegan package.
The phylogenetic community assembly processes were assessed with
the null-model analysis. The investigation into the differences in
phylogenetic and taxonomic diversity was carried out through the uti­
lization of null-model-based phylogenetic and taxonomic β-diversity
matrices. Specifically, two indices were used: the Bray–Curtis-based
Raup–Crick (RCbray) and the β-nearest taxon index (βNTI). To briefly
summarize, |βNTI| > 2 indicated the prevalence of deterministic pro­
cesses, with significantly more phylogenetic turnover than expected
(βNTI > 2) or less phylogenetic turnover than expected (βNTI < − 2),
representing variable selection and homogeneous selection, respec­
tively. On the other hand, |βNTI| < 2, RCbray < − 0.95, and RCbray > 0.95
denoted the impacts of homogeneous dispersal and dispersal limitation,
respectively. Additionally, |RCbray| < 0.95 signified the influence of the
“non-dominant” fraction, which primarily consisted of weak selection,
diversification, weak dispersal, and/or drift.
For ecological network construction, ASVs that existed in more than
1/5 of all samples were used to avoid spurious correlations (Faust,
2021). Spearman correlation between the ASVs was estimated with the
Hmisc package (Harrell and Dupont, 2017; Li et al., 2022). Correlations
meeting the criteria of |r| > 0.6 and FDRp < 0.01 were deemed signif­
icant and incorporated into the ecological networks (Zheng et al., 2021).
These ecological networks were then visualized with the Gephi software
(v.0.10.1, https://gephi.org/). Additionally, node-level topological
properties, including the number of nodes, edges, average clustering
coefficient (ACC), average path length (APL), average degree, diameter,
and modularity were calculated using igraph package.
Differences in soil physicochemical properties between the both
water regimes were determined using the Duncan’s test (p < 0.05) with
the assistance of SPSS software (v19.0, SPSS, Inc., Chicago, IL, USA).
Pearson correlation tests were conducted using the psych package to
evaluate the relationships between bulk soil and rhizosphere physico­
chemical properties versus the bacterial and fungal alpha diversities and
community compositions. The Mantel test was employed to examine the
impacts of environmental variables on the diversity and assembly pro­
cess of the bacterial and fungal communities under the water regimes.
3. Result
3.1. Agronomy traits, crop yield, and soil moisture content
Compared to drought treatment, the plant height, dry weight of leaf,
stem, and spike of rehydration regime increased significantly by
43.47%, 91.38%, 112.82%, and 242.26%, respectively (p < 0.01,
Fig. 1A, D-F). Meanwhile, the 1000-grain weight, spike length, grain
weight per spike, and yield of rehydration regime enhanced significantly
by 19.55%, 185.60%, 32.59%, and 183.43% than those of drought
treatment, respectively (p < 0.01, Fig. 1C, G-I).
On the 5th day after rehydration, except for 80–100 cm, the soil
moisture contents of rehydration regime were obviously greater than
that of drought treatment (p<0.05, Fig. 2A). On the 10th days after
rehydration, although the soil moisture content of both treatments
decreased, the soil moisture content of rehydration was still
L. Tian et al.
Fig. 1. Effects of rehydration treatment on agronomic traits and yield components of broomcorn millet under extreme drought conditions. The asterisk represents
significant differences (student’s t test, p < 0.05, n = 3) under water regimes. ns represents no significant; * represents p < 0.05; ** represents p < 0.01; ***
represents p < 0.001.
considerably greater than that of drought (p<0.05, Fig. 2B). On the 20th
and 30th days after rehydration, there was no obvious difference in soil
moisture content between both water regimes due to the high local
evaporation capacity (Fig. 2C and D).
3.2. Bulk soil and rhizosphere properties
Compared to drought treatment, rehydration regime significantly
increased the bulk soil and rhizosphere pH values, while there were no
obvious differences in the bulk soil and rhizosphere pH values between
sampling days (Table 1). We observed that rehydration did not signifi­
cantly impact the contents of OM and TN in the bulk soil, but signifi­
cantly reduced the OM and TN contents in the rhizosphere at different
sampling days (Table 1). For available nutrients, compared to drought
treatment, rehydration regime significantly decreased the AK content in
the rhizosphere and the AP content in the bulk soil and rhizosphere, and
also obviously decreased the NH+ - bacteriota and Firmicutes, but enhanced the relative abundance of
4 -N and NO3-N contents in the bulk soil
and rhizosphere (Table 1).
3.3. Bacterial and fungal community compositions and diversities
After bioinformatic processing, 788,628 and 1452,518 high-quality
4
Agricultural Water Management 295 (2024) 108781
sequences of bacteria and fungi were obtained from the 48 samples.
To facilitate further analysis of the sequences, the dataset was rarefied to
10,906 bacterial sequences and 18,768 fungal sequences. These se­
quences were then sorted into 5751 ASVs for bacteria and 1462 ASVs for
fungi. The dominant bacterial phyla among the bulk soil and rhizo­
sphere were Actinobacteriota, Proteobacteria, Firmicutes, and Acid­
obacteriota (Fig. S1A), and the dominant fungal classes were
Sordariomycetes, Dothideomycetes, and Eurotiomycetes (Fig. S1B).
LEfSe analysis showed that more biomarkers were enriched in bacterial
and fungal rhizosphere communities than in bulk soil (Fig. 3). In the
bacterial community of bulk soil, Acidobacteriota was significantly
enriched in the rehydration regime (Fig. 3A; Table S1). In the bacterial
community of rhizosphere, Actinobacteriota and Firmicutes were obvi­
ously enriched in the drought condition, while Proteobacteria was
significantly enriched in the rehydration regime (Fig. 3B), and rehy­
dration considerably decreased the relative abundances of Actino­
Proteobacteria than those of drought condition (Table S1). In the fungal
community of bulk soil more biomarkers were enriched in the drought
treatment (Fig. 3C), and rehydration regime considerably decreased the
relative abundance of Sordariomycetes, whereas considerably reduced
the relative abundances of Dothideomycetes and Mortierellomycetes on
L. Tian et al. Agricultural Water Management 295 (2024) 108781

Fig. 2. Effects of rehydration treatment on soil moisture content under extreme drought conditions.

Table 1
Effects of rehydration treatment on the bulk and rhizosphere soil properties under extreme drought conditions.
Compartment Sampling days Treatment pH OM TN AK AP NH+
4 -N NO—
3 N
(g kg− 1) (g kg− 1) (mg kg− 1) (mg kg− 1) (mg kg− 1) (mg kg− 1)

Bulk Day 5 Drought 8.82bc 11.35b 0.64a 85bc 17.45c 2.55a 67.12a
Rehydration 9.28a 11.96a 0.63ab 99.67a 16.55 cd 1.02b 11.95d
Day 10 Drought 8.79bc 11.55ab 0.61ab 85.33bc 16.54 cd 3.01a 63.23ab
Rehydration 9.18a 11.12b 0.61ab 88.00b 15.34d 0.97b 17.08d
Day 20 Drought 8.74c 11.14b 0.66a 89.00b 21.52a 2.89a 60.62b
Rehydration 9.14a 11.26b 0.62ab 80.33c 19.77b 1.09b 17.16d
Day 30 Drought 8.91b 11.27b 0.61ab 83.00bc 20.52ab 2.74a 37.6c
Rehydration 9.22a 11.16b 0.58b 73.00d 12.57e 1.29b 14.54d
Rhizosphere Day 5 Drought 8.43b 15.02a 0.86a 307.33b 52.60a 14.80b 63.35b
Rehydration 9.09a 12.16d 0.65b 106.67 f 24.28d 1.33d 23.71e
Day 10 Drought 8.61b 14.53b 0.83a 309.00b 46.70b 17.18a 27.67de
Rehydration 8.94a 12.10d 0.69b 155.33d 27.75c 1.14d 29.17d
Day 20 Drought 8.57b 13.10c 0.81a 335.33a 50.67a 16.06ab 74.05a
Rehydration 9.02a 12.25d 0.65b 131.00e 26.77 cd 0.95d 12.23 f
Day 30 Drought 8.55b 15.01a 0.87a 325.33a 52.77a 17.40a 37.93c
Rehydration 9.07a 14.10b 0.72b 194.00c 26.03 cd 6.48c 6.42 g
ANOVA
Compartment (C) 70.367*** 808.290*** 203.894*** 9941.176*** 2750.717*** 1246.193*** 5.284*
Sampling days (S) 1.319 28.140*** 0.270 27.965*** 10.300*** 18.199*** 101.866***
Treatment (T) 266.047*** 125.894** 86.106*** 3408.460*** 1182.848*** 1355.773*** 2167.333***
C*S 1.359 27.321*** 5.340** 85.592*** 6.426** 15.410*** 22.347***
C*T 3.710 139.131*** 52.497*** 3382.216*** 728.466*** 826.660 32.017***
S*T 2.464 10.701*** 0.806 30.957*** 14.695*** 8.695*** 85.371***
C*S*T 1.423 16.038*** 1.084 49.924*** 7.454** 5.554** 83.503***

Note: Different letters within the same column denote significant differences (p < 0.05, Duncan’s HSD post-hoc analysis, n = 3) under water regimes. * represents p <
0.05; ** represents p < 0.01; *** represents p < 0.001, the same below. OM, organic matter; TN, total nitrogen; AP, available phosphorus; AK, available potassium;
-
NH+4 -N, ammonium nitrogen; NO3-N., nitrate nitrogen;

5
L. Tian et al. Agricultural Water Management 295 (2024) 108781

Fig. 3. Enrichment of bacterial and fungal biomarkers in bulk and rhizosphere soils under drought and rehydration treatments. The taxa with significantly different
abundances in both treatments are symbolized by colored dots. From inside to outside, the six rings of the cladogram denote the kingdom, phylum, class, order,
family, and genus, respectively. A: bulk soil bacterial biomarkers; B, rhizosphere bacterial biomarkers; C: bulk soil fungal biomarkers; D, rhizosphere
fungal biomarkers.

the 5th day after rehydration (Table S2). In the rhizosphere fungal (Fig. 5C). Similar to community alpha-diversities, almost all rhizo­
community, Dothideomycetes was significantly enriched in the rehy­ sphere parameters obviously affected the beta-diversities of fungal and
dration regime (Fig. 3D), and the relative abundance of Dothideomy­ bacterial communities (Fig. 5D).
cetes in the rehydration regime was obviously higher than that of
drought on the four sampling days (Table S2).
3.4. Bacterial and fungal co-occurrence networks
We observed that rehydration regime significantly enhanced the
bacterial observed ASVs and Shannon index in the rhizosphere on the
We established co-occurrence networks of bulk soil and rhizosphere
5th and 30th days after rehydration (p < 0.05, Fig. 4A and B). Compared
to investigate the microbial interactions (Fig. S4), and the both networks
to drought treatment, the fungal Shannon index of rehydration regime
followed a scale-free degree distribution and showcased a non-random
significantly increased by 72.29%, 53.11%, and 46.70% on the 5th,
pattern as well as a scale-free network structure (Fig. S5A, B). The
10th, and 30th days after rehydration, respectively (p < 0.05, Fig. 4D).
rhizosphere network had higher numbers of edges, average degree, and
Furthermore, we found that bacterial alpha diversities were obviously
ACC, and lower APL and diameter value than that of bulk soil network,
negatively associated with bulk soil TN content, and fungal observed
- revealing that the rhizosphere network-enriched ASVs were more
ASVs was positively related to bulk soil NH+4 -N and NO3-N contents (p <
interconnected and more closed relationships (Table S4). Meanwhile, in
0.01, Fig. 5A). Except for pH, all rhizosphere parameters were obviously
the bulk soil and rhizosphere networks, bacterial taxa occupied a more
negatively associated with bacterial and fungal alpha diversities
important position. In the bulk soil network, the interactions between
(Fig. 5B).
fungi and fungi were higher than that between bacteria and bacteria.
PCoA result showed that the fungal and bacterial community struc­
However, in the rhizosphere network, the interactions between bacteria
tures in the bulk soil and rhizosphere were considerably separated along
and bacteria were more important (Table S4). Based on the rules of
axis 1. and the rhizosphere fungal and bacterial community structures in
choosing the main module, the first four modules were observed from
the rehydration and drought treatments were obviously separated along
the bulk soil and rhizosphere networks, respectively (Fig. S4A and B).
axis 2 (Fig. 4E, F). ANOSIM result showed that the contributions of
We observed that rehydration regime obviously enhanced the relative
compartment (R2= 0.887 and 0.728) to bacterial and fungal community
abundances of Module #1, but reduced the relative abundances of
structures were larger than those of regime (R2= 0.151 and 0.208), and
Module #3 and Module #4 in the bulk soil bacterial and fungal networks
PERMANOVA also revealed that compartment and regime also obvi­
(p < 0.001, Fig. S4A). Meanwhile, rehydration regime also considerably
ously affected the bacterial and fungal community structures (Table S3,
increased the relative abundances of Module #2-#4 in the rhizosphere
p < 0.001). Subsequently, db-RDA result revealed that bulk soil and
bacterial and fungal networks (p < 0.001, Fig. S4).
rhizosphere AK content were considerably related to the bacterial
Subsequently, we constructed eight sub-networks (Fig. 6). From the
community structures, respectively (Fig. S3A, B), and OM, AP, NH+ 4 -N,
perspectives of network node compositions, similar to the microbial
and NO-3-N contents of bulk soil and rhizosphere were obviously corre­
community compositions, rehydration regime reduced the proportion of
lated with the fungal community structures (Fig. S3C, D). Meanwhile,
Firmicutes and Actinobacteriota in the bulk soil and rhizosphere bac­
mantel test revealed that bulk soil pH and NH+ 4 -N content positively
terial networks, and increased the proportion of Chloroflexi and Acid­
influenced the fungal and bacterial community beta-diversities
obacteriota (Fig. S6A). In the rhizosphere fungal network, rehydration

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L. Tian et al. Agricultural Water Management 295 (2024) 108781

Fig. 4. The bacterial and fungal community diversities in bulk and rhizosphere soils under water regimes. Histogram showing the differences in the observed ASVs
and Shannon index of bacterial (A, B) and fungal (C, D) communities in bulk (A, C) and rhizosphere soils (B, D) under water regimes; Principal coordinates analysis
(PCoA) based on Bray-Curtis distances shows the bacterial (E) and fungal (F) community structures. The asterisk represents significant differences (student’s t test, p
< 0.05, n = 3) under water regimes. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001.

regime increased the proportion of unclassified_k_Fungi, but reduced the
proportion of Eurotiomycetes (Fig. S6B). We observed that the rhizo­
sphere bacterial network under rehydration regime had higher numbers
of nodes, edges, and average degree, suggesting that rehydration regime
increases node interconnectivity of the rhizosphere bacterial network.
Meanwhile, the rhizosphere fungal network under drought treatment
had higher numbers of ACC, and lower values of APL and diameter,
suggesting that the rhizosphere fungal network-enriched ASVs under
drought condition were closer relationships (Table 2).
3.5. Bacterial and fungal assembly processes
In comparison to the drought treatment, rehydration regime had
little effects on the βNTI values of the fungal and bacterial communities
in the bulk soil. However, it led to an obvious decrease in the βNTI value
of the bacterial community and an increase in the βNTI values of the
fungal community in the rhizosphere (Fig. 7). Specifically, compared to
drought treatment, rehydration regime obviously decreased the βNTI
value of the rhizosphere bacterial community after 5th days of rehy­
dration (Fig. S7B). Meanwhile, rehydration regime considerably
decreased the βNTI values of the bulk soil fungal community after 20th
and 30th days of rehydration (Fig. S7C). Mantel test showed that bulk
soil TN and NH+ 4 -N were key variables affecting the assembly processes
of bacterial and fungal communities, respectively, and we also noted
that all rhizosphere parameters shaped the bacterial assembly process
(Table 3). More importantly, rehydration regime had little influences on

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L. Tian et al. Agricultural Water Management 295 (2024) 108781

Fig. 5. Pearson’s rank correlation analysis between bacterial and fungal Shannon index and Richness index versus bulk soil (A) and rhizosphere (B) properties. The
correlations between bacterial and fungal communities versus bulk soil (C) and rhizosphere properties (D). Bacterial and fungal communities were related to each
environmental factor by Mantel tests. Edge width corresponds to the Mantel’s r statistic, and edge color denotes the statistical significance based on 999 permu­
tations. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001.

Fig. 6. Co-occurrence networks of the bacterial and fungal communities in bulk soil and rhizosphere under water regimes. The size of each node is proportional to
the number of connections (i.e., degree). Each bacterial and fungal node is marked at the phylum and class level, respectively. The edges indicate the correlation
between two nodes.

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L. Tian et al. Agricultural Water Management 295 (2024) 108781

Table 2
Topological properties of co-occurrence networks of bacterial and fungal communities in bulk and rhizosphere soils.
Microbe Compartment Treatment Edges Nodes ACC APL Modularity Diameter Average degree

Bacteria Bulk Drought 353 1277 0.98 1.03 0.98 3 0.55

Rehydration 278 1305 0.99 0.99 0.97 2 0.43
Rhizosphere Drought 128 799 1.00 0.98 0.97 1 0.32
Rehydration 319 1169 0.99 1.00 0.97 2 0.55
Fungi Bulk Drought 1724 462 0.89 6.28 0.75 23 7.46
Rehydration 1343 330 0.83 5.60 0.77 16 8.14
Rhizosphere Drought 120 130 0.93 1.49 0.59 6 1.85
Rehydration 202 185 0.71 3.62 0.81 10 2.18

APL: average path length; ACC: average clustering coefficient

Fig. 7. Effects of rehydration treatment on the βNTI values of the bacterial (A) and fungal (B) communities in bulk and rhizosphere soils under extreme drought
conditions. The asterisk represents significant differences (student’s t test, p < 0.05, n = 3) under water regimes. ns represents no significant; ** represents p < 0.01;
*** represents p < 0.001.

the assembly process of the bacterial community in the bulk soil and
rhizosphere (Fig. 8A), but rehydration regime increased deterministic
processes in the bulk soil fungal community and decreased deterministic
processes in the rhizosphere fungal community (Fig. 8B).
4. Discussion
4.1. Rehydration changed the compositions and diversities of rhizosphere
microbial communities under extreme drought conditions
Among various abiotic stresses, drought may be one of the abiotic
stresses with the highest influence on soil biota (Leng and Hall, 2019).
Drought stress not only affected the leaf water potential of plants,
thereby altering their physiological and morphological traits but also
increased soil heterogeneity and changed soil nutrient contents (Naylor
and Coleman-Derr, 2018; Wahab et al., 2022). Our results confirmed
that under extreme drought conditions, rehydration once at the
flowering stage can significantly improve the agronomic traits of the
aboveground, grain yield and its components of broomcorn millet,
indicating limited water supply alleviated severe drought stress to a
certain extent, promoted the recovery ability of broomcorn millet, and
thus improved crop productivity (Patanè et al., 2021). However, it is still
unclear how rewatering regulates plant-microbiome interactions under
extreme drought conditions.
In this work, we found that the compositions of bacterial and fungal
microbial communities exhibited obvious differences from bulk soil to
rhizosphere under water regimes, which were consistent with the pre­
vious observations, indicating the compartments were the biggest con­
tributors to compositional variations (Singer et al., 2019). Actually, the
bulk soil represents a microbial pool that affects the rhizosphere struc­
ture, and the rhizosphere selects microorganisms from bulk soil, and
plants can selectively collect some microorganisms to set them into root
tissues (Liu et al., 2021b; van der Heijden and Schlaeppi, 2015). Plants
have selective forces and powerful drivers that can adopt specific

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L. Tian et al. Agricultural Water Management 295 (2024) 108781

Table 3 Proteobacteria, significant decrease of genera Pseudomonas, Ensifer,

Spearman’s rank correlations between the βNTI values of bacteria/fungal Sphingomonas, Lysobacter, and Massilia was also found in the rhizosphere
communities and environmental variables (Euclidean distance) based on Mantel under drought condition. We also observed that Firmicutes was enriched
tests. in the rhizosphere under drought condition, which is a monoderm taxon
Compartment Variables Bacteria Fungi that can form specific characteristics in response to drought stress, such
r P r P as thick peptidoglycan cell walls. Additionally, rehydration also
consistently reduced the relative abundance of genus Bacillus compared
Bulk pH 0.001 0.225 0.282 0.001
OM -0.003 0.481 0.137 0.088
to drought treatment. Bacillus can improve plant drought tolerance by
TN 0.291 0.008 -0.055 0.690 activating stress adaptation responses and regulating cellular abscisic
AP 0125 0.111 0.010 0.439 acid levels (Zhou et al., 2016). The relative abundance of Sordar­
AK 0.151 0.075 -0.036 0.615 iomycetes on the 5th and 10th days after rewatering was significantly
NH+4 -N 0.110 0.039 0.401 0.001
lower than that under extreme drought conditions, and the previous
NO-3-N 0.144 0.030 0.341 0.002
Rhizosphere pH 0.640 0.001 0.144 0.024 study confirmed that Sordariomycetes was enriched in the drought
OM 0.403 0.001 0.027 0.321 condition (Huang et al., 2021; Xiao et al., 2020). The augmentation in
TN 0.585 0.001 0.070 0.131 the relative abundance of drought-tolerant Sordariomycetes could be
AP 0.683 0.001 0.215 0.002 attributed to the physiological response exhibited by fungi when sub­
AK 0.594 0.001 0.252 0.002
NH+
jected to water stress (Huang et al., 2021). However, Eurotiomycetes,
4 -N 0.617 0.001 0.098 0.058
NO-3-N 0.403 0.001 0.144 0.033 Dothideomycetes, Mortierellomycetes, Leotiomycetes showed the
opposite trend, revealing that these taxa are more sensitive to water, soil
water content can regulate plant community diversities and alter soil
strategies to recruit soil microbes, thereby helping them to cope with the nutrient contents, thus driving the adaptability of these taxa under water
challenges of drought. In this study, the bulk soil and rhizosphere conditions (Chen et al., 2017). Overall, extreme drought conditions
exhibited enrichment of three main phyla Actinobacteria, Proteobac­ recruited some drought-resistant microbes in the rhizosphere, while
teria, and Firmicutes under water regimes. Actinobacteria were enriched rehydration also modulated the changes in fungal and bacterial com­
in the rhizosphere under drought conditions. Actinobacteria, known as munity abundance.
plant growth-promoting bacteria, may be excellent bacteria to improve In this study, rehydration regime had a larger influence on the bac­
plant drought tolerance, which was used to screen drought-tolerant terial and fungal alpha diversities in rhizosphere than in bulk soil under
varieties quickly and reliably (Hu et al., 2023; Wipf et al., 2020; Xie extreme drought conditions, because the rhizosphere physicochemical
et al., 2021). Meanwhile, the relative abundance of genus Streptomyces properties were significantly associated with the bacterial and fungal
in the rhizosphere was also considerably decreased after rewatering alpha diversities, which is attributed to the fact that rehydration can
than in extreme drought conditions. Streptomyces is a drought-tolerant greatly alleviate soil drought conditions and benefit soil microorganisms
microorganism, which can improve the plant vigor and decrease the to be more active, thus improving the diversity and richness of micro­
damage of reactive oxygen species to plants in water-stressed soil (Leirós organisms in the rhizosphere, and higher alpha diversity can improve
et al., 2014). In our study, Proteobacteria in the rhizosphere exhibited the microbial metabolic capacity, which is beneficial for host plants to
the decreased trend under extreme drought conditions, this observation resist environmental stresses (Hu et al., 2023). In contrast, several
was in line with previous works (Yao et al., 2017; Zhang et al., 2022), studies have showed that drought stress obviously enhanced the
while other studies reported the opposite trends (Ullah et al., 2019; Xu alpha-diversity of rhizosphere-associated bacterial and fungal commu­
et al., 2018). The main reason for such results is that Proteobacteria nity (de Oliveira et al., 2020; Hu et al., 2023; Zhang et al., 2022). These
exhibit higher sensitivity to environmental changes due to the preva­ opposite results may be attributed to (i) differences in microbial com­
lence of non-dormant cells within their population. Consequently, such munity compositions in different habitats (Liu et al., 2022); (ii) differ­
changes have the potential to either increase or decrease their overall ences in rewatering stage and drought intensity (Xu et al., 2023; Zhang
abundance (Jones and Lennon, 2010). Correspondingly, within the et al., 2019).

Fig. 8. Summary of ecological processes that governed the bacterial and fungal community assembly in bulk and rhizosphere soils under water regimes. Percentages
are given for the relative contributions of each process to the community assembly, as indicated by different colors.

10
L. Tian et al.
This work also confirmed that the variations in rhizosphere proper­
ties had a larger impact on fungal and bacterial community structure
than bulk soil under water regimes. Compared to bulk soil, the rhizo­
sphere is physically connected to the plant roots, indicating that rhizo­
sphere is more susceptible to the impact of roots (Pang et al., 2017). The
release of root exudates had the ability to attract microorganisms pre­
sent in the rhizosphere. Furthermore, root exudates exerted a critical
influence on the diversity and structure of the microbial community in
the rhizosphere microbiome by regulating the rhizosphere physico­
chemical properties (Yang et al., 2017). Meanwhile, water regimes also
obviously affected the fungal and bacterial community structure of bulk
soil and rhizosphere, which was attributed to the selection of different
resistant taxa in this environment, and altered the soil properties and
diffusion rate of microbes (Naylor and Coleman-Derr, 2018). More
importantly, compared to the fungal community in the rhizosphere, the
bacterial community in the rhizosphere displayed considerable separa­
tion under rehydration and drought treatment, suggesting that fungal
communities exhibit higher tolerance to drought when compared to
bacterial communities (de Vries et al., 2018).
4.2. Rehydration altered the interconnected taxa in bacterial and fungal
networks under extreme drought conditions
The ecological network can reveal the complex relationships among
microbial taxa (Faust and Raes, 2012; Tian et al., 2022b). In this study,
the taxa enriched in the rhizosphere network are more interconnected
and more closely related than the bulk soil network. The rhizosphere is
closer to the roots and may be more directly affected by root exudates,
for which members within the rhizosphere community might develop
more cooperation to adapt to the extreme environments (He et al.,
2023b). Pathan et al. (2023) reported that the complexity of bacterial
networks decreased from roots to soil compartments, suggesting that
although host plants govern their own microbial selection, there are
other biotic or abiotic factors that may affect microbial selection and
their interactions. In contrast, Xiong et al. (2021) confirmed that the
network complexity gradually decreased from bulk soil to root to leaf
compartments. These different results may be due to differences in host
genetics or other related biotic or abiotic factors (Xiong et al., 2021).
Additionally, compared to drought treatment, rehydration increased the
taxa interconnectivity of the rhizosphere bacterial network, but reduced
taxa interactions in the rhizosphere fungal network. Several studies re­
ported that drought reduced the stability and complexity of protistan
(Chen et al., 2022) and bacterial (Pathan et al., 2023) networks, which
was due to the destabilization of microbial networks caused by drought
stress (de Vries et al., 2018). Nevertheless, drought enhanced the
complexity of pqqC-harboring bacterial network (Zhang et al., 2022),
but had no significant effect on the soil fungal network (de Vries et al.,
2018). These differences may be attributed to (i) different ecosystem
types have different soil properties and soil structures, which may lead
to different sensitivity of soil microorganisms to water regimes (Sol­
eimani et al., 2019); (ii) different tolerance of different organisms to
water regimes (Wu et al., 2018).
4.3. Rehydration altered the assembly processes of fungal communities
rather than bacteria under extreme drought conditions
To better understand how water regimes influence the microbial
community assembly processes in distinct compartments, it is crucial to
elucidate the ecosystem functions and biodiversity stability under water
regimes (Chu et al., 2020). Rehydration regime did not change the
bacterial assembly processes in bulk soil and rhizosphere, that is, all
samples were dominated by deterministic processes. The bacterial
communities, which have smaller cell sizes and faster diffusion rates,
were significantly affected by deterministic selection in comparison to
the fungi (Zhang et al., 2021), and the characteristics of rapid growth
rate and adaptive metabolism enable bacteria to colonize effectively in
11
Agricultural Water Management 295 (2024) 108781
various extreme environments, which will enable them to better ’track’
the appropriate environment from deterministic selection (Chen et al.,
2021; Jiao et al., 2020). However, the fungal stochastic process (i.e.,
dispersal limitation) in the rhizosphere under rehydration regime was
greatly decreased than under drought condition. Similarity, previous
studies also have showed that drought stress improved the contributions
of stochastic processes in governing the pqqC and protistan communities
(Chen et al., 2022; Zhang et al., 2022). The findings of this study
revealed that the dynamics of various microbial communities’ assembly
processes are influenced by external disturbances, highlighting the
importance of maintaining a balance in response to such disturbances
(Wu et al., 2018).
5. Conclusions
In our study, compared to drought condition, rehydration regime
reduced the nutrient contents of rhizosphere to varying degrees, but had
no influence on the OM and TN contents in bulk soil, and rehydration
regime also significantly increased the agronomic traits and grain yield
of broomcorn millet, and the grain yield was considerably increased by
183.43%. Rehydration regime significantly changed the compositions,
alpha diversities, and assembly processes of bacterial and fungal com­
munities in rhizosphere than those of drought treatment, and the AP and
AK contents in rhizosphere were important variables affecting bacterial
and fungal community structures and assembly processes, respectively.
Furthermore, rehydration regime increased the interconnected taxa in
the rhizosphere bacterial network, while reduced taxa interactions in
the rhizosphere fungal network compared to drought treatments.
Overall, this study provided a new perspective for better understanding
the effects of rehydration regime on the ecological functions of the mi­
crobial communities in rhizosphere under extreme drought conditions.
CRediT authorship contribution statement
Lixin Tian: Writing – original draft, Investigation, Data curation. Yu
Yang: Methodology, Formal analysis. Youhong Song: Writing – review
& editing, Validation. Baili Feng: Writing – review & editing, Supervi­
sion, 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.
Data availability
Data will be made available on request.
Acknowledgements
This work was funded by the China Agriculture Research System of
MOF and MARA (CARS-06-A26), Minor Grain Crops Research and
Development System of Shaanxi Province (NYKJ-2021-YL(XN)40), and
Foundation of High-level Talents of Anhui Agricultural University
(rc312305, rc312212).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.agwat.2024.108781.
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