agronomy
Article
The Effect of Rotational Cropping of Industrial Hemp
(Cannabis sativa L.) on Rhizosphere Soil Microbial Communities
Lili Tang 1,2,† , Chao Fan 1,3,† , Hongmei Yuan 2 , Guangwen Wu 2 , Jing Sun 2 and Shuquan Zhang 2, *
Citation: Tang, L.; Fan, C.; Yuan, H.;
Wu, G.; Sun, J.; Zhang, S. The Effect
of Rotational Cropping of Industrial
Hemp (Cannabis sativa L.) on
Rhizosphere Soil Microbial
Communities. Agronomy 2022, 12,
2293. https://doi.org/10.3390/
agronomy12102293
Received: 29 July 2022
Accepted: 19 September 2022
Published: 24 September 2022
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Agronomy 2022, 12, 2293. https://doi.org/10.3390/agronomy12102293
1 Heilongjiang Academy of Agricultural Sciences Postdoctoral Program, Harbin 150086, China
2 Institute of Cash Crops, Heilongjiang Academy of Agricultural Sciences, Harbin 150086, China
3 Institute of Crop Cultivation and Tillage, Heilongjiang Academy of Agricultural Sciences,
Harbin 150086, China
* Correspondence: txzz2021@126.com; Tel.: +86-0451−86677430
† These authors contributed equally to this work.
Abstract: Crop rotation affects soil properties and soil microbial diversity and structure. Currently, it
is not well understood how soil microbial diversity changes following different crop rotation systems
of industrial hemp, an ancient and economically important crop. Therefore, these changes were
analyzed in this study. Our results showed that different rotation systems significantly affected
the wilt disease incidence, plant height, yield, soil physicochemical properties and soil microbial
communities in the greenhouse. The rotation systems used in this study significantly reduced the
plant mortality and increased the yield compared with a monoculture system. The levels of alkaline
hydrolysis and available phosphorus in the soil decreased significantly compared with a monoculture
cropping system. Using MiSeq high-throughput sequencing, we showed that the soil diversity and
number of bacteria and fungi were significantly higher for rotation systems and controls compared to
the monoculture system. The relative abundance of pathogens increased with a monoculture system.
Redundancy analysis suggests that soil properties may also affect the soil microbial composition.
Taken together, different rotation systems used in this study significantly decreased the disease
incidence, increased plant yields and increased soil microbial diversity compared with monoculture
for industrial hemp. We believe that applying these rotation systems is an efficient and eco-friendly
approach to control soil borne pathogenic diseases and increase floral yields.
Keywords: industrial hemp; rotation system; soil microbes; Illumina MiSeq
1. Introduction
Soil microbes are a critical component of the terrestrial soil ecosystem. They participate
in the synthesis, decomposition, and transformation of many nutrients, which are important
for plant growth. The number and diversity of soil microorganisms are important indicators
of soil fertility and the microecological status [1]. The soil microbial community structure
and its changes reflect soil productivity and stability and act as an early indicator of
ecosystem change [2–4]. Beneficial microorganisms in the soil promote plant growth and
improve plant resistance to various diseases. Under normal circumstances, the species and
number of beneficial microorganisms present in the soil, including antagonistic bacteria,
nitrogen-fixing bacteria, and ammonia oxidizing bacteria, are far greater than that of
harmful microorganisms, such as soil-borne pathogens, and there is normally a dynamic
balance between beneficial and harmful microorganisms [5]. Antagonistic microorganisms
in the soil can inhibit the growth, survival, or infection of plant pathogens by secreting
antibiotic toxins, biosurfactants, extracellular cell-wall decomposing enzymes and other
substances. They also compete with pathogens for nutrition and mineral elements [6].
Originating in Central Asia, industrial hemp (Cannabis sativa L.) is an important
herbaceous plant that has been used for centuries in medicine, as a source of textile fiber,
and as a recreational drug [7,8]. In recent years, there has been continuous development and
https://www.mdpi.com/journal/agronomy
Agronomy 2022, 12, 2293 2 of 15

application of the medicinal uses of industrial hemp. In particular, the extract cannabidiol
(CBD), which comes from the flowers and leaves of the plant, has been used to treat
pain, depression, anxiety, and other related diseases [9–11]. Due to its health benefits, the
production of industrial hemp has become an important industry.
Industrial hemp is dioecious and mainly relies on wind for its pollination. It is, there-
fore, difficult to maintain the chemical profile and purity of its products [12]. The use of
greenhouses to grow industrial hemp becomes an effective and feasible way to prevent
pollen mixing and increase the quality of industrial hemp. However, previous studies
showed that continuous cropping in greenhouse results in crop yield decline, poor quality,
and increased occurrence of diseases and insect pests [13–15]. Our previous study found
that fusarium wilt occurred in industrial hemp after continuous cropping, and the pathogen
Fusarium oxysporum was identified [16]. The pathogen Fusarium oxysporum could lead to
monoculture watermelon decreases in crop yield and quality [17]. In recent years, more
studies have found that continuous cropping is the main factor in rhizosphere microecologi-
cal imbalance [18]. Guo et al. [19] revealed that the relative abundance of beneficial bacteria
was significantly reduced, while the potentially harmful bacteria increased, resulting in
changes in the bacterial community structure under industrial hemp monoculture system.
After continuous monocropping, the main reason for the disease occurrence is that
a certain type of pathogenic bacteria becomes the dominant microbe in the soil microbial
community, which increases the incidence of plant diseases. Studies have shown that a
rational rotation system could make pathogenic microorganisms lose their original hosts,
change the microbial community and diversity in soil, reduce the number of harmful
pathogens, and thus reduce the occurrence of diseases [20]. For example, strawberry
rotation with broccoli and lettuce could effectively control verticillium wilt and increase
strawberry quality and yield [21]; barley–ryegrass–potato rotations inhibited the incidence
of potato Rhizoctonia solani and Streptomyces scabies by 18.0 to 33.0% [22]; a black pepper–
vanilla system harbored a lower abundance of Fusarium oxysporum in the vanilla rhizosphere
soil and increased the putatively plant-beneficial fungal groups such as Trichoderma and
Penicillium genus [23].
In the present work, we found that plant mortality increased, growth potential became
weaker, and the yield of floral and leaf yields decreased in monoculture compared with
rotation systems of industrial hemp. We hypothesized that rotation systems could decrease
the incidence of industrial hemp fusarium wilt disease by increasing plant-beneficial
microorganisms and inhibiting the pathogen population or changing the composition of
industrial hemp rhizosphere bacterial and fungal communities. In addition, the effect
varies between different rotation systems. To test this hypothesis, the microbial community
compositions of 15 soil samples from the industrial hemp monoculture system, 3 rotation
systems and control (no crop planted) in 2021 at harvest stages were analyzed using
Illumina MiSeq targeted amplicon sequencing using the bacterial 16S rRNA gene and the
fungal internal transcribed spacer (ITS) gene of industrial hemp rhizosphere soil. This
study provides insights to soil micro-ecological environment for industrial hemp rotation
systems, which is important to the sustainable development of industrial hemp.

2. Materials and Methods

2.1. Experimental Location and Design
Experiments were conducted in four greenhouses at the Modern Agriculture Demon-
stration Area (45◦ 490 4400 N, 126◦ 480 5500 E), located at the southern bank of the Songhua
River, Harbin City, Heilongjiang Province. The greenhouses were filled with black soil and
the soil layer is more than 60 cm thick. The soil chemical properties were reported as the
following: pH 6.7–6.9; organic matter (OM) 31.9–34.1 g·kg−1 ; total potassium 2.68–2.93%;
total nitrogen 0.158–0.167%; available phosphorus (AP) 28.5–29.75 mg·kg−1 ; available
potassium (AK) 2.80–3.07 g·kg−1 ; alkaline hydrolysis (AN) 147.9–151.9 g·kg−1 .
The experiment was conducted over three years (2019–2021) in greenhouses built
at the experimental site. Each greenhouse was 12 m wide and 60 m long, and the area
Agronomy 2022, 12, 2293 3 of 15

was 720 m2 ; the roof was 3.4 m at its highest point and 1.2 m at the lowest point of the
arched plastic shed. The greenhouse used natural light and the temperature was between
25 ◦ C and 35 ◦ C. The relative humidity was 50~70%. The seeds used in this experiment
were industrial hemp (Cannabis sativa L.), watermelon (Citrullus lanatus (Thunb.) Mansfeld
and Nakai), potato (Ralstonia solanacearum) and bean (Phaseolus vulgaris L.). The source,
growth conditions and quantity of these plants are listed in Table S1. The different rotation
systems in our experiment were that industrial hemp (H) was cultivated in the first year
(2019), followed with watermelon (W), potato (P), or bean (B) in the second year (2020),
then industrial hemp (H) again in the third year (2021), that is, HWH-industrial hemp
(year 1), watermelon (year 2) and industrial hemp (year 3); HPH-industrial hemp (year 1),
potato (year 2) and industrial hemp (year 3); HBH-industrial hemp (year 1), bean (year 2)
and industrial hemp (year 3) (Table S2). The monoculture system in our experiment
was HHH-industrial hemp in all three years. The control was no crop planted in all
three years (empty spaces on either side of each greenhouse without any crops). The
plants were subjected to typical management procedures. No pest or disease controls
and manual weeding were applied during the whole experiment. The base fertilizer was
N:P:K = 15:15:15, 25 kg per greenhouse. The fertilizer was mixed with soil before planting
the crops. No additional fertilizer was used in the crop-growing stage. Sub-membrane
irrigation was used for planting. Rational irrigation was carried out according to the actual
water demand of different crops.

2.2. Measurement of Wilt Disease Incidence, Crop Height, and Yield of Industrial Hemp
To evaluate the growth of industrial hemp under different rotation systems (HWH,
HPH and HBH) and a monoculture system (HHH), plant height was measured at 1, 2 and
4 months after cultivation in year 1 and year 3. In October of year 1 and year 3, flowers
and leaves were harvested, respectively. The yields of flowers and leaves were compared
among different cultivation methods. The wilt disease incidence was measured throughout
the planting season in year 1 and year 3. Wilt disease incidence was expressed as the
proportion of symptomatic plants out of the total number of plants. The disease index
(DI) denotes healthy plants as “0” and fusarium wilt plants as “1”. Each treatment was
administered to 40 plants and was repeated three times.

2.3. Soil Sample Collection

Soil attached to plant roots of industrial hemp was collected at the harvest stage of
year 3. The planting area of each greenhouse was evenly divided into six parts. Five
plants were randomly selected from each part, and the roots were completely removed.
The rhizosphere soil of industrial hemp was obtained by strongly vibrating root and
brushing the roots with a brush. Rhizosphere soils from two parts were randomly selected
as a sample with three replicates per treatment. Collected soil samples from different
plants were mixed and sieved through a 2 mm mesh to thoroughly homogenize and
remove roots, plant residue and rocks. Each soil sample was divided into two parts and
placed in individual plastic bags, sealed, and transferred to the laboratory. Half of the
samples were stored at −80 ◦ C for DNA extraction; the other half was stored at −4 ◦ C for
chemical analysis.

2.4. Soil Physicochemical Properties Analysis

The intrinsic physical and chemical characteristics of the soil were analyzed as previ-
ously described [24]. Briefly, the soil pH was measured using a glass electrode in a soil water
suspension (1:2.5 w/v). Available potassium, available phosphorus and alkali-hydrolyzed
nitrogen were extracted with 2 M KCl, 0.5 M NH4+ OAc (pH = 7) and 1 M NaHCO3
(pH = 8.5), respectively. Soil filtrates were measured using a Continuous Flow Analyser
(SAN++, Skalar, Breda, The Netherlands). Contents of the soil organic matter were deter-
mined using the potassium dichromate oxidation method. The experiment was repeated
three times using three biological replicates.
Agronomy 2022, 12, 2293 4 of 15

2.5. DNA Extraction and MiSeq Sequencing

Total DNA was extracted using the E.Z.N.A. stool DNA Isolation Kit (Omega Biotek,
Norcross, GA, USA), according to the manufacturer’s instructions. DNA concentration and
purity were determined with a NanoDrop 2000 Spectrophotometer (ThermoFisher Scientific,
Waltham, MA, USA). The V3–V4 hypervariable region of bacterial 16S rRNA gene was am-
plified by PCR using the following primer pairs, forward: 50 -CCTACGGGNGGCWGCAG-30 ;
reverse: 50 -GGACTACHVGGGTATCTAAT-30 . The fungal ITS region was amplified
with the following primer pairs, forward: 50 -GATGAAGAACGYAGYRAA-30 ; reverse:
50 -GGACTACHVGGGTATCT AAT-30 .
PCR amplification was performed in 50 µL reaction solution consisting of 5 µL
10× Buffer, 5 µL dNTPs (2.5 mM), 1.5 µL forward and reverse primers (5 µM), 1 µL
polymerase (2 U/µL), and 100 ng template DNA. The PCR amplification condition was:
initial denaturation at 95 ◦ C for 2 min, followed by 35 cycles at 98 ◦ C for 15 s, 63 ◦ C for
30 min, 68 ◦ C for 30 s, and a final extension at 72 ◦ C for 10 min for the V3–V4 region of the
16S rRNA gene; 3 min of initial denaturation step at 95 ◦ C, followed by 35 cycles at 94 ◦ C
for 30 s, 55 ◦ C for 30 s and 72 ◦ C for 45 s, and a final extension at 72 ◦ C for 10 min for the ITS
genes. PCR products were detected by electrophoresis using 2% agarose gel (ThermoFisher
Scientific) and recovered using an AxyPrep DNA Gel kit (Axygen Biosciences, Union City,
CA, USA). The quality and concentration of the PCR products were assessed using an
Agilent 2100 Bioanalyzer (Agilent Technologies Co., Ltd., Santa Clara, CA, USA) and KAPA
Library quantitative kit (KAPA Biosystems, Wilmington, CA, USA), respectively. The
amplified products were purified and normalized to form a sequencing library. Illumina
MiSeq high-throughput sequencing was performed by Shanghai Shenggong Biological
Company (Shanghai, China).

2.6. Sequencing Data Analysis

Raw sequencing data were analyzed using the QIIME software (v1.9.0, J Gregory
Caporaso, Denver, CO, USA) and the UPARSE pipeline as previously described [25].
Quality control was carried out on the raw data based on the following criteria: (1) removing
reads with more than 10% of unknown nucleotides; (2) removing reads that have a Q20
below 80%; (3) removing reads with a mismatch error rate up to 2% of the readings
using FLASH 55 (v1.2.11, Tanja Mago ˇc∗ and Steven L. Salzberg, Baltimore, MD, USA);
(4) removing redundant sequences using QIIME56 (V1.9.1) [26–28]. The raw data were
deposited in the NCBI database (https://www.ncbi.nlm.nih.gov, accessed on 25 May 2022)
with the deposition number PRJNA841395.
High-quality reads were compared with the reference database (http://drive5.com/
uchime/uchime_download.html, accessed on 21 January 2022) and pruned using the
UCHIME software (http://www.drive5.com/usearch/manual/uchimealgo.html, accessed
on 26 January 2022). UPARSE was used for cluster analysis. Sequences with 97% homology
were combined into operational taxonomic units (OTUs). The sequence with the highest
abundance score in each cluster was selected as the representative sequence.
The bacterial and fungal α-diversity estimation for Chao1 richness, ACE index, Shan-
non index, Simpson index, Shannon even index and coverage were calculated using QI-
IME [27]. Welch’s t-test and the Wilcoxon signed-rank test were used for quantitative
analysis of α-diversity between groups. Both PCoA analysis and UPGMA clustering analy-
sis were based on the Binary Jaccard algorithm to reveal the relationship of microbiome
composition. Heatmap analysis was used to compare the top 50 classified genera in each
sample of two growing seasons using the gplot package in R (R v.3.2.5) [29]. Redundancy
analysis (RDA) was used to examine microbiome composition and microbial abundance
in relation to environmental factors. RDA was also used to examine possible connections
between the OTU frequencies and measured soil variables among different samples.
 Agronomy 2022, 12, 2293 5 of 15

2.7. Statistical Analysis

One-way analysis of variance (ANOVA) was used to analyze the plant height, disease
incidence and yield using SAS 9.3 software (SAS Institute Inc., Carry, NC, USA). The soil
physiochemical properties, the bacterial and fungal taxa, and the microbial α-diversity
indices were compared using the Student’s t-test. A p value of <0.05 was considered
statistically significant.

Agronomy 2022, 12, 2293
3. Results
3.1. Wilt Disease Incidence, Crop Height, Floral and Leaf Yields of Industrial Hemp Grown in Greenhouses
To examine whether different rotation systems affect plant production of industrial
hemp, we compared the wilt disease incidence, plant height, floral and leaf yields. The wilt
disease incidence, plant height, floral and leaf yields of the industrial hemp were measured
and compared in year 1 and year 3. Our results showed that the rotation systems (HWH,
HPH and HBH) and monoculture system (HHH) had the same growth at the different
growth stages, including seedling stage (1 month), rapid growth stage (2 months) and
pre-flowering stage (4 months) in year 1 (Figure 1A). In year 3, the plant height of the HHH
at seedling stage industrial hemp was lower than that of other treatments, but the difference
was not significant. Plant height of HHH was significantly lower at 2 and 4 months than
other planting methods (Figure 1A). In year 1, there was no significant difference in the
wilt disease incidence all groups, while in year 3, the wilt disease incidence of HHH was
significantly higher than that of the rotation systems (Figure 1B). Analysis of floral and leaf
yields showed that there was no significant difference between groups in year 1, while in
year 3, the floral and leaf yields per plant of HHH were significantly lower than the rotation6 of 15
systems (Figure 1C). The results showed that the wilt disease incidence increased, growth
potential became weaker, and the yield of floral and leaf yields decreased in a monoculture
compared with rotation systems of industrial hemp.

Figure 1. Effects of monoculture or rotation systems on plant height (A), wilt disease incidence (B) and
Figure 1. Effects of monoculture or rotation systems on plant height (A), wilt disease incidence (B)
floral and leaf yield (C) of industrial hemp. Data are the mean ± standard error of 20 replicates. Letters
and floral and leaf yield (C) of industrial hemp. Data are the mean ± standard error of 20 replicates.
Letters above bars (a, b, or c) represent differences between groups according to Tukey’s test, p <
0.05 level. HWH, industrial hemp (year 1)—watermelon (year 2)—industrial hemp (year 3); HPH,
industrial hemp (year 1)—potato (year 2)—industrial hemp (year 3); HBH, industrial hemp (year
1)—bean (year 2)—industrial hemp (year 3); HHH, industrial hemp in all three years (monoculture
 Agronomy 2022, 12, 2293 6 of 15

above bars (a, b, or c) represent differences between groups according to Tukey’s test, p < 0.05 level.
HWH, industrial hemp (year 1)—watermelon (year 2)—industrial hemp (year 3); HPH, industrial
hemp (year 1)—potato (year 2)—industrial hemp (year 3); HBH, industrial hemp (year 1)—bean
(year 2)—industrial hemp (year 3); HHH, industrial hemp in all three years (monoculture system).

3.2. Soil Physicochemical Properties

The nutrient content of soil is an indicator of soil quality and soil fertility. To examine
the possible influence of different rotation systems to soil quality, we measured the soil
pH, total phosphorus, total potassium, and organic matter after rotation and monoculture
systems were completed. Our results showed no significant differences between the rotation
systems, monoculture system and the control (CK—no crop was planted each year) in soil
pH (Figure 2A), total phosphorus (Figure 2C), total potassium (Figure 2D) and organic
matter (Figure 2H). The total nitrogen content in HWH was significantly lower than CK,
HPH and HHH, but not significantly different from HBH (Figure 2B). The amounts of
alkali-hydrolyzed nitrogen (AN) (Figure 2E), available phosphorus (AP) (Figure 2F) and
available potassium (AK) (Figure 2G) in HHH were significantly lower than those in the
CK, HWH, HPH and HBH. The levels of AN (Figure 2E) and AP (Figure 2F) in HWH, HPH
and HHH were significantly lower than those in the CK, while the levels of AK (Figure 2G)
in HWH, HPH and HHH were not significantly different from those in CK. These results
Agronomy 2022, 12, 2293 7 of 15
indicate that the AK, AP and AN of soil were significantly reduced after monoculture
cropping of industrial hemp, suggesting that a monoculture system reduces the soil quality.

Figure
Figure 2. Effects
2. Effects of soil
of soil physicochemical
physicochemical properties.
properties. (A) (A)
SoilSoil
pH.pH. (B) Total
(B) Total nitrogen
nitrogen for soil.
for soil. (C) Total
(C) Total phosphorus for soil. (D) Total potassium for soil. (E) Alkaline hydrolysis
phosphorus for soil. (D) Total potassium for soil. (E) Alkaline hydrolysis nitrogen for soil. (F) Avail- nitrogen for
soil.
able (F) Available
phosphorus forphosphorus for soil. (G)
soil. (G) Available Availablefor
potassium potassium
soil. (H)for soil. (H)matter
Organic Organicformatter for soil.indus-
soil. HWH,
trial hemp
HWH, (year 1)—watermelon
industrial (year 2)—industrial
hemp (year 1)—watermelon hemp (year
(year 2)—industrial hemp3); (year
HPH,3);industrial hemp (year
HPH, industrial
1)—potato
hemp (year (year 2)—industrial
1)—potato hemp (yearhemp
(year 2)—industrial 3); HBH,
(yearindustrial hemp (year
3); HBH, industrial hemp1)—bean (year 2)—in-
(year 1)—bean
dustrial hemp (year 3);
(year 2)—industrial HHH,
hemp (yearindustrial
3); HHH,hemp in allhemp
industrial threeinyears; CK,
all three no crop
years; CK, noplanted. Data are ex-
crop planted.
pressed as expressed
Data are mean ± standard ± standard
as mean error (n = 20).
errorLetters onLetters
(n = 20). top ofon bars
top(a,
of b, or(a,
bars c) b,
indicate the differences
or c) indicate the
between groups
differences according
between groupstoaccording
Tukey’s to test (p < 0.05).
Tukey’s test (p < 0.05).

3.3. Soil Microbial Diversity

The abundance and diversity of microorganisms in soil can be reflected by α-diver-
sity analysis. In this study, MiSeq sequencing was performed on the V3–V4 region of bac-
terial 16S rRNA and the ITS2 highly variable region of fungal ITS from 15 samples. A total
Agronomy 2022, 12, 2293 7 of 15

3.3. Soil Microbial Diversity

The abundance and diversity of microorganisms in soil can be reflected by α-diversity
analysis. In this study, MiSeq sequencing was performed on the V3–V4 region of bacterial
16S rRNA and the ITS2 highly variable region of fungal ITS from 15 samples. A total of
1,465,412 and 1,458,347 sequences were obtained from bacteria and fungi, respectively.
There were 29,353 bacterial sequences and 29,834 fungal sequences per sample on average.
There were at least 20,134 sequences for bacteria and 21,345 sequences for fungi. The
average fragment length of bacteria and fungi was 421 bps and 348 bps, respectively. To
reduce the differences caused by high-throughput sequencing, the minimum sequence
number (20,134 for bacteria and 21,345 for fungi) was used for statistical analysis.
Microbial diversity is an important index for measuring the biological composition of
a community. Comparative analyses of bacterial diversity indices showed that the Chao1,
ACE, Shannon and Shannon even index of HHH were significantly lower than CK, HWH,
HPH and HBH, while the Simpson index was significantly higher than CK, HWH, HPH
and HBH (Figure 3 and Table S3). There was no significant difference in the coverage index
between CK and HWH, HPH, HBH or HHH (Table S3). Similar with the bacterial diversity
indices, analysis of the rhizosphere soil fungal diversity index showed that ACE, Shannon
and Shannon even indices of HHH were significantly lower than CK, HWH, HPH and
HBH (Figure 3 and Table S4). The Simpson index of HHH was significantly higher than CK,
Agronomy 2022, 12, 2293 HWH, HPH and HBH; the Chao1 and coverage indices showed no significant differences 8 of 15

among all samples (Figure 3 and Table S4).

Figure 3. The α−diversityofofbacteria

The α−diversity bacteriaand
andfungi
fungiininthe
therhizosphere
rhizosphereofofindustrial
industrial hemp
hemp under
under different
different
cultivation
cultivation patterns,
patterns, displayed
displayed using
using with violin plots. The
The black
black line
line running
running through the violin
diagram up and down represents the interval from min to max, 95% confidence interval. The lower
end
end of
of the
the line
line represents the upper
represents the upper and
and lower
lower limit,
limit, and
and the
the data
data beyond
beyond this
this range
range is
is abnormal.
abnormal.

3.4. Soil Microbial Composition

Principal coordinate
coordinateanalysis
analysis(PCoA)
(PCoA)cancanbebeused
usedto to
better understand
better understand thethe
relationship
relation-
between
ship multiple
between samples.
multiple Our Our
samples. results showed
results showedthat that
biological replicates
biological werewere
replicates clustered
clus-
together,
tered indicating
together, that thethat
indicating bacterial community
the bacterial structure
community of each planting
structure system showed
of each planting system
good repeatability.
showed There were
good repeatability. significant
There differences
were significant among HWH,
differences among HPH, HBH,
HWH, HHH
HPH, and
HBH,
CK (Figure 4A). Unweighted Pair Group Method with Arithmetic
HHH and CK (Figure 4A). Unweighted Pair Group Method with Arithmetic Mean (UP-Mean (UPGMA) cluster
analysiscluster
GMA) showed a similar
analysis result to
showed PCoA analysis
a similar result to(Figure
PCoA 4B). PCoA(Figure
analysis and UPGMA analyses
4B). PCoA and
of the fungal community composition showed a clear separation
UPGMA analyses of the fungal community composition showed a clear separation between CK and HHHbe-
but a significant
tween CK and HHH overlap
but aamong HWH,
significant HPH among
overlap and HBH HWH,(Figure
HPH 4C,D).
and HBH (Figure 4C,D).
Agronomy 2022, 12, 2293 9 of 15
Agronomy 2022, 12, 2293 8 of 15

Figure
Figure4.4. PCoA
PCoA and UPGMAanalyses
and UPGMA analysesofof bacterial
bacterial (A,B)
(A,B) andand fungal
fungal (C,D)(C,D) community
community structure.
structure.
Based
BasedononPCoA
PCoA analysis andUnweighted
analysis and Unweighted Pair
Pair Group
Group Method
Method withwith Arithmetic
Arithmetic mean (UPGMA)
mean (UPGMA) of of
the
thebinary
binaryJaccard algorithm.
Jaccard algorithm.

3.5. Soil Bacterial and Fungal Abundance

3.5. Soil Bacterial and Fungal Abundance
A total of 13 bacterial phyla were annotated by high-throughput sequencing. The
A total
relative of 13 bacterial
abundance phylaAcidobacteria
of Proteobacteria, were annotated by high-throughput
and Bacteroidetes were higher insequencing.
all groups. The
relative
They accounted for 28.4–34.4%, 19.4–23.2% and 10.3–12.9% of the total soil bacteria, higher
abundance of Proteobacteria, Acidobacteria and Bacteroidetes were respec- in all
groups. They accounted
tively (Figure 5A and Table forS5).
28.4–34.4%,
The relative19.4–23.2%
abundance and 10.3–12.9%was
of Proteobacteria of the
the total
lowest soil
in bacte-
HHH, compared with HWH, HPH, HBH or CK. The relative
ria, respectively (Figure 5A and Table S5). The relative abundance abundance of Acidobacteria
Proteobacteria was
and
the Chloroflexi
lowest in CKcompared
in HHH, was significantly higher than
with HWH, HPH, that
HBHin HWH,
or CK.HPH,
The HHH andabundance
relative HWH, of
while no significant difference was found between HWH, HPH and HBH.
Acidobacteria and Chloroflexi in CK was significantly higher than that in HWH, HPH, HHH The relative
abundance of Bacteroidetes in HBH was significantly lower than CK, HWH, HPH and HHH.
and HWH, while no significant difference was found between HWH, HPH and HBH. The
The relative abundance of Actinobacteria, Verrucomicrobia and Nitrospirae for HHH was
relative abundance of Bacteroidetes in HBH was significantly lower than CK, HWH, HPH
significantly lower than CK, HWH, HPH and HBH. The relative abundances of Gemmati-
and
monadetes inThe
HHH. CK relative
and HBHabundance of Actinobacteria,
were significantly higher than Verrucomicrobia
in HWH, HPH and and Nitrospirae
HHH. The for
HHH was
relative significantly
abundance lower than
of Firmicutes was CK, HWH, HPH
significantly higherand HBH.
in HBH The
than CK,relative
HWH,abundances
HPH
ofand
Gemmatimonadetes
HHH. No significant in CK and HBH
difference were for
was found significantly
the relative higher
abundancethanof in HWH, HPH and
Planctomycetes,
HHH. The relative abundance
Candidatus-Saccharibacteria, of Firmicutes was
candidate-division-wps1 and significantly higher in(Figure
Cyanobacteria-Chloroplast HBH than5A CK,
and Table
HWH, HPH S5).
and HHH. No significant difference was found for the relative abundance of
A total of Candidatus-Saccharibacteria,
Planctomycetes, five fungal phyla were annotated by high-throughputand
candidate-division-wps1 sequencing. The
Cyanobacteria-Chlo-
relative abundance of Ascomycota was higher in CK and HWH than in HPH, HBH and
roplast (Figure 5A and Table S5).
HHH. The relative abundance of Ascomycota, Basidiomycota and Mortierellomycota in the
A total of five fungal phyla were annotated by high-throughput sequencing. The rel-
rotation systems and monoculture system were significantly lower than in CK, except that
ative abundance
the relative of Ascomycota
abundance was in
of Ascomycota higher
HWHin CKnot
was and HWH than
significantly in HPH,
different HBH
from CK.and
The HHH.
The relative
relative abundance
abundance of Ascomycota,
of Mucoromycota andBasidiomycota
Chytridiomycota and
wasMortierellomycota in the rotation
not significantly different
systems and monoculture system were significantly
between HWH, HPH, HBH, HHH and CK (Figure 5B and Table S6). lower than in CK, except that the rel-
ative abundance of Ascomycota in HWH was not significantly different from CK. The rel-
ative abundance of Mucoromycota and Chytridiomycota was not significantly different be-
tween HWH, HPH, HBH, HHH and CK (Figure 5B and Table S6).
gronomy 2022, 12, 2293 10 of

Agronomy 2022, 12, 2293 10 of 15

Agronomy 2022, 12, 2293 9 of 15

Figure 5. The relative abundance of bacterial (A) and fungal (B) communities at the phylum lev
under different culture conditions.

Figure
Figure
Figure 5. The 6 relative
5. Thedemonstrated
relativeabundancetheof
abundance influence
ofbacterial
bacterial(A)of rotational
and
(A) fungal
and (B) systems,
fungal a monoculture
communities at the phylum
(B) communities system
level
at the phylum an
leve
CK under
at the
under different
genera
different culture conditions.
levelconditions.
culture of soil bacterial and fungal communities. The relative abundan
of the soil microbial
Figure communities
6 demonstrated fromofhigh
the influence to low
rotational is represented
systems, a monoculture by system
purpleand to white
green. Figure
CK Of 6 demonstrated
thegenera
at the bacterial the
level ofcommunity, influence
soil bacterial and of rotational
rotational systems,
cropping and
fungal communities. a monoculture
CK increased
The relative system
abundancethe and
of relativ
CK the
at the
abundance genera
soil microbial level
of Subdivision, of 3soil
communities bacterial
high and
fromincertae
genera to lowfungal communities.
is represented
seds, Pedobacter, by purpleThe relative
to white
Chryseolinea, abundance
to green.
Nitrospira and Te
of the
Of soil
the microbial
bacterial communities
community, from
rotational high
cropping to
and low
CK is represented
increased
rimonas but displayed reduced the abundance of Sphingomonas and Lysobacter (Figure 6Athe by
relativepurple
abundanceto white to
of Subdivision,
green. Of soil 3 genera
the bacterial incertae seds,
community, Pedobacter, Chryseolinea,
rotational cropping Nitrospira
and CK and Terrimonas
increasedand but
theSetophom
relative
Regarding fungal genera, Cladorrhinum, Zopfiella, Fusarium,
displayed reduced the abundance of Sphingomonas and Lysobacter (Figure 6A). Regarding
Mortierella
abundance
had relatively of Subdivision,
high abundances 3 genera inincertae seds, Pedobacter,
the monoculture (HHH).Chryseolinea, Nitrospira
Plectosphaerella and Teri
displayed
soil fungal genera, Cladorrhinum, Zopfiella, Fusarium, Mortierella and Setophoma had relatively
rimonas
creased but
highabundancedisplayed
abundances ininthe reduced
the the abundance
rotational(HHH).
monoculture cropping of Sphingomonas
systems,displayed
Plectosphaerella compared and Lysobacter
monoculture6A)
(Figure
with abundance
increased (Fi
Regarding
ure soil fungal
in the rotational
6B). genera,
cropping Cladorrhinum,
systems, compared with Zopfiella, Fusarium,
monoculture Mortierella
(Figure 6B). and Setophoma
had relatively high abundances in the monoculture (HHH). Plectosphaerella displayed in
creased abundance in the rotational cropping systems, compared with monoculture (Fig
ure 6B).

Figure 6. Heatmap
Figure 6. Heatmapof of
soil
soilbacterial (A)and
bacterial (A) andfungal
fungal
(B)(B) communities
communities at thelevel
at the genus genus level
under under differe
different
culture conditions.
culture conditions.
Figure 6. Heatmap of soil bacterial (A) and fungal (B) communities at the genus level under differen
3.6. Correlation
culture of Physicochemical Properties with Microbial Diversity
conditions.
Redundancy analysis (RDA) is used to analyze the possible influence of soil prope
ties on changes of
3.6. Correlation inPhysicochemical Properties
the soil microbial with Microbial
community, such as Diversity
changes in structure and comp
sition.Redundancy
RDA revealed that soil
analysis pH iswas
(RDA) highly
used correlated
to analyze with disease
the possible index.
influence Theproper
of soil relativ
 Agronomy 2022, 12, 2293 10 of 15

Agronomy 2022, 12, 2293 3.6. Correlation of Physicochemical Properties with Microbial Diversity 11 of 15
Redundancy analysis (RDA) is used to analyze the possible influence of soil properties
on changes in the soil microbial community, such as changes in structure and composition.
RDA revealed that soil pH was highly correlated with disease index. The relative abun-
disease
dancesindex for bacteriaGp3,
of Flavobacterium, (Figure 7A).and
Nitrospira TheGp4
relative abundances
were positively of Aspergillus,
correlated Fusarium,
with the disease
Cercophora
index for bacteria (Figure 7A). The relative abundances of Aspergillus, Fusarium, Cercophora (Fig-
and Heydenia were positively correlated with the disease index for fungi
ureand
7B).Heydenia were positively correlated with the disease index for fungi (Figure 7B).

Figure 7. Redundancy
Figure 7. Redundancyanalysis
analysis (RDA) showinginteractions
(RDA) showing interactions between
between soilsoil characteristics
characteristics and
and the the top
top
10 bacterial genera
10 bacterial genera(A)
(A) or
or fungal genera(B).
fungal genera (B).
pHpH denotes
denotes the pH;
the soil soilAP
pH;denotes
AP denotes available
available phospho-
phosphorus;
rus;AK
AK denotes
denotes available
available potassium;
potassium; AN denotes
AN denotes alkaline
alkaline hydrolysis;
hydrolysis; OM matter;
OM organic organicDImatter;
denotesDI de-
notes disease index (healthy plants as “0” and disease plants
disease index (healthy plants as “0” and disease plants as “1”). as “1”).

4. Discussion
4. Discussion
In agriculture and soil ecology, the composition and structure of soil microbe com-
In agriculture
munities are central and to soil ecology,the
understand therelationship
composition and structure
between of soil microbe
rotation systems and crop com-
munities are central to understand the relationship between
health. Crop rotation could improve both the soil microbial ecosystems and nutrients rotation systems and crop
health.
and isCrop rotation
believed to becould improve both
environmentally the soil
friendly for microbial
agricultural ecosystems
management and nutrients
[30–32]. In and
is believed to be environmentally
contrast, monoculture cropping of the friendly
same cropfor agricultural
often reducesmanagement
both the yield and [30–32]. In con-
quality
of crops [33]. Monoculture cropping also increases plant-species-specific
trast, monoculture cropping of the same crop often reduces both the yield and quality of pathogens in
the soil
crops [33].and is therefore not
Monoculture sustainable
cropping also for long-term
increases practice [34–36]. A previous
plant-species-specific pathogensstudyin the
showed that the crop yield, growth condition, soil physicochemical properties and pH
soil and is therefore not sustainable for long-term practice [34–36]. A previous study
are improved by rotation systems [37]. In this study, we focused on the interrelationship
showed that the crop yield, growth condition, soil physicochemical properties and pH are
among rotation systems, soil microbe communities, and industrial hemp fusarium wilt
improved
rate. We byfound rotation
that crop systems
rotation[37]. In this
systems (HWH, study,
HPH, weorfocused on the interrelationship
HBH) significantly decreased
among
industrial hemp disease, improved the plant height and increased floralhemp
rotation systems, soil microbe communities, and industrial and leaffusarium
yields wilt
rate. We found
(Figure that cropwith
1). Compared rotation systems
the rotation (HWH,
systems, HPH, or HBH)
monoculture croppingsignificantly
(HHH) caused decreased
industrial hemp
higher plant disease,
mortality, improved
weaker the plant
plant growth, andheight
lower cropandyields
increased
(Figurefloral andresults
1). These leaf yields
were likely
(Figure caused by improved
1). Compared soil physicochemical
with the rotation properties andcropping
systems, monoculture microbial(HHH)
ecologicalcaused
higher plant mortality, weaker plant growth, and lower crop yields (Figure[38].
environment in the rotation systems, which has been reported in previous studies Our re-
1). These
results further showed that the soil AP, AK and AN content were significantly decreased in
sults were likely caused by improved soil physicochemical properties and microbial eco-
the monoculture system (HHH) (Figure 2).
logical environment in the rotation systems, which has been reported in previous studies
Soil microorganisms are an important component of the soil, and the structure and
[38]. Our results
diversity of thefurther
microbial showed
communitythat theare soil AP, AK
important forand AN content
maintaining were significantly
soil ecological bal-
decreased in the monoculture system (HHH) (Figure 2).
ance [39]. One factor that influences the structure and diversity of the microbial community
Soilplant
is the microorganisms
species presentare anThe
[40]. important
greater the component of the
diversity index soil,
and the and
morethe structure
complex the and
diversity of the microbial community are important for maintaining soil ecological
structure of the microbial community, the stronger its stability and the greater its ability to bal-
cope
ance with
[39]. One thefactor
environment [41]. Our current
that influences study showed
the structure that bothofbacterial
and diversity and fungal
the microbial commu-
diversity was higher in the soil with three rotation crops compared
nity is the plant species present [40]. The greater the diversity index and the more complex to monoculture crop.
the structure of the microbial community, the stronger its stability and the greater its abil-
ity to cope with the environment [41]. Our current study showed that both bacterial and
fungal diversity was higher in the soil with three rotation crops compared to monoculture
crop. There was no significant difference between the three rotation systems and the con-
Agronomy 2022, 12, 2293 11 of 15

There was no significant difference between the three rotation systems and the control
system (Figure 3). One of the important findings is that the community diversity was
negatively correlated with industrial fusarium wilt rate. However, there was no direct
relationship between biodiversity and function, but the soil with the highest biodiversity
was more resistant to stress than soil with impaired biodiversity [42]. We speculated that
compromised microbial diversity could lead to the soil microbial community becoming
unbalanced, leading to the invasion of pathogenic bacteria. There is evidence that soil micro-
bial diversity confers protection against soil-borne disease and is important for agricultural
sustainability [43]. Therefore, our results support the hypothesis that rotation systems with
high biodiversity were more resistant to infection of pathogen than monoculture cropping.
The soil microbial community is affected both by the types of soil and the types of plant
species. Previous studies have shown that the microbial community is also affected by crop
rotation or monoculture cropping of the same plant [44–46]. In this study, we found that the
microbial community is significantly different between monoculture cropping and rotation
systems or CK (Figure 5). The abundances of Proteobacteria and Verrucomicrobia were higher
in the rotation systems and CK than with monoculture cropping (HHH), and the abundance
of Actinobacteria was significantly was lower in HHH (Figure 5). Proteobacteria is important
for predicting changes in soil microecology [47]. Verrucomicrobia is widely distributed in
nature and can degrade refractory organic matter [48,49]. Actinobacteria secretes several
secondary metabolites that promote plant growth but inhibit pathogen growth [50–52]. The
relative abundance of Actinobacteria was higher in Rhizoctonia (root rot)-suppressive soil [53].
This can be relevant to industrial hemp health as enhanced plant vigor is important in
pathogen resistance.
Some potential industrial hemp fusarium wilt-suppressing bacteria were also detected
in this study. Our heatmap assessment showed that the dominant taxa for the rotation
systems and CK, but not the monoculture system, were Proteobacteria, Chloroflexi, Rhizo-
bium, Bacteroides, Actinobacteria, Pseudomonas, Flavobacterium, Saccharibacteria and Nitrospirae
(Figure 6). A previous study showed that Pseudomonas can inhibit the growth and infection
of plant pathogens by secreting antibiotics, toxins, biosurfactants, extracellular cell-wall
decomposition enzymes and other substances [54]. Firmicutes and Flavobacterium can in-
hibit pathogen growth [55]. Flavobacterium can inhibit the growth of the blight-causing
Phytophthora capsici in pepper plants [56]. Rhizobium played an important role in plant root
colonization [57]. In conclusion, the rotation systems help increase the growth of beneficial
microorganisms.
Ascomycota and Basidiomycota are two large and diverse groups of fungi that play an
important role in maintaining the soil ecosystem. For example, Preussia can inhibit fungal
diseases, while Penicillium and Chaetomium can degrade organic matter [58–60]. In this study,
we found that the relative abundances of Ascomycota, Basidiomycota and Mortierellomycota
were significantly higher in CK than in other systems (Figure 6), suggesting that cropping,
whether rotation or monoculture, can affect the overall fungal community diversity. An
early study showed that certain species of the fungi genus Fusarium were pathogenic and
could cause fusarium wilt disease in watermelon monoculture [61]. Fusarium oxysporum was
also isolated from rhizosphere soils of industrial hemp where fusarium wilt occurred [16].
Therefore, we hypothesize that the increased disease incidence in industrial hemp after
monoculture cropping was mainly due to the abundance of Fusarium oxysporum. However,
other pathogens may cause wilt in industrial hemp, such as Fusarium brachygibbosum,
Pythium aphanidermatum, Fusarium solani, and Fusarium equiseti [62]. Future studies will
verify the pathogenicity of these strains to industrial hemp.
We conducted PCoA and RDA analyses to profile the soil microbial community struc-
ture. Our study showed that there was a significant change among rotation systems,
monoculture system and CK in bacterial and fungal communities. However, the fungal
community structure was also significantly altered in the different rotation systems (HWH,
HPH and HBH) (Figures 4 and 7). Previous studies have shown that the most prosperous
growth for Fusarium oxysporum occurred at pH values between 6 and 7 [63]. The results
Agronomy 2022, 12, 2293 12 of 15

showed that the soil AP, OM, and pH were associated with soil microbiota composition and
had close relationships with DI (Figure 7). Other studies have shown that Sphingomonas
could promote crop resistance against multiple pathogens [64]. The Gemmatimonas genus
has been shown to be related to the formation of organic soil content and has a significant
role in promoting the degradation of cellulose and enhancing pathogenic infections [65].
Our study confirmed that the rotation of industrial hemp could alleviate fusarium wilt.
There was a complex interaction among industrial hemp fusarium wilt, soil physico-
chemical and microbial communities. Further studies are required to better understand
these interactions.

5. Conclusions
Our study demonstrated that rotation systems could enhance the resistance of indus-
trial hemp plants to pathogens by affecting the composition and function of soil bacterial
and fungal communities. We believe that the application of rotation system is an efficient
and eco-friendly approach to control destructive soil-borne pathogens and diseases for
industrial hemp. It is also sustainable and improves the floral and leaf yield and quality
while protecting the environment.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/agronomy12102293/s1, Table S1: Source of seeds and growth
condition; Table S2: The time table of the cultivation during the 3 years; Table S3: Diversity and
richness indexes of bacterial communities under crop rotation systems; Table S4: Diversity and
richness indexes of fungal communities under crop rotation systems; Table S5: The relative abundance
of bacterial communities at the phylum level; Table S6: The relative abundance of fungal communities
at the phylum level.
Author Contributions: L.T. and C.F. performed the sample collection, library preparation and
microbial annotation, and participated in the bioinformatics and statistical analyses; they also wrote
the original draft and performed the review and editing. H.Y. and G.W. took charge of the software
and methodology of the manuscript. S.Z. and J.S. oversaw project administration. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by the Heilongjiang Provincial Scientific Research Institute
Scientific Research Business Expense Project (CZKYF2021-2-C010).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Raw readings were submitted to the Sequence Read Archive (SRA) for
the NCBI database (Accession Number: PRJNA841395).
Conflicts of Interest: The authors declare that they have no conflict of interest.

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