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Frontiers in Microbiology | www.frontiersin.org 1 May 2022 | Volume 13 | Article 850917
ORIGINAL RESEARCH
published: 12 May 2022
doi: 10.3389/fmicb.2022.850917
Edited by:
Prem Lal Kashyap,
Indian Institute of Wheat and Barley
Research (ICAR), India
Reviewed by:
Omar Abdelraouf Hewedy,
University of Menoua, Egypt
Akansha Jain,
Bose Institute, India
*Correspondence:
Min Zhao
82191513@163.com
Hongyan Yang
yanghy@nefu.edu.cn
Specialty section:
This article was submitted to
Microbiological Chemistry and
Geomicrobiology,
a section of the journal
Frontiers in Microbiology
Received: 08 January 2022
Accepted: 02 March 2022
Published: 12 May 2022
Citation:
Wang Y, Wang L, Suo M, Qiu Z,
Wu H, Zhao M and Yang H (2022)
Regulating Root Fungal Community
Using Mortierella alpina for Fusarium
oxysporum Resistance in Panax
ginseng.
Front. Microbiol. 13:850917.
doi: 10.3389/fmicb.2022.850917
Regulating Root Fungal Community
Using Mortierella alpina for Fusarium
oxysporum Resistance in Panax
ginseng
YanWang
1,2, LiweiWang
1,2, MengSuo
1,2, ZhijieQiu
1,2, HaoWu
1,2, MinZhao
1,2* and
HongyanYang
1,2*
1 College of Life Sciences, Northeast Forestry University, Harbin, China, 2 Key Laboratory for Enzyme and Enzyme-like Material
Engineering of Heilongjiang, Harbin, China
Plant-associated microbes play important roles in plant health and disease. Mortierella
is often found in the plant rhizosphere, and its possible functions are not well known,
especially in medical plants. Mortierella alpina isolated from ginseng soil was used to
investigate its effects on plant disease. The promoting properties and interactions
with rhizospheric microorganisms were investigated in a medium. Further, a pot
experiment was conducted to explore its effects on ginseng root rot disease.
Physicochemical properties, high-throughput sequencing, network co-occurrence,
distance-based redundancy analysis (db-RDA), and correlation analysis were used
to evaluate their effects on the root rot pathogen. The results showed that Mortierella
alpina YW25 had a high indoleacetic acid production capacity, and the maximum yield
was 141.37 mg/L at 4 days. The growth of M. alpina YW25 was inhibited by some
probiotics (Bacillus, Streptomyces, Brevibacterium, Trichoderma, etc.) and potential
pathogens (Cladosporium, Aspergillus, etc.), but it did not show sensitivity to the
soil-borne pathogen Fusarium oxysporum. Pot experiments showed that M. alpina
could signicantly alleviate the diseases caused by F. oxysporum, and increased the
available nitrogen and phosphorus content in rhizosphere soil. In addition, it enhanced
the activities of soil sucrase and acid phosphatase. High-throughput results showed
that the inoculation of M. alpina with F. oxysporum changed the microbial community
structure of ginseng, stimulated the plant to recruit more plant growth-promoting
bacteria, and constructed a more stable microbial network of ginseng root. In this
study, wefound and proved the potential of M. alpina as a biocontrol agent against
F. oxysporum, providing a new idea for controlling soil-borne diseases of ginseng by
regulating rhizosphere microorganisms.
Keywords: Mortierella alpina, Panax ginseng, microbial community, Fusarium oxysporum, resistance
Wang et al. M. alpina Regulate Ginseng Root Community
Frontiers in Microbiology | www.frontiersin.org 2 May 2022 | Volume 13 | Article 850917
INTRODUCTION
Ginseng (Panax ginseng C. A. Meyer), a member of the Araliaceae
family, is a valuable medicinal plant with multiple functions,
such as enhancing organ functioning, inhibiting inammation,
preventing tumors and diseases, and potentially inhibiting
COVID-19 (Han et al., 2018; Li et al., 2020b; Lee and Rhee,
2021). It is estimated that the global ginseng market, including
ginseng root and the processed products, is worth $2,084
million (Baeg and So, 2013). Ginseng is cultivated in many
countries, such as China, United States, and South Korea. As
a perennial plant, ginseng grows in cold and humid environments
and is prone to various diseases during growth. F. oxysporum
can cause many plant diseases, such as tomato wilt, potato
dry rot, and soybean root rot (Palmieri etal., 2020; Han et al.,
2021; Ren etal., 2021). Root rot is a serious soil-borne disease
of ginseng that damages ginseng of all ages and can lead to
crop failure in severe cases (Farh et al., 2018). F. oxysporum
is one of the main pathogens causing root rot in ginseng
(Punja et al., 2008).
Currently, ginseng soil-borne diseases are mainly controlled
by chemical agents. e frequent use of chemical fungicides
has led to many problems, including increased pathogen resistance
to fungicides, destruction of the soil microenvironment, high
levels of toxic substances in ginseng, and environmental pollution
(Wang et al., 2021a). Biological control is an environmentally
friendly method of controlling plant diseases using benecial
microorganisms to regulate the microbiological composition
of the soil. is eectively protects plants from pathogenic
microorganisms while gradually leading to positive microbial
community succession. A large number of commercial microbial
agents have been developed from endophytic and rhizospheric
microorganisms, such as Bacillus, Pseudomonas, and Trichoderma
(John et al., 2010; You et al., 2016; De Silva et al., 2019).
Dierent plants have dierent physiological characteristics and
rhizospheric soil microdomains. Consequently, screening
microorganisms from native plants and rhizospheric soil can
easily enhance the eectiveness of biocides (Bagy et al., 2019;
Azabou et al., 2020). erefore, it can become an eective
means to control soil-borne diseases.
Mortierella, due to its ability to degrade organic pollutants,
can be used for soil remediation. Mortierella has also been
detected in the rhizosphere and bulk soils of many plants (Liu
et al., 2021; Tong et al., 2021; Xiang et al., 2021; Zhou et al.,
2021). Some studies have indicated that Mortierella is related
to soil disease inhibition, and may inhibit the diseases caused
by Fusarium and participate in the transformation of phosphorus
in soil. is is benecial for soil health and nutrient absorption
in plants from soil (Li etal., 2020a; Liu etal., 2020a). However,
another study reported that Mortierella was a dominant plant
pathogen (Guo et al., 2021). is controversy suggests that
the eects of Mortierella on plants may be species-specic. In
addition, the current analysis of Mortierella disease inhibition
has not reached a consensus on whether Mortierella achieves
inhibition and growth promotion in pathogenic microorganisms
by aecting the bacterial or fungal community in soil or plants
(Li et al., 2020a; Guo et al., 2021). erefore, it is vital to
discuss possible plant-specic probiotic eects of microbial
species. is will contribute to better dening the scope of
action and functions of biocontrol microorganisms.
Mortierella has also been detected in the rhizosphere of
Panax ginseng (Li etal., 2018; Liu et al., 2020b). However, its
possible function during Panax ginseng cultivation remains
unclear. Our previous research showed that Mortierella accounted
for dierent proportions of fungal communities under dierent
soil planting conditions, with the highest proportion in forest
soil and the lowest in 4-year ginseng-cultivated soil. is
indicates that there is a positive correlation between Mortierella
and the health of ginseng cultivation soil (Wang et al., 2021b).
However, it is unclear whether it can be used as a possible
biocontrol fungus to improve the resistance of ginseng to soil-
borne diseases.
Here, an M. alpina strain YW25 isolated from ginseng
rhizosphere soil was inoculated into the ginseng rhizosphere
to test its possible pathogen resistance and biocontrol potential
during Panax ginseng cultivation. F. oxysporum strain YFW32,
which causes ginseng root rot, was used as the pathogen. In
this study, weaimed to determine (1) the eects of inoculation
of native M. alpina on ginseng and rhizosphere soil; (2) whether
M. alpina has the ability to help plants resist the invasion of
pathogens; and (3) if it does, how is the underlying mechanism?
MATERIALS AND METHODS
Microbial Strains
Mortierella alpina YW25 was isolated from ginseng rhizosphere
soil, and F. oxysporum YFW32 was isolated from diseased
ginseng roots. e above strain sequences were been stored
in DDBJ/EMBL/GenBank using DDBJ quick annotation and
submission tool (DFAST),1 and their login numbers were
LC663965 and LC656545, respectively. e strains were stored
at −80°C and then streaked on PDA plates, cultured at 28°C
for 7 days, and transferred twice for subsequent tests.
Analysis of Growth-Promoting Potential of
Mortierella alpina
e Salkowski colorimetric method (Gordon and Weber, 1951)
was used to evaluate the IAA production capacity of M. alpina
YW25. Briey, six PDA plugs with M. alpina YW25 mycelia
(5 mm diameter) were inoculated in asks containing 100 ml
PDB liquid medium and 3 mM tryptophan. e asks were
maintained at 28°C for 2–7 days at 180 rpm. Uninoculated
medium was used as the control. en, 2 ml of culture was
centrifuged at 10,000 rpm and 4°C for 10 min. e supernatant
was mixed with Salkowski reagent in equal volumes, and the
reaction was developed at 25°C in the dark for 30 min. e
absorbance was measured at 535 nm. A calibration curve was
established for calculating IAA concentration (5–100 mg/L) at
535 nm using pure IAA. e values were averaged over triplicates.
1
https://dfast.nig.ac.jp/
Wang et al. M. alpina Regulate Ginseng Root Community
Frontiers in Microbiology | www.frontiersin.org 3 May 2022 | Volume 13 | Article 850917
e solubility of M. alpina YW25 inorganic phosphorus
was evaluated using Pikovskaya’s (PVK) medium (Pikovskaya,
1948). e 1 L medium consisted of 10 g glucose, 0.3 g NaCl,
0.3 g KCl, 0.5 g (NH4)2SO4, 0.3 g MgSO4·7H2O, 0.03 g
MnSO4·4H2O, 0.03 g FeSO4·7H2O, 5 g Ca3(PO4)2, 18 g agar, and
1 L distilled water, and was adjusted to pH 7.0–7.2. M. alpina
YW25 was inoculated into plates containing PVK agar medium.
e inoculated plates were incubated in the dark at 28°C for
7 days. Clear halos were observed around the colonies, which
indicated that the isolate solubilized inorganic phosphate. Lecithin
(P7443, Sigma-Aldrich, United States) was used instead of
Ca3(PO4)2 to evaluate its ability to dissolve organophosphorus
(Wei et al., 2018). is was carried out by the same process
as inorganic phosphorus. e phosphate solubility index (SI),
which is the whole diameter zone (diameter of halo + diameter
of colony) ÷ colony diameter, was used to evaluate the phosphorus
solubility of the strain. e values were averaged over triplicates.
Chrome Azurol S (CAS; Schywn and Nielands, 1987) was
used to evaluate the siderophore production capacity of M. alpina
YW25. PDA plugs with M. alpina YW25 mycelia (5 mm
diameter) were inoculated on CAS plates and incubated at
28°C for 7 days. e formation of an orange halo around the
colony was observed. Larger halos had darker colors, which
indicated a higher yield of siderophores. Six PDA plugs with
M. alpina YW25 mycelia were inoculated in 100 ml of PDA
liquid medium. e asks were maintained at 28°C for 7 days
at 180 rpm. Subsequently, 2 ml of culture at 4°C was centrifuged
at 10,000 rpm for 10 min. e supernatant was mixed with
CAS solution in equal volumes, and the reaction was carried
out at 25°C in the dark for 1 h. e absorbance was detected
at 630 nm (A), and the uninoculated medium was used as
the control (Ar). Siderophores produced by the isolate were
measured as percent siderophore units (% SU), and were
calculated according to the following formula: % SU = (Ar–A)
÷ Ar × 100. e values were averaged over triplicates (Machuca
and Milagres, 2003).
PDA plugs containing M. alpina YW25 mycelia were
inoculated on PDA plates containing 0.2% soluble starch, 0.5%
carboxymethyl cellulose, 0.5% xylan, 1% pectin, and 1% skim
milk powder and cultured at 28°C for 7 days to evaluate the
activity of amylase, cellulase, xylanase, pectinase, and protease,
respectively. e plate containing 0.2% soluble starch was treated
with a 1% iodine solution. A transparent halo around the
colony indicated amylase activity. Congo red solution (0.2%)
was added to the plates containing 0.5% carboxymethyl cellulose
and 0.5% xylan. Following this, the plates were washed with
1 M NaCl. Yellow halos were observed around the colonies,
which indicated cellulase and xylanase activities, respectively.
When 1% cetyl trimethyl ammonium bromide (CTAB) was
added to the plate containing 1% pectin, a transparent halo
appeared around the colony, which indicated pectinase activity.
Aer the fungi were cultured on PDA plates containing 1%
skim milk powder, a transparent hydrolytic halo appeared
around the colony, which indicated protease activity (Sopalun
and Iamtham, 2020; Liu et al., 2020c; Sopalun et al., 2021).
Six PDA plugs with M. alpina YW25 mycelia (5 mm diameter)
were inoculated in asks containing 100 ml YM liquid medium
(Papagianni and Moo-Young, 2002). e asks were maintained
at 28°C for 5 days at 180 rpm. Mix 1 ml culture supernatant
in equal volume with a phosphate buer (pH 7.0) containing
1% (w/v) casein, and incubated for 10 min at 30°C. Two
milliliter of 0.4 M trichloro acetic (TCA) acid was added to
terminate the reaction. e mixture containing the culture
supernatant was incubated for 30 min at 25°C followed by
centrifugation at 10,000 rpm for 5 min. Five microliter of 0.4 M
Na2CO3 was then mixed with the supernatant (1 ml) and aer
10 min, 1 ml of Folin reagent was added to each tube. e
tubes were allowed to stand for 30 min at 30°C and then the
absorbance was measured at 660 nm. Similar approach was
used to prepare the control except casein was added only aer
the reaction was stopped. 1 U = the amount of enzyme required
to liberate one microgram (1 μg/ml) of tyrosine under the
assay conditions described (Chimbekujwo et al., 2020). e
values were averaged over triplicates.
In vitro Analysis of Interactions Between
Mortierella alpina and Rhizosphere
Microorganisms
e interaction between M. alpina YW25 and ginseng
rhizosphere microorganisms was evaluated in vitro using the
plate confrontation method (Cong etal., 2019). e 17 fungi,
15 bacteria, and two actinomycetes used for confrontation
were isolated from ginseng rhizosphere soil. M. alpina YW25
and rhizosphere fungi were symmetrically and equidistantly
inoculated on a PDA plate 2.5 cm away from the center, and
cultured at 28°C in the dark for 7 days. M. alpina YW25
was placed in the center of LB and Gao’s No.1 plates, and
bacterial and actinomycete colonies, respectively, were picked
out with sterilized toothpicks. e bacterial and actinomycete
colonies were inoculated symmetrically and equidistantly at
a distance of 2.5 cm from the M. alpina YW25 block on the
plate, and cultured at 28°C in the dark for 5 days. e plate
inoculated with M. alpina YW25 was used as the control.
All processing settings were triplicated. Inhibition rate
(%) = (colony radius of control group − colony radius of
treatment group)/colony radius of control group × 100 (Cong
etal., 2019). e inhibition of M. alpina YW25 by rhizosphere
microorganisms was divided into four grades: − (no inhibition),
+ (inhibition rate < 30%), ++ (inhibition rate 30–60%), and
+++ (inhibition rate > 60%).
Experimental Design and Sample
Collection
e PDA plugs containing mycelia of M. alpina YW25 and
F. oxysporum YFW32 with a diameter of 5 mm were cultured
for 7 days at 28°C in PDA liquid medium separately at 180 rpm.
e mycelium was ltered using gauze and diluted with sterile
water to prepare a 1.2 × 107/ml spore suspension, which was
used for pot inoculation of ginseng. ree treatment groups
were established: single inoculation of M. alpina YW25 (MA),
single inoculation with F. oxysporum YFW32 (FO), and
inoculation with M. alpina YW25 and F. oxysporum YFW32
(MA_FO).
Wang et al. M. alpina Regulate Ginseng Root Community
Frontiers in Microbiology | www.frontiersin.org 4 May 2022 | Volume 13 | Article 850917
Potted soil (not autoclaved) contains 25.05 mg/kg nitrate
nitrogen, 0.69 mg/kg ammonium nitrogen, 1.18 mg/kg available
phosphorus, 292.25 mg/kg available potassium, total nitrogen
10.2 mg/g, total phosphorus 8.59 mg/g, total potassium 20 mg/g,
and organic matter 0.35 g/g and the pH was 6.96. ree-year-old
ginseng seedings were planted in each pot and inoculated by
root irrigation. In MA and FO treatments, each pot (1.5 kg
ower soil) was inoculated with 10 ml spore suspension. In
MA_FO treatment, M. alpina YW25 and F. oxysporum YFW32
spore suspensions were inoculated with 5 ml each, and 10 ml
sterile water was used as the control (CK). e setup for each
treatment was repeated ve times.
Ginseng was harvested aer 70 days of pot planting. It
was carefully uprooted and gently shaken to remove loosely
adhered soil from the roots. Subsequently, all ginseng
rhizosphere soil samples from the same treatment were mixed,
and the rhizosphere soil sample of the treatment was formed.
e rhizosphere soil samples were divided into two parts,
and one of these was immediately stored in a −80°C refrigerator
for the detection of soil microbial diversity. e other was
air-dried indoors and stored at room temperature aer ltering
through a 2 mm sieve for determination of various soil physical
and chemical properties. Aer washing and drying ve ginseng
plants in each treatment, the length and fresh weight of
ginseng plants were measured by scale and balance. e
ginseng plants were then divided into root and aboveground
parts. Aer surface disinfection, the samples were quickly
frozen in liquid nitrogen and then stored at −80°C for the
detection of ginseng microbial diversity and plant
defense enzymes.
Measurement of Soil Physicochemical
Properties and Plant Defense Enzymes
Soil pH was measured using a pH meter (S010, Horiba, Japan).
Nitrate and ammonium nitrogen were determined by 2 mol/L
KCl extraction spectrophotometry (Li et al., 2021b). Available
phosphorus was determined by NaHCO3 extraction and
molybdenum–antimony resistance spectrophotometry (Yua n
et al., 2020). Kjeldahl was used to determine total nitrogen
(Arunrat et al., 2022). Total phosphorus was determined by
sodium hydroxide alkali fusion–molybdenum–antimony anti
spectrophotometry (Liu et al., 2022). Total potassium and
available potassium were determined by ame atomic absorption
spectrophotometry (Li et al., 2021a), and organic matter was
determined by the loss-of-burning method (Salehi etal., 2011).
Soil urease activity was determined by indophenol colorimetry
(Adetunji et al., 2021), and the activities of soil catalase, acid
phosphatase, and sucrase were determined using kits (Suzhou
Grace Biotechnology Co. Ltd.; Zhou et al., 2020).
Harvested fresh ginseng root tissue was used to detect plant
defense enzymes. e activities of peroxidase (POD), polyphenol
oxidase (PPO), lipoxygenase (LOX), and phenylalanine ammonia
lyase (PAL) were determined using microplate kits (NO. G0107W,
NO. G0113W, NO. G0906W, and NO. G0114W, respectively,
Suzhou Grace Biotechnology Co. Ltd.; Cheng et al., 2020;
Yang et al., 2020).
High-Throughput Sequencing and Analysis
of 16S rDNA and Internal Transcribed
Spacer Regions
High-throughput Illumina sequencing was used to characterize
the microbial community structure in the soil and plant samples
(Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China).
e V3-V4 regions of the soil bacterial 16S rRNA genes were
amplied using the primers 338F (5′-ACTCCTACGGGAGGC
AGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′;
Xu et al., 2019). To assess the ginseng bacterial community,
two sets of primers targeting the V3-V4 region of 16S rRNA
gene were designed. e rst-round reaction was amplied with
primers 799F (5′-AACMGGATTAGATACCCKG-3′) and 1392R
(5′-ACGGGCGGTGTGTRC-3′; Cui et al., 2020). e second-
round reaction was amplied with primers 799F (5′-AACMGGATT
AGATACCCKG-3′) and 1193R (5′-ACGTCATCCCCACCTT
CC-3′; Bulgarelli et al., 2015). e ITS1F-ITS2R region of the
ginseng fungal gene was amplied using the primers ITS1F
(5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCT
GCGTTCTTCATCGATGC-3′; Sun etal., 2018). Specic primers
with barcodes were synthesized according to the designated
sequencing region, and then the samples were amplied using
a thermocycler (GeneAmp® 9700, ABI, United States). e raw
reads were deposited into the NCBI sequence read archive (SRA)
under the submission ID SUB10895992.2
Bacterial PCR reactions were performed in triplicate, with
4 μl 5× FastPfu Buer, 2 μl 2.5 mM dNTPs, 0.8 μl 5 μM forward
primer, 0.8 μl 5 μM reverse primer, 0.4 μl FastPfu Polymerase,
0.2 μl bovine serum albumin (BSA), and 10 ng template DNA
in a 20 μl reaction volume. e thermal cycling conditions for
prokaryotic 16S rRNA gene from soil bacteria fragment
amplication were as follows: 3 min at 95°C, 30 cycles of 30 s
at 95°C, 30 s at 55°C, 45 s at 72°C, and 10 min at 72°C. e
16S rRNA gene from ginseng bacterial fragments was amplied
in two rounds, and the thermal cycling conditions of amplication
were as follows: rst round: 3 min at 95°C, 27 cycles of 30 s
at 95°C, 30 s at 55°C, 45 s at 72°C; and 10 min at 72°C; second
round: 3 min at 95°C, 13 cycles of 30 s at 95°C, 30 s at 55°C,
45 s at 72°C, and 10 min at 72°C. Fungal PCR reactions were
performed in triplicate with 2 μl 10× rTaq Buer, 2 μl 2.5 mM
dNTPs, 0.8 μl 5 μM forward primer, 0.8 μl 5 μM reverse primer,
0.2 μl rTaq polymerase, 0.2 μl BSA, and 10 ng template DNA
in a 20 μl reaction volume. e thermal cycling conditions for
prokaryotic ITS gene fragment amplication were as follows:
3 min at 95°C, 30 cycles of 30 s at 95°C, 30 s at 55°C, 45 s at
72°C, and 10 min at 72°C. e PCR products were identied
by 2% agarose gel electrophoresis, puried using an AxyPrep
DNA gel extraction kit (Axygen, Corning, NY, United States),
and quantied using a QuantiFluor™-ST Blue Fluorescence
Quantication System (Promega).
e amplied sub-library was sequenced on an Illumina
PE250 platform (Biozeron, Shanghai, China). e eective
sequences of all samples were obtained according to the barcode,
and Trimmomatic (version 0.36; Lohse et al., 2012) ltration
2
https://submit.ncbi.nlm.nih.gov/subs/sra/SUB10895992/overview
Wang et al. M. alpina Regulate Ginseng Root Community
Frontiers in Microbiology | www.frontiersin.org 5 May 2022 | Volume 13 | Article 850917
was used to remove reads with an average mass of less than
20 in 50 bp. Sequences were assembled using FLASH with a
minimum overlap of 10 bp and a maximum mismatch ratio
of 0.2 (Magoc and Salzberg, 2011). e RDP classier Bayesian
algorithm (Wang etal., 2007; version 2.2) was used to classify
the representative sequences of each operational taxonomic
unit (OTU) with 97% similarity.3 e bacterial 16S rRNA
comparison database was Silva (Release138; Quast etal., 2013)4
and the fungal ITS comparison database was Unite (Release
8.0; Koljalg et al., 2013).5
Co-occurrence Network Analysis
A co-occurrence network based on the Spearman correlation
coecient matrix was constructed by NetworkX to study the
relationship and interaction between bacteria and fungi in the
aboveground and root of ginseng under dierent inoculation
treatments. OTUs with relative abundance greater than 0.01%
in each treatment were screened for OTU with subsequent
correlation network construction. e most important interaction
was highlighted, and the Spearman correlation threshold was
set to 0.7, p < 0.05. Each node represents an OTU, and each
edge represents a strong and signicant correlation between
the dierent nodes. Networks were visualized using the Gephi
platform.6 Topological features (average degree and modularity)
of the networks were calculated using NetworkX on the free
online platform of Majorbio Cloud Platform.7
3
http://sourceforge.net/projects/rdpclassier/
4
http://www.arb-silva.de
5
http://unite.ut.ee/index.php
6
http://gephi.github.io/
7
http://www.majorbio.com
Statistical Analysis
GraphPad Prism 8.3.0 was used to draw line and bar charts.
SPSS 19.0 was used for one-way analysis of variance (ANOVA),
and the signicance level was p < 0.05. For the high-throughput
Illumina sequencing data, Adonis test, Student’s t-test along
with alpha diversity, db-RDA, and linear regression analyses
were performed using the online platform of Majorbio Cloud.8
RESULT
Growth-Promoting Potential of Mortierella
alpina YW25
To detect the plant growth-promoting potential of M. alpina
YW25, the capacities of IAA and siderophore production,
phosphorus solubilization, and hydrolase activity of M. alpina
YW25 were determined. e results showed that it had a high
ability to produce IAA, and the highest IAA concentration in
PDB liquid medium containing 3 mM tryptophan reached
141.37 mg/L at 4 days (Figure 1). e ability to produce
siderophores and dissolved phosphorus was not detected in
M. alpina YW25. No hydrolase activities of M. alpina YW25,
except for protease activity was 5.5 U/ml aer 5 days.
In vitro Analysis of Mortierella alpina YW25
and Rhizosphere Microbes Interactions
e interaction between M. alpina YW25 and a variety of
microorganisms in the ginseng rhizosphere were studied by
plate confrontation experiments (Table S1 and Figure2). Bacillus
species (Bacillus siamensis, B. velezensis, B. toyonensis, B. cereus,
and B. zhangzhouensis) signicantly inhibited the growth of
M. alpina YW25 among the 15 bacterial strains isolated from
the ginseng rhizosphere. Streptomyces tricolor and Brevibacterium
frigoritolerans (actinomycetes) also showed signicant inhibition
of M. alpina YW25. Fungi isolated from ginseng rhizosphere,
such as Trichoderma koningiopsis, T. viridescens, T. harzianum,
T. velutinum, Rhizopus oryzae, Penicillium citrinum,
P. chrysogenum, Aspergillus ochraceus, A. avus, Cladosporium
anthropophilum, and C. cladosporioides, also inhibited the growth
of M. alpina YW25. However, there was no obvious interaction
between F. oxysporum YFW32 and M. alpina YW25.
Effects of Inoculation With Mortierella
alpina YW25 on Panax ginseng
To determine the eects of M. alpina YW25 inoculation on
ginseng, plant height, root length, and fresh weight of both
aboveground and root regions were measured (Figure 3). e
defense enzymes of ginseng roots were also determined (Table1).
In the FO treatment, leaves withered and roots were infected
and decomposed (Figure3A). Ginseng in the other treatments
showed no disease symptoms. e root length of FO was
signicantly lower than that of the other treatments (p < 0.05),
and there was no signicant dierence between MA and
CK. ere was no signicant dierence in fresh weight of
8
https://cloud.majorbio.com/
FIGURE1 | Effects of incubation time on the production of IAA by Mortierella
alpina YW25.
Wang et al. M. alpina Regulate Ginseng Root Community
Frontiers in Microbiology | www.frontiersin.org 6 May 2022 | Volume 13 | Article 850917
ginseng aboveground. e fresh weight of ginseng roots in
FO was signicantly lower than that in the other treatments.
e fresh weight of ginseng roots in CK was signicantly
higher than that in MA, but there was no signicant dierence
between CK and MA (Figure 3B). In the FO treatment, POD
and PPO activities were 133.09 and 213.33, respectively, which
were signicantly higher than those in CK (p < 0.05). LOX
activity was signicantly lower, whereas PAL activity showed
no signicant change. Compared with CK, the activities of
PPO, LOX, and PAL in the MA_FO were higher, but the
activities of the four plant defense enzymes in the MA and
MA_FO treatments were not signicantly dierent (Table 1 ).
Effects of Mortierella alpina YW25
Inoculation on Soil Physicochemical and
Enzymatic Properties
Soil pH, ammonium nitrogen (NH4+-N), nitrate nitrogen (NO3−-
N), total nitrogen (TN), available phosphorus (AP), total
phosphorus (TP), available potassium (AK), total potassium
(TK), urease (Urease), catalase (CAT), sucrase (SC), and acid
phosphatase (ACP) were measured in ginseng roots (Tab le 2).
As shown in the table, there were no signicant dierences
in pH, TN, and TP among the MA, MA_FO, and CK treatments.
e NO3−-N and TK content in MA rhizosphere soil was
signicantly lower than those in CK, and the NH4+-N, AP,
and AK content was signicantly higher than those in CK
(p < 0.05). In the FO treatment, soil pH was signicantly lower
than that in CK, but the soil AK content was signicantly
higher than that of the other treatments (p < 0.05). e content
of NO3−-N, AP, and AK in the MA_FO rhizosphere soil was
signicantly lower, but the content of NH4+-N was signicantly
higher than that in FO (p < 0.05). e available nitrogen (NH4+-N
and NO3−-N) and AP content in the MA treatment was
signicantly higher than those in the MA_FO treatment. In
addition, the activity of urease, SC, and ACP in MA soil was
signicantly higher, while the activity of CAT was signicantly
lower than that in CK (p < 0.05). e activities of CAT and
SC in the MA_FO treatment were signicantly higher, while
the activity of ACP was signicantly lower than that in FO.
Effects of Mortierella alpina YW25
Inoculation on Plant and Soil Microbiome
e Shannon index was used to evaluate the soil microbial
diversity of ginseng aboveground, roots, and rhizosphere under
dierent treatments (Figure 4). e bacterial diversity in the
ginseng rhizosphere soil was higher than that in the plant
(p < 0.05) in all treatments. In the FO and MA treatments,
soil fungal diversity was signicantly lower than that in the
CK and MA_FO treatments (p < 0.05). ere was no signicant
dierence between MA and CK in the diversity of bacteria
and fungi in the aboveground parts of ginseng. In the MA
treatment, the fungal diversity was signicantly lower in the
roots of ginseng than in CK and FO. Further, FO and MA_FO
showed no signicant dierences in the diversity of bacteria
in the aboveground parts of ginseng. Compared with FO, the
bacterial diversity signicantly increased, and the fungal diversity
signicantly decreased in the roots of ginseng in MA_FO
(p < 0.05).
ere were also signicant dierences in the bacterial (Adonis,
R2 = 0.2943, p = 0.001) and fungal (Adonis, R2 = 0.4483, p = 0.001)
community structures in dierent parts of ginseng. Visual circos
of microorganisms in aboveground and root of ginseng were
ABCD
EF
GH
FIGURE2 | Results of confrontation between Mortierella alpina YW25 and rhizosphere microorganisms. As the control, Mortierella alpina YW25 was inoculated
separately on LB (A), Gao’s No.1 (C) medium for 5 days, and on PDA medium 7 days (E). Mortierella alpina YW25 was co-cultured with Bacillus velezensis (B) on
LB medium, Streptomyces tricolor (D) on Gao’s No.1 medium at 28°C for 5 days, and with Fusarium oxysporum YFW32 (F), Penicillium citrinum (G), Aspergillus
ochraceus (H) on PDA medium at 28°C for 7 days.
Wang et al. M. alpina Regulate Ginseng Root Community
Frontiers in Microbiology | www.frontiersin.org 7 May 2022 | Volume 13 | Article 850917
constructed using bacteria and fungi genera, respectively, with
relative abundance greater than 1% to evaluate the relationship
between microorganisms and samples in dierent parts of
ginseng (Figure 5).
e relationship between bacteria of aboveground ginseng
parts and dierent treatments is shown in Figure 5A.
Sphingomonas, Ralstonia, Amnibacterium, and Polaromonas were
the main bacterial genera in the aboveground ginseng. e
proportions of Sphingomonas and Polaromonas in MA_FO
treatment were 34% and 51%, respectively, and Ralstonia had
the highest distribution in MA treatment (41%). Compared with
the FO treatment, the relative abundance of Sphingomona,
Oxalobacteraceae, Fimbriimonadaceae, and Comamonadaceae in
the MA_FO treatment was signicantly increased (Figure 5B).
Clostridium is the main bacterial genus of FO-treated ginseng
roots and had a relative abundance of 96%. e main bacterial
genera in CK roots were Acidovorax, Flavobacterium, and
Dechioromonas with relative abundances of 28%, 9.9%, and 11%,
respectively (Figure 5A). e bacterial diversity in ginseng roots
between the MA and MA_FO treatments was signicantly higher
than that between the CK and FO treatments (p < 0.05), and
there was no signicant dierence between the MA and MA_FO
treatments. Compared to FO, the relative abundances of
Pseudomonas, Comamonadaceae, Polaromonas, Novosphingobium,
Dokdonella, Apia, Rhizobium, Sphingomonas, Mycobacterium, and
Parablastomonas increased signicantly in ginseng roots aer
MA_FO treatment (Figure 5B).
Knua and Didymella are the main fungal genera in the
aboveground parts of ginseng. e relative abundance of Knua
in the MA treatment was 15%, which was signicantly lower
than that in the other treatments (p < 0.05; Figure 5A). In
MA_FO, the species of potential plant pathogens in the
aboveground parts of ginseng, such as Didymella, Cercospora,
Boeremia, and Alternaria, were less than that of FO, and the
relative abundance of Vishniacozyma was signicantly higher
than that of FO (p < 0.05; Figure 5B). Among the ginseng
roots treated with CK, MA, and MA_FO, Tetracladium, Helotiales,
Cadophora, and Alatospora were the main fungal genera. e
relative abundances of Helotiales (1.3% and 8.8%) and Cadophora
(22% and 20%) in MA and MA_FO were lower than those
in CK, and Tetracladium (41% and 24%) and Alatospora (9.9%
and 17%, respectively) were signicantly higher than those in
CK (Figure 5A). e relative abundance of Cadophora and
Alatospora in the FO treatment was signicantly lower than
that in the MA_FO treatment (p < 0.05; Figure 5B). However,
in terms of the distribution of fungi in dierent treatments,
Aspergillus, Plectosphaerella, Candida, Cladosporium, and
Cladophialophora had the highest distribution proportion in
the FO treatment (89%, 100%, 83%, 91%, and 56%; Figure5A).
e co-occurrence networks of bacterial and fungal
communities signicantly varied in dierent parts of ginseng
and among the dierent treatments (Figure6, Tables 3 and 4).
Except for FO treatment, the bacterial network structure of
ginseng root was generally more complex (based on the number
of edges and nodes, and average degree) than that of the
aboveground parts of ginseng. Among all bacterial networks,
the FO-treated bacterial network of ginseng root was the
simplest (nodes: 13; edges: 32; average degree: 4.932). Compared
with FO treatment, MA_FO had a higher proportion of negative
correlation between aboveground and root bacterial networks
(aboveground: 42.88%; root: 39.6%) and modularity
(aboveground: 0.713; root: 0.646). e number of edges and
nodes, average degree, and modularity of the bacterial networks
in the aboveground and root of ginseng in MA were higher
than those of the control, but the proportion of negative
correlation was lower than that of the control. MA treatment
A
B
FIGURE3 | Growth status of ginseng under different inoculation treatments.
(A) Photos of ginseng under different inoculation treatments. (B) The length
and fresh weight of different parts of ginseng under different inoculation
treatments. CK: control, FO: Fusarium oxysporum YFW32, MA: Mortierella
alpina YW25, MA_FO: Mortierella alpina YW25 and Fusarium oxysporum
YFW32, the same as below. The length and fresh weight of aboveground and
root of ginseng under different treatments were analyzed for difference
signicance separately, and different lowercase letters indicated signicant
difference (p < 0.05).
TABLE1 | Plant defense enzyme activities of ginseng root treated by differential
inoculation.
POD/U PPO/U LOX/U PAL/U
CK 31.55±5.31 b 107.73±20.62 b 2093.3±177.6 ab 30.06±3.11 a
MA 29.97±3.65 b 175.47±7.74 ab 1386.0±373.5 ab 34.40±7.61 a
FO 133.09±15.25 a 213.33±30.08 a 1329.0±174.2 b 31.94±3.57 a
MA_FO 26.79±0.96 b 162.27±43.55 ab 2341.3±676.3 a 37.73±7.75 a
POD, peroxidase; PPO, polyphenol oxidase; LOX, lipoxygenase; PAL,
phenylalanine ammonia lyase. Different lowercase letters indicated significant
difference (p < 0.05).
Wang et al. M. alpina Regulate Ginseng Root Community
Frontiers in Microbiology | www.frontiersin.org 8 May 2022 | Volume 13 | Article 850917
had a more complex bacterial network (based on the number
of edges and nodes, and average degree) than MA_FO, but
MA_FO might have a more stable bacterial network (based
on the negative correlation ratio).
e fungal network of ginseng is simpler since it has fewer
nodes and edges compared to the bacterial network (Figure6;
Tabl e 4). In contrast to the bacterial networks, the fungal
network structure of the aboveground parts of ginseng is
generally more complex than that of the root (based on the
number of edges and nodes, and average degree); FO being
the exception. e number of edges and nodes, average degree,
and modularity of the fungal network in the aboveground
parts of ginseng treated with MA_FO were higher than those
in FO, while the opposite trend was observed in the root
network of ginseng. However, the proportion of negative
correlation was lower than that of FO. e fungal network of
ginseng roots had a very high positive correlation ratio under
the treatment of FO (99.08%). e number of edges and nodes,
and average degree of the fungal networks in the aboveground
ginseng treated with MA were higher than those of the control,
but the proportion of negative correlation and modularity were
lower than those of the control. e fungal networks of ginseng
roots showed the opposite trend.
e bacterial community structure of ginseng rhizosphere
soil under FO treatment was signicantly dierent from that
under the other treatments (Figure7A). Pseudarthrobacter was
the bacterium with the highest relative abundance in the
rhizosphere soil of ginseng (5.5%–9.6%). e relative abundances
of Flavobacterium, Marmoricola, Gaiella, and Ellin6070 in FO
were signicantly higher than those in the other treatments
(p < 0.05; Figure 8). ere were no signicant dierences in
soil fungal diversity and community structure between CK
and MA_FO, but the diversity of FO and MA soils decreased
signicantly, and the fungal communities of FO and MA soil
were signicantly separated in CAP1 (36.02%). is suggests
that FO and MA soils had dierent community structures
TABLE2 | Physicochemical and enzyme activity characteristics of ginseng rhizosphere soil under different inoculation treatments.
CK MA FO MA_FO
pH 6.56±0.31 a 6.31±0.02 ab 6.06±0.04 b 6.37±0.02 ab
NO3−-N/(mg/kg) 22.48±0.06 a 17.58±0.25 b 11.69±1.79 c 4.48±0.43 d
NH4+-N/(mg/kg) 0.38±0.09 c 14.22±1.51 a 2.38±0.48 c 9.27±1.58 b
AP/(mg/kg) 1.45±0.11 b 1.99±0.02 a 1.37±0.04 b 1.08±0.04 c
TN/(mg/g) 8.76±0.15 ab 8.45±0.12 b 8.83±0.17 a 8.51±0.28 ab
TP/(mg/g) 0.88±0.01 a 0.84±0.03 a 0.87±0.04 a 0.87±0.02 a
TK/(mg/g) 20.95±0.18 a 19.95±0.27 b 19.4±0.16 c 19.68±0.08 bc
AK/(mg/kg) 311.00±1.87 c 346.75±4.49 b 363.00±2.12 a 351.00±4.53 b
SOM(g/g) 0.22±0.01 a 0.12±0.07 a 0.14±0.07 a 0.13±0.01 a
Urease/(μg/g) 11.50±0.72 b 14.55±1.35 a 13.61±1.26 ab 13.94±0.42 a
CAT/(μmol/h/g) 293.06±1.09 a 207.27±2.29 c 285.34±1.71 b 289.82±0.60 a
SC/(mg/d/g) 41.11±2.75 b 46.54±0.65 a 35.48±0.57 c 45.76±2.83 ab
ACP/(μmol/h/g) 1.43±0.01 bc 2.08±0.19 a 1.59±0.13 b 1.31±0.03 c
NO3−-N, nitrate nitrogen; NH4+-N, ammonium nitrogen; AP, available phosphorus; TN, total nitrogen; TP, total phosphorus; TK, total potassium; AK, available potassium; SOM,
organic matter; Urease, urease activity; CAT, catalase activity; SC, sucrase activity; ACP, acid phosphatase activity; the same as below. Different lowercase letters indicated
signicant difference (p < 0.05).
AB
FIGURE4 | Shannon diversity index of bacterial (A) and fungi (B) community structure at different ecological niches (Aboveground: aboveground of ginseng; Root:
root of ginseng; Soil: rhizosphere soil). Different lowercase letters indicate signicant difference (p < 0.05).
Wang et al. M. alpina Regulate Ginseng Root Community
Frontiers in Microbiology | www.frontiersin.org 9 May 2022 | Volume 13 | Article 850917
AE
BF
CG
DH
FIGURE5 | Analysis of microbial composition and differences in ginseng. Effects of different inoculation treatments on internal bacteria ((A) aboveground of
ginseng; (B) root of ginseng) and fungi ((C) aboveground of ginseng; (D) root of ginseng) of ginseng. The left semicircle represents the different inoculation
treatments. The right semicircle represents the dominant genera and proportions of each genus in different samples. Student’s t-test was used to test the
signicance of differences between FO and MA_FO at the genus level ((E) aboveground bacteria of ginseng; (F) root bacteria of ginseng; (G) aboveground fungi of
ginseng; (H) root fungi of ginseng). The y-axis represents the species names at the genus level, the x-axis represents the average relative abundance in different
groups of species, and the columns with different colors represents different groups. The far right is the value of p, *p < 0.05; **p < 0.01; ***p < 0.001.
Wang et al. M. alpina Regulate Ginseng Root Community
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FIGURE6 | Analysis of microbial co-occurrence network in ginseng under different inoculation treatments. The nodes are colored according to bacterial and fungal
phylum. Node size indicates the relative abundance of OTU. Edge color represents positive (blue) and negative (orange) correlations.
TABLE3 | Key topological features of bacterial networks in aboveground and root of ginseng under different inoculation treatments.
Aboveground Root
CK MA FO MA_FO CK MA FO MA_FO
Nodes 313 395 264 265 316 472 13 479
Edges 9,096 14,360 6,999 6,402 15,415 20,297 32 25,101
Positive edges ratio (%) 57.26 67.14 58.34 57.12 88.23 61.37 62.5 60.4
Negative edges ratio (%) 42.74 32.86 41.66 42.88 11.77 38.63 37.5 39.6
Average degree 58.121 72.709 53.023 48.317 97.563 86.004 4.923 104.806
Modularity 0.709 0.716 0.693 0.713 0.531 0.751 0.225 0.646
Wang et al. M. alpina Regulate Ginseng Root Community
Frontiers in Microbiology | www.frontiersin.org 11 May 2022 | Volume 13 | Article 850917
(Figure 7B). Fusarium and Mortierella could colonize soil and
become the dominant genera in FO and MA, with relative
abundances of 41.3% and 57%, respectively. e relative
abundance of Fusarium in MA_FO was signicantly lower
than that in FO, but the abundance of Pseudeurotium and
Schizothecium was signicantly higher (Figure 8).
Correlation Between Soil Microorganisms
and Physicochemical and Enzymatic
Factors in Ginseng Rhizosphere
db-RDA and linear regression analyses were used to analyze
the eects of soil physicochemical factors and enzyme activities
on the soil microbial community structure. Using the variance
ination factor (VIF) to judge the collinearity between dierent
soil physicochemical factors, the physicochemical factors with
VIF > 10in soil physical and chemical indicators were screened
and removed. NO3−-N (VIF = 40.01) and AK (VIF = 72.16)
were removed because they strongly correlated with other
physicochemical factors.
e selected physicochemical factors were compared with
soil bacteria (Figure 7A) and fungi (Figure 7B) for db-RDA
analysis based on the Bray–Curtis distance. e results showed
that pH (r2 = 0.5745, p = 0.011), NH4+-N (r2 = 0.9168, p = 0.001),
TN (r2 = 0.5517, p = 0.032), and TK (r2 = 0.631, p = 0.023) were
signicantly correlated with bacterial community structure.
Further, pH (r2 = 0.5775, p = 0.002), AP (r2 = 0.738, p = 0.007),
and TK (r2 = 0.6945, p = 0.011) were signicantly correlated with
fungal community structure.
Linear regression analysis was used to evaluate the degree
of explanation of the activity of sucrase, urease, ACP, and
catalase to the variation in soil bacterial and fungal community
structure (Supplementary Figure S1). Sucrase activity was
signicantly correlated with the community structure of soil
bacteria (R2 = 0.5216, p = 0.008) and fungi (R2 = 0.4544, p = 0.0162).
ere was no signicant correlation between urease, ACP, and
catalase, and soil bacterial and fungal community structure.
DISCUSSION
Growth-Promoting Characteristics of
Mortierella alpina YW25 and Its in vitro
Interaction With Rhizospheric
Microorganisms
Plant-associated microbes are known to play important roles
in plant health and disease (Kwak etal., 2018). Roots absorb
TABLE4 | Key topological features of fungal networks in aboveground and root of ginseng under different inoculation treatments.
Aboveground Root
CK MA FO MA_FO CK MA FO MA_FO
Nodes 175 174 143 243 120 110 130 86
Edges 2,917 2,984 2,228 5,428 1,442 1,028 3,056 628
Positive edges ratio (%) 64.38 65.45 58.44 68.24 86.62 86.19 99.08 63.06
Negative edges ratio (%) 35.62 34.55 41.56 31.76 13.38 13.81 0.92 36.94
Average degree 33.337 34.299 31.161 43.193 22.185 18.691 47.015 14.605
Modularity 0.742 0.687 0.681 0.794 0.783 0.79 0.342 0.772
AB
FIGURE7 | db-RDA analysis of soil microbial community and environmental factors. (A) bacterial community; (B) fungal community. Arrows represent
environmental factors.
Wang et al. M. alpina Regulate Ginseng Root Community
Frontiers in Microbiology | www.frontiersin.org 12 May 2022 | Volume 13 | Article 850917
water and inorganic nutrients from the soil and secrete organic
exudates to shape the microbial diversity and structure of
the soil (Bulgarelli et al., 2013). Exploring the interaction
between plant and soil microbes and rhizospheric
microorganisms is vital to prevent and suppress diseases,
promote plant growth, or improve plant stress resistance
(Mendes et al., 2013).
In this study, ginseng rhizosphere microorganisms were
selected for the plate confrontation test to preliminarily
study the interaction between M. alpina YW25 and the
rhizosphere microorganisms. The results showed that the
mycelial growth of M. alpina YW25 was inhibited by some
probiotics in the rhizosphere, such as Bacillus, Streptomyces,
Brevibacterium, Trichoderma, and Penicillium (Lee etal., 2021;
Zhao etal., 2021a,b), as well as by some potential pathogens,
such as Cladosporium and Aspergillus (Carolina Virginia
et al., 2021; Tan et al., 2021). Antimicrobial substances
(lipopeptides, antibiotics, and volatile organic compounds)
secreted by Bacillus and metabolites of Streptomyces (e.g.
quercetin) may inhibit the growth of M. alpina YW25 mycelia
on plates (Awla and Rashid, 2020; Chen etal., 2020). However,
it showed no sensitivity to other microorganisms, such as
Fusarium, Bjerkandera adusta, Trametes, Trichaptum abietinum
(Supplementary Table S1), and the soil-borne pathogen
F. oxysporum during co-cultivation. The results of the pot
experiment showed that Mortierella was negatively correlated
with Fusarium and Trichoderma in ginseng rhizosphere soil
(Spearman, −0.95 and −0.72). These negative correlations
AB
FIGURE8 | Analysis of microbial composition and differences in ginseng rhizosphere soil. (A) Relative abundance at the genus level of bacteria and fungi, where
“others” represents species with relative abundance less than 1% in all samples; (B) One-way univariate analysis of variance (ANOVA) was used to test the
signicance of differences between groups at the genus level of bacteria and fungi. The y-axis represents the species names at the genus level, the x-axis represents
the average relative abundance in different groups of species, and the columns with different colors represents different groups. Far right is the value of p, *p < 0.05;
**p < 0.01; ***p < 0.001.
Wang et al. M. alpina Regulate Ginseng Root Community
Frontiers in Microbiology | www.frontiersin.org 13 May 2022 | Volume 13 | Article 850917
between Mortierella and Fusarium have also been observed
in other systems (Hong et al., 2020; Xiang et al., 2021),
which indicated that Mortierella did not directly inhibit the
growth of Fusarium.
Effects of Inoculation With Native
Mortierella alpina YW25 on
Physicochemical Properties of Ginseng
Plants and Rhizosphere Soil
In this study, when M. alpina YW25 was singularly inoculated,
the leaves of the ginseng plants expanded, brous roots
developed, and it did not show any disease symptoms. ere
was no signicant dierence between the aboveground and
root lengths of ginseng compared with CK (Figure 3B). is
indicates that inoculation of M. alpina YW25 in ginseng
rhizosphere did not result in ginseng root disease. Weevaluated
the growth-promoting characteristics of M. alpina YW25 and
found that it had a high IAA production capacity, with a
maximum value of 141.37 mg/L, which was much higher than
the IAA yield of reported strains (Bader etal., 2020; Galeano
et al., 2021). However, no obvious growth-promoting eect
was observed in the ginseng plants. A study involving M. capitata
inoculation showed that it could increase maize biomass and
promote plant growth (Li et al., 2020a). is dierence may
be attributed to the dierent microbial or plant species in
this study.
Interactions with microbial species and network modularity
aect the community stability (Coyte etal., 2015). Compared
with CK, MA signicantly increased the diversity of root
bacteria and signicantly decreased the diversity of root fungi,
but there was no signicant dierence in the microbial diversity
of the aboveground parts of ginseng (Figure 4). In the
co-occurrence network (Figure 6), MA was more complex
than CK. Compared with CK, ginseng roots in MA had more
edges and nodes in the bacterial network and fewer edges
and nodes in the fungal network. Moreover, in the MA treatment,
the ginseng root microbial network had a higher negative
correlation ratio and modularity. e results showed that
inoculation of M. alpina YW25 could increase the complexity
of the bacterial community structure in ginseng root, while
reducing the complexity and improving the stability of the
ginseng root fungal community.
The results of this study showed that a single inoculation
of M. alpina YW25 had significant effects on some nutrient
content in ginseng rhizosphere soil. Phosphorus can enhance
drought and disease resistance in plants and promote their
growth and development. A lack of phosphorus leads to a
significant decrease in crop yield (Elhaissoufi et al., 2021).
The results of this study showed that the AP content and
ACP activity of ginseng rhizosphere soil treated with MA
were significantly higher than those in other treatments.
Hence, inoculation with Mortierella increased the AP content
in soil (Spearman, 0.70). This was the same as observed
in previous studies (Li et al., 2020a; Guo et al., 2021), and
indicated that Mortierella could dissolve inorganic phosphorus
in soil. In addition, oxalates are also synthesized and released
to help plants or mycorrhizal fungi obtain phosphorus (Qiang
et al., 2021). However, M. alpina YW25 did not show
phosphorus solubility in the PVK plate. The difference
between plate cultivation and pot experimentation might
be because dissolving phosphorus in the pot experiment
was realized by regulating rhizosphere microorganisms.
Compared with CK, Actinobacteria (Pseudarthrobacter,
Microbacterium, and Microlunatus) and Rhizobium were
significantly enriched in MA (Supplementary Figure S2).
These microorganisms have their own biophosphorus
conversion activity (Pindi, 2012). In addition, the content
of soil available nitrogen (NH4+-N and NO3−-N) after M. alpina
YW25 inoculation was significantly higher than that in
FO. The AP content in the rhizosphere soil was positively
correlated with the available nitrogen content (NH4+-N and
NO3−-N; Spearman, 0.26 and 0.62), but negatively correlated
with the available potassium content (Spearman, −0.36). The
results showed that nitrogen and phosphorus availabilities
were driven by each other between plants and soil (Xu
etal., 2020). Phosphorus in soil also increases the retention
of nitrogen in soil–plant systems, thereby reducing nitrogen
loss due to soil leaching (Mehnaz et al., 2019).
Mortierella alpina YW25 Could Aid in Plant
Resistance Against Pathogenic Invasion
After MA_FO Treatment
In FO treatment, ginseng plant showed the typical
characteristics of root rot disease with wilted leaves, brown
and rotten root (Punja etal., 2008). However, in the MA_FO
treatment, the leaves of ginseng expanded and the roots
did not show browning symptoms. This indicates that the
treatment with MA_FO in ginseng rhizosphere could
effectively resist root rot caused by F. oxysporum YFW32.
Plant defense enzymes (POD, PPO, and PAL) in ginseng
roots were detected while exploring the reason for disease
resistance (Tab l e 1), but these results are different from
those of previous studies (Nandhini et al., 2018). Pattern
recognition receptors located on the surface of plants can
recognize microbe- or pathogen-associated molecular patterns,
and this recognition can then stimulate cascade defense
signals resulting in induced systemic resistance (ISR) in
plants (Bukhat et al., 2020). ISR is associated with defense
enzymes, such as POD, PPO, and PAL. When plants are
under biotic stress, these enzymes are induced to help resist
pathogens (Appu et al., 2021). Invasion of F. oxysporum
YFW32 significantly increased POD and PPO activities in
ginseng roots. However, the activities of these enzymes were
not significantly increased in MA_FO, which suggests that
M. alpina YW25 may not induce plant resistance of ginseng
to resist pathogen invasion.
Furthermore, we investigated the plant-associated
microbiomes. The structure of the plant microbiome is
influenced by complex interactions between the host,
microorganisms, and related environmental factors, such as
climate, soil, and cultivation practices (Kmgd et al., 2020).
Treatment with MA_FO had a significant effect on the
Wang et al. M. alpina Regulate Ginseng Root Community
Frontiers in Microbiology | www.frontiersin.org 14 May 2022 | Volume 13 | Article 850917
ginseng microbiome (Figure5). Compared with FO, treatment
with MA_FO significantly increased bacterial diversity and
decreased fungal diversity in ginseng roots. The relative
abundances of Pseudomonas, Comamonadaceae (e.g.,
Polaromonas), Sphingomonadaceae (Sphingomonas and
Novosphingobium), and Rhizobium in MA_FO were
significantly higher than those in FO (Figure 5B). This
result is similar to that of previous research on American
ginseng with Trichoderma atroviride inoculation (Li et al.,
2022). These root microbiotas showed high antagonistic
ability against root-associated fungi (Duran et al., 2018).
In addition, the relative abundance of some potential plant
growth-promoting microorganisms, such as Vishniacozyma,
Cadophora, and Alatospora, was higher in the MA_FO
treatment than in FO (Bizabani and Dames, 2015; Artigas
et al., 2017; Lutz et al., 2020). Therefore, we speculated
that M. alpina YW25 may enrich plant growth-promoting
microorganisms by stimulating ginseng plants, and absorbing
more nutrients for plant growth while inhibiting the invasion
and proliferation of potential pathogens.
The plant microbial community structure of ginseng
treated with MA_FO was different from that treated with
FO, and the effect of MA_FO treatment on root microbial
community was greater than that of aboveground ginseng
(Figure 6). In the ginseng root, the number of nodes and
edges of the bacterial network in FO was much lower than
that in the control, and the number of nodes and edges
in the fungal network was higher than that in CK. This
indicated that FO reduced the complexity of the bacterial
network, but increased the complexity of the fungal network
in the ginseng root. The same results were observed in
co-occurrence networks of peppers infected with Fusarium
wilt disease (Gao et al., 2021). The complexity of microbial
networks may berelated to alpha diversity (Fan etal., 2018).
The modularity and negative correlation of the bacterial
and fungal networks of ginseng roots treated with FO were
also much lower than those of the control. Low modularity
and negative correlation may increase the unstable effect
of the community (Grilli et al., 2016; Hernandez et al.,
2021). Compared with FO, MA_FO increased the complexity
of the root bacterial network, reduced the complexity of
the root fungal network (based on the number of edges,
nodes, and average degree), and improved the stability of
the root microbial community (based on modularity and
negative correlation ratio).
In this study, wefound that ginseng rhizosphere-inoculated
fungi had an effect on soil properties and the rhizosphere
soil microbial community (Table 2 and Figure 8). First,
pH was significantly correlated with the changes in
rhizosphere soil bacterial and fungal communities (R2 = 0.5745
and 0.5775, respectively; Figure 7), which was the main
factor affecting soil microbial diversity and community
structure (Kang etal., 2021). Second, soil NH4+-N content
had the strongest correlation with soil bacterial community
structure (R2 = 0.9168; Figure 7A), and the soil physical
and chemical factors had the greatest influence on the
composition of the rhizosphere bacterial community
(Liu et al., 2020a). Compared with the FO treatment, the
activities of NH4+-N, sucrase, and catalase in soil increased
significantly in the MA_FO treatment. This suggested that
when treated in MA_FO, MA may help improve soil fertility,
provide more nutrients for plants and soil microorganisms,
and bioremediate the soil (Stepniewska etal., 2009; Sellami
et al., 2022).
e fungal diversity of the rhizosphere soil in MA_FO was
signicantly higher than that in the FO treatment, but there
was no signicant change in bacterial diversity (Figure 4).
Mortierella inoculation with Fusarium signicantly reduced the
relative abundance of Fusarium in soil (Figure 8B), and there
was no signicant dierence in the soil fungal community
structure between the two treatments (Figure 7B). Previous
studies have also shown that the abundance of Mortierella in
soil was signicantly negatively correlated with diseases in
plants, such as apple (Wang etal., 2018), vanilla (Xiong et al.,
2017), eggplant (Ogundeji etal., 2021), celery, and watermelon
(Liu et al., 2020a). In conclusion, inoculation with M. alpina
YW25 signicantly inhibited the proliferation of F. oxysporum
in ginseng rhizosphere soil but did not aect the health of
the rhizosphere soil.
To conclude, M. alpina YW25 had the maximum yield
of IAA at 4 days (141.37 mg/L). Inoculation of M. alpina
in ginseng rhizosphere significantly alleviated the pathogenicity
of F. oxysporum in ginseng plants, increased the content of
available nitrogen and phosphorus in rhizosphere soil, and
improved the activities of soil sucrase and ACP. M. alpina
inoculation with F. oxysporum had the greatest effect on
the microbial community in the ginseng roots and had a
greater effect on the fungal community than on the bacterial
community. M. alpina inoculation helped ginseng recruit
more plant growth-promoting microorganisms, change the
microbial structure of ginseng roots, and build a more stable
microbial network of ginseng roots. Thus, it inhibited potential
pathogens, effectively prevented the invasion of pathogens,
and ensured healthy plant growth. Therefore, M. alpina
helped Panax ginseng resist F. oxysporum infection by mainly
regulating the fungal community in the root.
DATA AVAILABILITY STATEMENT
e datasets presented in this study can be found in online
repositories. e names of the repository/repositories and
accession number(s) can be found at: https://www.ncbi.nlm.
nih.gov/, Biosample No. SAMN24474502.
AUTHOR CONTRIBUTIONS
YW and HY conceived and designed the experiment. YW
and LW performed the experiment. YW, LW, and MS analyzed
the data. YW wrote the paper. HW, MZ, and HY guided the
research work and thoroughly reviewed and corrected English
language of the manuscript. All authors contributed to the
article and approved the submitted version.
Wang et al. M. alpina Regulate Ginseng Root Community
Frontiers in Microbiology | www.frontiersin.org 15 May 2022 | Volume 13 | Article 850917
FUNDING
is study was supported by Fundamental Research Funds for
the Central Universities (No. 2572020DR08 and No. 2572020DP07).
ACKNOWLEDGMENTS
e authors would like to thank Qiang Ye (Yanbian Korean
Autonomous Prefecture Academy of Agricultural Sciences) for
providing the ginseng seedings. e authors would also like to
thank Hongyan Zhao (College of Agronomy, Yanbian University,
Yanji, China) for the detection of physicochemical indexes.
SUPPLEMENTARY MATERIAL
e Supplementary Material for this article can befound online
at: https://www.frontiersin.org/articles/10.3389/fmicb.2022.850917/
full#supplementary-material
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