Abstract
Fusarium oxysporum is a widely distributed soil-borne pathogenic fungus
that can cause medicinal herbs and crops to wither or die, resulting in
great losses and threat to public health. Due to the emergence of
drug-resistance and the decline of the efficacy of antifungal
pesticides, there is an urgent need for safe, environmentally friendly,
and effective fungicides to control this fungus. Plant-derived natural
products are such potential pesticides. Extracts from seeds of Peganum
harmala have shown antifungal effects on F. oxysporum but their
antifungal mechanism is unclear. In vitro antifungal experiments showed
that the total alkaloids extract and all five β-carboline alkaloids
(βCs), harmine, harmaline, harmane, harmalol, and harmol, from P.
harmala seeds inhibited the growth of F. oxysporum. Among these βCs,
harmane had the best antifungal activity with IC[50] of 0.050 mg/mL and
MIC of 40 μg/mL. Scanning electron microscopy (SEM) and transmission
electron microscopy (TEM) results revealed that the mycelia and spores
of F. oxysporum were morphologically deformed and the integrity of cell
membranes was disrupted after exposure to harmane. In addition,
fluorescence microscopy results suggested that harmane induced the
accumulation of ROS and increased the cell death rate. Transcriptomic
analysis showed that the most differentially expressed genes (DEGs) of
F. oxysporum treated with harmane were enriched in catalytic activity,
integral component of membrane, intrinsic component of membrane, and
peroxisome, indicating that harmane inhibits F. oxysporum growth
possibly through damaging cell membrane and ROS accumulation via
regulating steroid biosynthesis and the peroxisome pathway. The
findings provide useful insights into the molecular mechanisms of βCs
of P. harmala seeds against F. oxysporum and a reference for
understanding the application of βCs against F. oxysporum in medicinal
herbs and crops.
Keywords: Fusarium oxysporum, antifungal, Peganum harmala, β-carboline
alkaloids, harmane, transcriptome
1. Introduction
High-quality medicinal herbs are the material basis for the inheritance
and development of traditional Chinese medicine and are strategic
resources related to the national economy and people’s livelihood. In
recent years, with the growing demand for high-quality medicinal herbs
at home and abroad, the species and area of artificially cultivated
medicinal herbs have increased significantly [[28]1]. However, problems
such as root rot and fusarium wilt are becoming more and more serious
in the process of planting medicinal herbs, such as Panax ginseng
[[29]2], Codonopsis radix [[30]3], Panax quinquefolius [[31]4], and
Crocus sativus [[32]5], resulting in the decline of yield and quality
of medicinal herbs [[33]6]. Fusarium oxysporum, a widely distributed
soil-borne pathogenic fungus with strong destructiveness, is the main
pathogen causing root rot or fusarium wilt of the medicinal plants
[[34]7]. It can infect more than 150 crops, such as banana, tomato,
soybean, and wheat before harvest [[35]8,[36]9,[37]10], and it was
listed as one of the top ten plant pathogenic fungi in the world in
2012 [[38]11]. A recent prediction showed that the banana wilt caused
by F. oxysporum worldwide would cause economic losses of more than 10
billion dollars by 2040 [[39]12].
In addition, F. oxysporum can produce some secondary metabolites in the
process of infection of crops, such as fusaric acid, fumonisins, and
beauvericin [[40]13,[41]14]. These toxins may cause nausea, diarrhea,
dizziness, fever and food-poisoning leukopenia, which pose a potential
threat to livestock [[42]15] and human health [[43]16].
At present, chemical antimicrobial agents, such as azoxystrobin and
thiophanate methyl, are often used to prevent and treat plant diseases
caused by agriculture fungal pollution [[44]17,[45]18]. Azoxystrobin is
a broad-spectrum fungicide with good activity against almost all fungal
diseases and it is the best-selling fungicide in the world. However,
long-term heavy use of such chemicals would cause drug resistance of
pathogenic fungi, pollutes the environment, and has potential food
safety risks, threatening human health, which does not meet the needs
of the sustainable development of modern agriculture [[46]19].
Therefore, it is urgent to develop green, safe and effective natural
antimicrobial agents to control soil-borne diseases caused by F.
oxysporum.
Many natural plant active compounds have attracted much attention due
to their excellent antimicrobial activities, such as chlorogenic acid
[[47]20], allicin [[48]21], eugenol [[49]22], and curcumin [[50]23].
Peganum harmala, a perennial herb from the Zygophyllaceae family, is
widely distributed in arid grasslands in desert areas, lightly
salinized sandy land on the edge of oasis, loamy low hillsides or river
valley dunes of Central Asia, Europe, and southern South America. It is
commonly used in folk medicine to treat fever, cough, diarrhea,
hypertension, asthma, jaundice, and skin diseases [[51]24]. It is rich
in β-carboline alkaloids (βCs), the content of which in seeds reaches
up to 10%, including harmine, harmaline, harmalol, harmol, and harmane
([52]Figure 1) [[53]25,[54]26,[55]27]. Studies have shown that extracts
from seeds of P. harmala have broad spectrum activities against fungi,
such as F. oxysporum, Aspergillus niger, Cryptococcus neoformans,
Alternaria sp., and Epidermophyton floccosum [[56]27,[57]28]. However,
studies on the antifungal activity of βCs from P. harmala against F.
oxysporum are limited and the antifungal mechanism has not been
elucidated.
Figure 1.
[58]Figure 1
[59]Open in a new tab
Structures of the five main βCs in P. harmala seeds.
In this paper, the potential antifungal effect of βCs, especially
harmane on F. oxysporum was investigated. Scanning electron microscopy
(SEM), transmission electron microscopy (TEM), and transcriptome
analysis were conducted to explore the inhibition mechanism, which
showed that harmane inhibits the mycelial growth of F. oxysporum
possibly through regulating the expression of genes related to steroid
biosynthesis and peroxisome metabolism. This study provides a reference
for understanding the application of βCs in medicinal herbs and crops.
2. Materials and Methods
2.1. Isolation and Identification of F. oxysporum
F. oxysporum was isolated according to the previously reported methods
[[60]29]. Briefly, fungal pathogens were isolated from root of
Codonopsis radix with root rot collected in Gansu province of China,
and grown on potato dextrose agar (PDA). After 5 days of culturing, the
colony was convex flocculent, pinkish white, slightly purple. The
mycelium was white and dense. It was identified as F. oxysporum by
morphological characteristics and 16S rRNA sequence analysis (Genbank
[61]MK966308).
Spore suspension was prepared according to the literature with slight
modifications [[62]30]. In short, the spore suspension was collected by
flooding the surface of the 7-day-old culture plates with sterile water
and filtering with sterile degreasing cotton. Then, the F. oxysporum
spore suspension was diluted to a concentration of approximately 1.0 ×
10^6 CFU/mL, using a hemocytometer.
2.2. Chemicals
Harmine (CAS NO. 442-51-3, purity 98%), harmaline (CAS NO. 304-21-2,
purity 98%), harmalol (CAS NO. 525-57-5, purity 98%), harmol (CAS NO.
487-03-6, purity 98%), and harmane (CAS NO. 486-84-0, purity 98%), and
total alkaloid extracts were isolated from P. harmala seeds by our
laboratory [[63]31]. The content of harmine and harmaline in total
alkaloid extracts was 55.3%. The structures of βCs included in the
project are shown in [64]Figure 1. Azoxystrobin (CAS NO. 131860-33-8,
purity 98%) was purchased from Beijing Norma Standard Technology Co.,
Ltd (Beijing, China).
2.3. Inhibition of Total Alkaloids on Mycelial Growth
The inhibition effect of total alkaloid extracts from P. harmala seeds
against F. oxysporum were tested by agar diffusion method [[65]32].
Alkaloid extracts from P. harmala were mixed with PDA, and the final
concentrations were 0.05, 0.1, 0.2, 0.4, and 0.5 mg/mL. Azoxystrobin at
dose of 0.4 mg/mL was used as a positive control. F. oxysporum was
inoculated on the PDA and cultured at 28 °C for 5 days. PDA without
alkaloid was used as a control. The mycelial growth diameter of F.
oxysporum colony was measured and the inhibition rate was calculated
according to the following Formula (1).
[MATH:
Inhibition rate (%)=the
diameter of control − the diameter of<
mtext> treatmentthe
diameter of control×100%<
/mo> :MATH]
(1)
2.4. Inhibition of Five βCs on Mycelial Growth and IC[50] CALCULATION
The inhibition effect of the five βCs on F. oxysporum was tested in the
same way as total βCs. The IC[50] was analyzed using SPSS (version
25.0, Norman H. Nie, C. Hadlai (Tex) Hull and Dale H. Bent, CA, USA).
2.5. Determination of Minimal Inhibitory Concentration (MIC)
According to the American Society for Clinical and Laboratory Standards
(CLSI) standard, the MIC of βCs against F. oxysporum was determined by
tube double dilution method in a 96-well plate [[66]33]. βCs were
separately mixed with Potato Dextrose Broth (PDB) in the concentration
range of 0.625–50 μg/mL, and 4 mL of the mixed solution was added into
20 μL of the conidial suspension. Then, each concentration of the mixed
solution was successively distributed to three wells of the 96-well
plate. PDB without βCs was used as the control group. The MIC was
defined as the lowest drug concentrations that caused complete visible
inhibition of growth.
2.6. Scanning Electron Microscopy (SEM)
The morphology of F. oxysporum after harmane treatment was observed
with SEM according to the literature [[67]34]. The spore suspension was
added into PDB and cultured at 28 °C (120 rpm) for 48 h. After
centrifugation at 4000× g for 5 min, the mycelium was suspended again
in PBS (pH 7.2). The βCs were added to the buffer solution to make the
concentration MIC and incubated at 28 °C for 12 h, with anhydrous
ethanol as the control group [[68]35].
The samples were fixed in 2.5% glutaraldehyde, washed with PBS three
times, 15 min each time, fixed with 1% osmic acid solution for 1 h,
washed three times, 15 min each time. The samples were dehydrated with
ethanol solution of five concentration gradients (including 30%, 50%,
70%, 80%, 90% and 95%). Each concentration was treated for 15 min, and
then 100% ethanol was used twice, 20 min each time. The sample was
treated with the mixture of ethanol and isoamyl acetate for 30 min, and
then treated with pure isoamyl acetate for 1 h, dried, coated and
examined by SEM (×10.0K and ×20.0K, U8010, Hitachi, Tokyo, Japan).
2.7. Transmission Electron Microscopy (TEM)
For TEM, mycelia were treated the same way as SEM and slightly
modified. In short, the treated samples were fixed in 2.5%
glutaraldehyde and washed three times with PBS for 15 min each time.
The samples were dehydrated with ethanol solution of five concentration
gradients (including 30%, 50%, 70%, 80%, 90% and 95%). Each
concentration of the sample was treated for 15 min and then treated
twice with 100% ethanol for 20 min each time. The samples were embedded
for 3 h and sliced in an ultra-thin cutting machine (UC7, Leica,
Wetzlar, Germany). The samples were stained with lead citrate solution
and 50% ethanol saturated solution of uranium dioxide acetate for 5
min, respectively, and then examined by TEM (H-7650, Hitachi).
2.8. Evaluation of Release of Cell Components
The release of cell components was evaluated using OD[260] determined
with UV spectrophotometry [[69]35]. To do that, the 1 × 10^6 CFU/mL
suspension was mixed with PDB and cultured at 28 °C (120 rpm) for 48 h.
After centrifugation at 4000× g for 15 min, the mycelia were collected
and washed with sterile water three times. Then, the mycelia were
suspended in phosphate buffer solution (PBS, pH 7.2), supplied with
harmane at the final concentration of 0.5 MIC and MIC, then incubated
at 28 °C for 4 h, 8 h, and 12 h, respectively. Samples were centrifuged
at 4000× g for 5 min to collect supernatant for OD[260] measurement.
PBS (pH 7.2) was used as the control.
2.9. Measurement of Electrical Conductivity
The influence of harmane on electrical conductivity of F. oxysporum was
measured according to the literature [[70]32]. The sample was treated
in the same way as for cell component assay. The conductivity of the
supernatant of different samples was determined using conductivity
meter (DDS-11D, JingKe, Shanghai, China).
2.10. ROS Assay
The content of ROS in cells was evaluated by Reactive Oxygen Species
assay kit (Beyotime, Shanghai) combined with fluorescence microscopy.
The method of culture and treatment of samples was described in SEM.
The DFCH-DA probe was added into the treated samples and incubated at
37 °C for 30 min. After centrifugation, the supernatant was washed
twice with PBS, and the precipitation was collected and observed under
bright light and green light by fluorescence microscopy (×10, Olympus
IX81, Tokyo, Japan).
2.11. Annexin V-FITC/PI Double Staining Assay
The cell death rate was analyzed using Annexin V-FITC Apoptosis
detection kit (Beyotime, Shanghai, China) combined with fluorescence
microscopy, which could also discriminate types of cell death
(apoptotic or necrotic cell death) [[71]30]. The method of culture and
treatment of samples was described in SEM. Briefly, a total of 500 μL
of the treated sample was mixed with 5 μL of Annexin V-FITC and then 5
μL of propidium iodide (PI) was added, incubated at 25 °C for 10 min,
and imaged under fluorescence microscopy (Olympus IX81).
2.12. Transcriptomic Analysis
The total RNA of the treated samples was extracted with TRIzol® Reagent
(Invitrogen, Carlsbad, CA, USA), according the manufacturer’s
instructions, and genomic DNA was removed using DNase I (TaKara, Kyoto,
Japan). Its concentration, purity and integrity were detected by
Nanodrop2000 (NanoDrop Technologies, Waltham, MA, USA). The
transcriptome library was prepared following Truseq^TM RNA sample
preparation kit from Illumina (San Diego, CA, USA) using 1 μg of total
RNA. Then, the synthesized cDNA was subjected to end-repair,
phosphorylation and ‘A’ base addition according to Illumina’s library
construction protocol. Libraries were size selected for cDNA target
fragments of 300 bp on 2% Low Range Ultra Agarose followed by PCR
amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After
quantified by TBS380, paired-end RNA-seq sequencing library was
sequenced with the Illumina NovaSeq 6000 sequencer (2 × 150 bp read
length). The original sequencing data was subjected to quality control
using SeqPrep ([72]https://github.com/jstjohn/SeqPrep, accessed on 15
November 2021) and Sickle ([73]https://github.com/najoshi/sickle,
accessed on 15 November 2021) software to obtain clean data. These
clean data were compared with the reference genome (Fusarium_oxysporum,
[74]http://fungi.ensembl.org/Fusarium_oxysporum/Info/Index, accessed on
15 November 2021) using HiSat2
([75]http://ccb.jhu.edu/software/hisat2/index.shtml, accessed on 15
November 2021) to obtain mapped data for subsequent transcript
assembly, expression amount calculation, and others. The RSEM
([76]http://deweylab.biostat.wisc.edu/rsem/, accessed on 22 November
2021) software was used to perform progressive analysis on the
expression levels of genes and transcripts to obtain read counts, and
DESeq2 ([77]http://bioconductor.org/packages/stats/bioc/DESeq2/,
accessed on 22 November 2021) software was used to identify
differentially expressed genes (DEGs) between samples using FDR < 0.05
& |log2FC| ≧ 1 as the standard. DEGs were annotated and analyzed for
enrichment in the GO database ([78]http://www.geneontology.org,
accessed on 3 July 2022) and the KEGG database
([79]http://www.genome.jp/kegg/, accessed on 3 July 2022),
respectively.
2.13. Statistical Analysis
Three independent experiments were performed for each assay. All
statistical analyses were performed using GraphPad Prism 9.0.0 (Harvey
Moltusky, San Diego, CA, USA), and regression analysis was used to
determine the significant differences with 95% confidence (p < 0.05).
3. Results
3.1. Inhibition of Total Alkaloid Extracts from P. harmala on Mycelial Growth
Results revealed that total alkaloids exhibited inhibition on mycelial
growth ([80]Figure 2A). The inhibitory effect of total alkaloids on
mycelial growth was concentration-dependent. The mycelial growth
inhibition rates at concentrations of 0.05, 0.1, 0.2, 0.4, and 0.5
mg/mL were 16.3%, 21.4%, 32.2%, 51.3% and 56.3%, respectively
([81]Figure 2B). The mycelial growth inhibition rate of the positive
control group at dose of 0.4 mg/mL was 84.2%. These results showed that
total alkaloid extracts from P. harmala can inhibit the growth of F.
oxysporum.
Figure 2.
[82]Figure 2
[83]Open in a new tab
The inhibitory effects of total alkaloid extracts from P. harmala
against F. oxysporum. (A) The inhibitory effects of total alkaloids on
mycelial growth of F. oxysporum. (B) The inhibition rate of total
alkaloids against F. oxysporum.
3.2. Inhibition of Five Target βCs on Mycelial Growth
To further explore the effect of total alkaloids, five main alkaloids
were cultured with F. oxysporum. As shown in [84]Figure 3A, all the
five βCs had obvious inhibitory effect on F. oxysporum and the
inhibition zone increased with the concentration of βCs from 0.05 to
0.5 mg/mL, indicating that the antifungal effect of βCs against F.
oxysporum was in a concentration-dependent manner. Among the five βCs,
harmane had the most significant inhibitory effect. When the
concentration was 0.5 mg/mL, the mycelia nearly stopped growing, and
the inhibitory rate reached 100% ([85]Figure 3B).
Figure 3.
[86]Figure 3
[87]Open in a new tab
The inhibitory effects of the five βCs on F. oxysporum. (A) The
inhibitory effect of the five βCs on mycelial growth of F. oxysporum.
(B) The inhibition rate of five βCs against F. oxysporum.
The IC[50] of the five βCs from low to high were 0.050 mg/mL (harmane),
0.143 mg/mL (harmine), 0.161 mg/mL (harmol), 0.331 mg/mL (harmaline),
and 0.798 mg/mL (harmalol) ([88]Table 1). Harmane showed the best
antifungal activity and was investigated in subsequent experiments.
Table 1.
IC[50] of the five βCs on F. oxysporum.
βCs Harmaine Harmaline Harmalol Harmane Harmol
IC[50] (mg/mL) 0.143 0.331 0.798 0.050 0.161
[89]Open in a new tab
3.3. MIC
By observing the clarification of different concentrations, we found
that when the concentration of harmane was 40 μg/mL, the fungal liquid
was clear, and when the concentration was 20 μg/mL and lower, the
fungal liquid was turbid. OD[600] values are shown in [90]Figure 4. It
was determined that the MIC of harmane was 40 μg/mL.
Figure 4.
[91]Figure 4
[92]Open in a new tab
The value of OD[600] of F. oxysporum cultures with different
concentrations of harmane.
3.4. SEM
The results of SEM analyses of F. oxysporum spores are shown in
[93]Figure 5. It can be observed that the morphology of hyphae and
spores had undergone significant changes. From the control group, it
can be seen that mycelia and spores are with a smooth surface and plump
in shape, with no wrinkles and have a normal growth ([94]Figure 5A,B).
The surface of mycelia and spores in the treatment group was wrinkled,
depressed, shriveled, and deformed where the red arrows pointed
([95]Figure 5C,D). It can be seen that inhibition of harmane against F.
oxysporum mainly affects cell morphology and leads to cell atrophy.
Figure 5.
[96]Figure 5
[97]Open in a new tab
Morphology of F. oxysporum under SEM, (A,B) Morphology of normal growth
of mycelia and spores in the control group, (C,D) Morphology of mycelia
and spores induced by harmane. (A,C) ×10K, bar = 5.00 μm, (B,D) ×20K,
bar = 5.00 μm.
3.5. TEM
The ultrastructural changes of F. oxysporum were further observed by
TEM and results are shown in [98]Figure 6. In the control group, the
cell boundary was clear, the cell wall was complete, the thickness was
uniform, the cell morphology was elliptical, the organelles were
arranged neatly, and the cell growth was normal ([99]Figure 6A,B). The
mycelia in the treatment group were dissolved in irregular oval shape,
the integrity of cell wall was destroyed, and the cytoplasm was blurred
where the red arrows pointed ([100]Figure 6C,D). This result confirmed
that the permeability or integrity of cell membrane was destroyed.
Figure 6.
[101]Figure 6
[102]Open in a new tab
Ultrastructure of mycelia and spores under TEM, (A,B) Ultrastructure of
mycelia and spores in the control group, (C,D) Ultrastructure of
mycelia and spores induced by harmane. (A,C) Longitudinal section
through the mycelia, (B,D) Tangential section through the mycelia.
(A,B) ×25K, bar = 0.5 μm, (C,D) ×50K, bar = 0.2 μm.
3.6. Detection of Release of Cell Components and Electrical Conductivity
As shown in [103]Figure 7, at the concentrations of 0, 0.5 MIC, and
MIC, harmane significantly increased the release of cell components of
F. oxysporum. The OD[260] was 0.43 at the concentration of MIC after
incubation for 12 h ([104]Figure 7A), which was significantly higher
than that in the control group (p < 0.05).
Figure 7.
[105]Figure 7
[106]Open in a new tab
The effect of harmane on cellular component release and electrical
conductivity of F. oxysporum, (A) Influence of harmane on OD[260], (B)
Influence of harmane on electrical conductivity.
With the increase of processing time, the electrical conductivity also
showed an increasing trend ([107]Figure 7B). After 12 h, the electrical
conductivity of the control group was the lowest (16.13 μS/cm), and the
electrical conductivity of the MIC was highest (46.6 μS/cm) compared
with that of the control, with significant differences (p < 0.05),
indicating that harmane possibly disrupted the cell membrane of F.
oxysporum and increased its permeability.
3.7. Harmane Induced Accumulation of ROS
DCHF-DA staining was used to evaluate the content of ROS levels in the
cells after incubation with harmane. According to the literature
[[108]36], the green fluorescence brightness is positively correlated
with the content of ROS in the cell. In the control group (CK), few
spores with weak fluorescence were found. When the concentration of
harmane was MIC, induced intracellular accumulation of ROS was noticed.
The proportion of spores producing fluorescence increased in a
concentration-dependent manner after treatment of harmane ([109]Figure
8). These results suggested that harmane could cause outbreak of ROS in
F. oxysporum.
Figure 8.
[110]Figure 8
[111]Open in a new tab
Harmane induced intracellular accumulation of ROS in F. oxysporum.
Bright-field was the results of DCFH-DA staining of F. oxysporum under
bright light (×10). Green-field was the results of DCFH-DA staining of
F. oxysporum under green light (×10).
3.8. Cell Death Analysis
The antifungal mechanism of harmane against F. oxysporum was
investigated using Annexin V-FITC/PI double staining. As shown in
[112]Figure 9, after Annexin V-FITC/PI staining, spores in the control
group (CK) rarely show green or red fluorescence with weak fluorescence
intensity. With the increase of harmane content, the green and red
fluorescence intensity and percentage of the cells were higher. Most
cells in the MIC group showed fluorescence, indicating that the
membrane permeability of F. oxysporum was damaged, leading to cell
death.
Figure 9.
[113]Figure 9
[114]Open in a new tab
Harmane induced cell death of F. oxysporum. Annexin V-FITC was the
results of Annexin V-FITC staining of F. oxysporum under green light
(×10); PI was the results of PI staining of F. oxysporum under red
light (×10).
3.9. Effect of Harmane on the Transcriptome of F. oxysporum
Transcriptome sequencing was performed to further reveal the antifungal
mechanism of harmane. We collected differently treated mycelia (0, MIC)
for RNA sequencing. Principal component analysis showed that the
repetitions of each sample clustered together, while different groups
were separated at PC1 and PC2 levels. There were significant
differences in gene expression between the two groups after treatment
of alkaloid. These data demonstrated that the accuracy and reliability
of RNA-sequencing for later analysis. Through the analysis of the DEGs
of the two groups, a total of 8624 identical genes were obtained
between the control and MIC groups. A total of 300 genes were specific
to the control group, and 630 genes were specific to the harmane group.
After treatment of harmane, 1883 genes were differentially expressed of
which 1137 genes were up-regulated and 746 genes were down-regulated.
To analyze the specific differences caused by harmane, DEGs were
classified according to molecular function, biological process and
cellular component in GO database. Eight terms in cellular component
and six terms in biological process and molecular function were
affected in F. oxysporum under harmane treatment. Among the terms,
“membrane part”, “metabolic process” and “catalytic activity” were most
significantly enriched in these three categories, respectively.
Similar to the GO annotation analysis, the GO term enrichment analysis
showed that DEGs related to catalytic activity, integral component of
membrane and intrinsic component of membrane were the most enriched
pathways ([115]Figure 10A) in which a unigene encoding C-5 sterol
desaturase (ERG3) was significantly down-regulated.
Figure 10.
[116]Figure 10
[117]Open in a new tab
Cluster analysis and enrichment analysis in GO and KEGG databases,
(A,B) Enrichment analysis of DEGs in GO and KEGG databases.
KEGG pathway enrichment analysis showed that the DEGs belonged to
peroxisome pathway were the most enriched ([118]Figure 10B) in which
unigenes encoding peroxisomal catalase (CAT) and superoxide dismutase
(SOD) were significantly decreased after harmane treatment.
4. Discussion
Over the years, the long-term heavy use of pesticides has made the
development of new natural antimicrobial agents with good antifungal
effect more and more popular [[119]37]. P. harmala is a drought
tolerant plant that is widely distributed in the world [[120]24].
Extracts from seeds of this plant have antimicrobial effects on a
variety of fungi, bacteria, and viruses [[121]27]. However, there are
few in-depth studies on the antifungal activity and mechanism of the
total βCs or the five β-carboline alkaloids against F.oxysporum. In
this study, the antifungal effect of βCs from P. harmala seed extract
and the mechanism of harmane against F. oxysporum was investigated in
order to provide evidence for the development of new, green agents
against F. oxysporum.
The mycelial growth test of the total alkaloids showed that total
alkaloids had an obvious inhibitory effect on mycelial growth. This
indicated that the total alkaloids were the antifungal components in
the extract of P. harmala seed. The results of the further mycelial
growth inhibition test of five βCs showed that these βCs from P.
harmala extract had different degrees of inhibition on F. oxysporum,
and harmane showed the strongest antifungal activity, with IC[50] of
0.050 mg/mL, which was lower than that of mancozeb, hymexazol and
palmatine [[122]38,[123]39]. The double dilution method is commonly
used to measure IC[50] in general. The inhibition rates of harmine,
harmaline and harmol were with significant difference at 0.4 mg/mL and
0.5 mg/mL. Yet, there was no difference of harmane at 0.4 mg/mL and 0.5
mg/mL of which the inhibition rate was 100%. In overall consideration,
we made a slight modification of tube double dilution method and chose
0.5 mg/mL for the maximum concentration.
Azoxystrobin is often used as a pesticide to prevent root rot of C.
radix in agriculture. It is a commonly used as a positive control in
the study of inhibiting F. oxysporum [[124]40]. At the concentration of
0.4 mg/mL, the antifungal effect of harmane is better than that of
azoxystrobin, and harmane has the potential to be developed into an
antifungal drug.
The MIC of harmane was 40 μg/mL, comparable to that of amphotericin B
[[125]41]. Harmane has the potential to be developed as a drug against
F. oxysporum. At the same time, it is necessary to study the antifungal
spectrum, which will be conducive to the development of broad-spectrum
antifungal drugs. These results indicated that harmane had good
antifungal potential and could be used as a potential fungicide against
F. oxysporum in the future.
SEM and TEM results showed that after harmane treatment, the boundary
of F. oxysporum cells was blurred; the cell membrane and cell wall are
dissolved or even ruptured in some places, and the cytoplasm is
disordered. It was proved that harmane damaged the cell membrane
integrity of F. oxysporum. The increased permeability, the released
cell components, and the increased extracellular electrical
conductivity also supported this point.
There was no significant difference of OD[260] at 4 h, 8 h, and 12 h,
indicating that the intracellular nucleic acid was released within 4 h.
The electrical conductivity was with significantly difference at 4 h, 8
h, and 12 h, indicating that the release process of a large number of
sugars, proteins, nucleic acids, inorganic salts and other contents in
the cells was relatively slow. Within 12 hours, their leakage increased
linearly with time. This trend was consistent with previous reports
[[126]42]. OD[260] and electrical conductivity have been proved to be
important indicators of cell membrane damage [[127]35]. Previous
studies have proved that the butan-1-ol extract of P. harmala seeds
could cause cell membrane damage [[128]43].
βCs could induce accumulation of ROS in plant pathogenic fungi
(Penicillium digitatum and Botrytis cinerea) [[129]44]. The
fluorescence microscopy results in this study also demonstrated that
harmane induced ROS accumulation in F. oxysporum. High concentrations
of ROS can slow down cell growth and even lead to cell death through
cellular oxidative stress [[130]45,[131]46]. Thus, the cell death
detected by Annexin V-FITC/PI staining after harmane treatment was
possibly partially resulted from the accumulation of ROS.
Further transcriptomic analysis revealed that harmane down-regulated
the expression level of ERG3, CAT and SOD in F. oxysporum. ERG3, a key
enzyme in the biosynthesis of ergosterol is involved in steroid
biosynthesis [[132]47]. The disruption of ergosterol biosynthesis
resulted in increased cell membrane permeability [[133]48]. The
decrease of ERG3 expression affected the growth of fungi, resulting in
the inability to produce ergosterol and destruction of membrane
integrity [[134]49]. It appears that the harmane-caused damage of cell
membrane of F. oxysporum was possibly related with the downregulation
of ERG3. Cells generate ROS through a variety of pathways, which can be
cleared by SOD and CAT, thereby maintaining a dynamic balance of
intracellular ROS [[135]50]. The accumulation of ROS in F. oxysporum
caused by harmane was likely related to the reduced expression of SOD
and CAT and the ROS could not be removed normally.
According to the results of cellular component release and electrical
conductivity, the cell membrane damage may occur before 4 h. It would
be better to verify the expression level of key unigenes earlier.
5. Conclusions
In summary, it was demonstrated that the alkaloid extract and βCs from
P. harmala could inhibit the mycelial growth of F. oxysporum. Among
these βCs, harmane had the best antifungal activity and caused damage
of the morphology of mycelia and spores of F. oxysporum, the integrity
of cell membrane, accumulation of intracellular ROS, and cell death.
Combined with transcriptome analysis, harmane may disrupt the integrity
of the cell membrane by regulating steroid biosynthesis and interfering
with ergosterol metabolism via down-regulating genes, such as ERG3,
causing cell wall dissolution and the damage of cell membrane
integrity, resulting in cell death. On the other hand, harmane
interferes with the metabolism of ROS by down-regulating CAT and SOD,
leading to the accumulation of ROS and damage to cells, which may also
cause cell death. βCs has the potential to control F. oxysporum
pollution as an antimicrobial agent. Therefore, future research is
needed to make out the anti-F. oxysporum effects in fields. Our results
provide important insights into the potential mechanism of βCs
inhibiting fungal growth, which may be helpful for future applications
of P. harmala in planting medicinal herbs and crops.
Author Contributions
Z.Z. performed the experiments and drafted the manuscript. S.Z.
provided technical assistance. S.Z. and C.W. designed the experiments,
revised the manuscript, and provided supervision and project
administration. All authors have read and agreed to the published
version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Not applicable.
Conflicts of Interest
The authors declare no conflict of interest.
Funding Statement
This research was funded by the National Key Research and Development
Program of China: 2018YFC1706304; 2022YFC3501701; National Natural
Science Foundation of China: 82173885; Project of Shanghai Science and
Technology: 21DZ2202200; Three-year Action Plan for the Development of
Traditional Chinese Medicine of Shanghai: ZY (2021-2023)-0215; Graduate
Student Innovation Ability Project of Shanghai University of
Traditional Chinese Medicine: Y2021004.
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References