Abstract
Mentha haplocalyx essential oil (MEO) has demonstrated inhibitory
effects on Fusarium oxysporum. Despite its environmentally friendly
properties as a natural product, the limited water solubility of MEO
restricts its practical application in the field. The use of
nanoemulsion can improve bioavailability and provide an eco-friendly
approach to prevent and control Panax notoginseng root rot. In this
study, Tween 80 and anhydrous ethanol (at a mass ratio of 3) were
selected as carriers, and the ultrasonic method was utilized to produce
a nanoemulsion of MEO (MNEO) with an average particle size of 26.07 nm.
Compared to MTEO (MEO dissolved in an aqueous solution of 2% DMSO and
0.1% Tween 80), MNEO exhibited superior inhibition against F. oxysporum
in terms of spore germination and hyphal growth. Transcriptomics and
metabolomics results revealed that after MNEO treatment, the expression
levels of certain genes related to glycolysis/gluconeogenesis, starch
and sucrose metabolism were significantly suppressed along with the
accumulation of metabolites, leading to energy metabolism disorder and
growth stagnation in F. oxysporum. In contrast, the inhibitory effect
from MTEO treatment was less pronounced. Furthermore, MNEO also
demonstrated inhibition on meiosis, ribosome function, and ribosome
biogenesis in F. oxysporum growth process. These findings suggest that
MNEO possesses enhanced stability and antifungal activity, which
effectively hinders F. oxysporum through inducing energy metabolism
disorder, meiotic stagnation, as well as ribosome dysfunction, thus
indicating its potential for development as a green pesticide for
prevention and control P. notoginseng root rot caused by F.oxyosporum.
Keywords: Nanometer emulsion, Ultrasonic method, Antifungal mechanism,
Metabolism pathways, Prevention of root rot
Subject terms: Biological techniques, Biotechnology, Chemical biology,
Microbiology
Introduction
Panax notoginseng, a valuable Chinese herbal medicine derived from
roots, is well-known for its therapeutic effects on cardiovascular and
cerebrovascular diseases. Saponins, the main active medicinal
ingredients, are recognized as valuable components in medicinal
products^[36]1. In recent years, there has been an increasing demand
for P. notoginseng. However, suitable land resources for its
cultivation have diminished due to the “continuous cropping obstacle”
and other factors^[37]2. During the growth process of P. notoginseng,
it is susceptible to various diseases, with root rot caused by F.
oxysporum, F. solani, and P. herbarum being the most common and
destructive soil-borne disease affecting its cultivation and
contributing to the continuous cropping obstacle^[38]3. Typical
symptoms include yellowing of the leaves, collapsing stems, root rot
and softening, and reduced numbers of smaller root hairs^[39]4.
Statistics indicate an annual loss of about 5–20% due to root rot
disease of P. notoginseng; severe cases can reach 70–85%, significantly
hindering industry development^[40]5.
Currently, chemical pesticides are primarily used to control root rot
disease in P. notoginseng cultivation. However, fungicides applied
through root irrigation can lead to heavy metal pollution and pesticide
residues that seriously impact quality and pharmaceutical value^[41]6.
Physical control and microbial control are more environmentally
friendly options. However, physical control is mainly preventive, while
microbial control has the disadvantages of a slow effect and being
susceptible to environmental influences, which are insufficient for
preventing and controlling root rot in P. notoginseng. Plant essential
oils (EOs), as secondary metabolites of plants, have demonstrated
potent inhibitory effects on pathogenic fungi such as F. oxysporum and
F. solani responsible for root rot^[42]2. The complex chemical
composition of plant EOs confers them with a broad spectrum of
antifungal activity, posing challenges for pathogenic fungi in
developing resistance. Furthermore, plant EOs can function as signaling
molecules in the natural environment to safeguard plants from
damage^[43]7. Additionally, plant EOs readily decompose in natural
environments and exert minimal impact on the environment, even aiding
in mitigating heavy metal pollution in agricultural land^[44]8.
Consequently, they represent promising candidates for the development
of novel eco-friendly antifungal pesticides^[45]9.
Mentha haplocalyx, a perennial aromatic herb of the Lamiaceae family,
is widely utilized in pharmaceutical and food industries due to its
broad-spectrum antifungal activity exhibited by its EO^[46]10. Numerous
studies have demonstrated the significant antifungal effects of M.
haplocalyx EO (MEO) against various agricultural fungi. For instance,
MEO effectively inhibits the proliferation of Botryotinia fuckeliana,
Curvularia hawaiiensis, and F. oxysporum, thereby preventing crop
infection and decay^[47]11,[48]12. Furthermore, MEO significantly
suppresses the growth of Rhizopus stolonifer, thereby reducing
strawberry and peach fruit rot caused by R. stolonifer^[49]13, as well
as papaya anthracnose induced by Colletotrichum gloesporioides and
Colletotrichum brevisporu^[50]14. The potential for MEO to replace
traditional fungicides in controlling fungus-induced spoilage of
agricultural products makes it a promising candidate for development
into a green fungicide that reduces reliance on chemical pesticides.
Like most EOs, MEOs are lipophilic liquids that are typically insoluble
in water; this limits their bioavailability^[51]15. Additionally, the
volatile nature of MEO components may reduce their antifungal efficacy
due to low persistence^[52]16. Consequently, researchers have
incorporated EOs into suitable delivery systems to enhance water
solubility and antimicrobial activity^[53]17. Among these systems,
water-in-oil nanoemulsions outperform traditional emulsions not only in
enhancing active ingredient stability but also in ensuring good water
solubility to increase the antimicrobial activity of EOs^[54]18.
Therefore, an increasing number of researchers are dedicated to
developing nanoemulsions of EOs. Nanoemusilfied EOs such as rosemary,
eucalyptus, basil, and copaiba EOs have been used as larvicides and
antimycotic agents^[55]19,[56]20.
Nanoemulsions, consisting of two immiscible phases (oil and water) with
particle sizes ranging from 10 to 100 nm, demonstrate enhanced
stability compared to traditional emulsions regarding polymerization,
flocculation, and precipitation^[57]21. Research has shown that the
smaller particle size and larger particle area of nanoemulsions result
in increased contact area with pathogens, thereby improving their
antifungal activity^[58]22. The stability and antifungal efficacy of
nanoemulsions are dependent on the choice of surfactants and the
preparation method used. For instance, Tween 80, a synthetic surfactant
widely utilized in producing nanoemulsions containing EOs, enhances
both solubility and antifungal activity^[59]23. Furthermore,
high-quality nanoemulsion preparation requires the use of high-energy
sources such as ultrasound or high pressure to generate strong
disruptive forces during homogenization process, which play a crucial
role in determining particle size, stability, and functionality^[60]24.
While nanoemulsions can enhance the solubility and stability of MEO, it
is important to consider the concentration, frequency, and dosage of
MEO nanoemulsions (MNEO) for achieving optimal control effect.
The diverse compositions of plant EOs confers upon them a wide range of
antifungal activities by inhibiting multiple targets within pathogens.
Some progress has been achieved in investigating their antifungal
mechanisms. It has been documented that plant EOs and their primary
components gradually disrupt fungal pathogens by inducing mycelial
morphological changes, vacuole fusion, shedding of cell wall fibrous
layer, and destruction of organelles^[61]25,[62]26. The degradation and
dissolution of the nucleus and mitochondria lead to the decomposition
of cytoplasmic contents, ultimately causing the death of
pathogens^[63]25. However, most studies on the antifungal mechanisms of
plant EOs are currently limited to the cellular level; therefore,
further understanding is required regarding their modes of action at
microscopic levels, such as fungal gene expression levels and
metabolite accumulation. This will facilitate a deeper understanding of
the pathways through which plant EOs affect pathogens and how pathogens
respond to stimulation by plant EOs.
The present study utilized Tween 80 and anhydrous ethanol as
surfactants for the preparation of MNEO through ultrasonication to
enhance their bioavailability. Various parameters were employed to
evaluate the stability of the nanoemulsion and its inhibitory effect on
F. oxysporum. Transcriptomics and metabolomics analyses were performed
to investigate the inhibitory mechanism of MNEO and MEO dissolved in an
aqueous solution of 2% DMSO and 0.1% Tween 80 (MTEO) against F.
oxysporum, as well as their common and specific antifungal mechanisms,
in order to elucidate the superior inhibitory effects of MNEO against
F. oxysporum. In vivo experiments on P. notoginseng seedlings and
rhizomes were also conducted to validate the practical effectiveness of
MNEO in suppressing the causal agent of root rot in P. notoginseng.
Results
Stability of MNEO
We prepared MNEO and MTEO according to the graphical method to prepare
for subsequent experiments (Fig. [64]1). The particle size of the MNEO
was measured, and the average particle size was 26.07 nm (Fig. [65]2A),
which was in line with the particle size range specified by the MNEO.
The particle size of MTEO was 375.54 nm, much larger than that of MNEO
(Fig. [66]2B). The results show that the nanoemulsion had a good
preparation effect, and the polymer dispersity index (PDI) was less
than 0.1, which indicates that the particle size was very uniform and
concentrated (Fig. [67]2C–D).
Figure 1.
[68]Figure 1
[69]Open in a new tab
Flow chart of the production of emulsions. (A) The production process
of MNEO. (B) The production process of MTEO.
Figure 2.
[70]Figure 2
[71]Open in a new tab
Particle size and transmission electron microscopy (TEM) images of MNEO
and MTEO at 25 mg/mL. (A) The particle size distribution of MNEO was
generated by particle size and its corresponding strength. (B) The
particle size distribution of MTEO. (C) In the TEM image of MNEO, the
acceleration voltage was 100 keV, and the morphology of MNEO was
observed at a rate of 100,000 X. (D) In the TEM image of MTEO, the
acceleration voltage was 100 keV, and the morphology of MNEO was
observed at a rate of 50,000 X.
The light transmittance of MNEO treated at − 20 °C, 4 °C, 30 °C, 40 °C,
and 50 °C was not significantly different from that of the control
group (20 °C), indicating that different temperatures had little effect
on the stability of MNEO (Figure [72]S2A). The light transmittance at
1%, 2%, 3%, 4%, and 5% salt concentrations was not significantly
different from that of the control group (Figure [73]S2B). It is well
known that salt will aggregate the particles in the nanoemulsion and
have a negative impact^[74]27. MNEO was still stable at different salt
concentrations, indicating that MNEO can resist available salt
concentrations (Figure [75]S2C). The nanoemulsion remained clear and
transparent after centrifugation at different speeds for 10 min. The pH
of MNEO was approximately 6.8, which was neutral. When the pH was
adjusted between 5 and 9 by adding acid and alkali (Figure [76]S2D), no
stratification or precipitation was observed, which indicated
stability. In the actual use of pesticides, as light is one of the most
important factors affecting their activity, we measured light’s impact
on MNEO and treated them at four different light intensities of 0%,
30%, 60%, and 90% for 48 h (Figure [77]S2E–F). The results showed that
after treatment, the appearance of MNEO was still clear and
transparent. The antifungal effect had no significant change,
indicating that the nanoemulsion had good light resistance.
The stability analysis mentioned above indicated that MNEO exhibited a
smaller particle size than MTEO, meeting the necessary particle size
specifications for a nanoemulsion. It has the advantages of high
optical transparency, good physical properties, and prevention of
gravity separation. No emulsification or phase separation was found in
long-term storage, indicating that nanoemulsions were successfully
prepared.
Chemical component analysis of MEO, MTEO and MNEO
MEO has many beneficial biological functions, including antiviral,
antioxidant, and antifungal effects^[78]28, and one of the most
important parameters determining its antifungal activity is its
chemical composition^[79]17. The chemical compositions of MEO, MTEO,
and MNEO were analyzed by GC‒MS analysis. The total ion chromatograms
were quantified using peak area normalization, and the relative
percentage content of each chemical was calculated (Fig. [80]3).
Twenty-four chemical components of MEO were identified, including
l-Menthol (72.82%), l-Menthone (12.381%), l-Menthyl acetate (3.348%)
and d-Limonene (2.386%). Our results were consistent with those of
other studies where l-Menthol and l-Menthone were the most abundant
components of MEO^[81]29.
Figure 3.
[82]Figure 3
[83]Open in a new tab
The chemical constituent analyses of MEO, MNEO, and MTEO were performed
using GC‒MS. (A) Clustering heatmaps for each of the three groups using
the base log scale of the GC‒MS peak area. (B) Venn diagram of the main
chemical components in the three groups.
Twenty-four chemical components were identified in MNEO, accounting for
98.29% of the emulsion content, with l-Menthol (77.852%), l-Menthone
(10.429%), l-Menthyl acetate (2.848%) and d-Limonene (2.02%) being the
main components. Compared with MEO, there were 19 common components
with the same abundance and varieties, indicating that the nanoemulsion
had little effect on the composition of MEO.
A total of 18 major compounds were identified in the MTEO, accounting
for only 42.897% of the emulsion content, with most of the remainder
being solvents. l-Menthol (32.294%) and l-Menthone (3.279%) accounted
for the most significant proportion of the overall composition, with a
greater chemical composition variation than MEO. Due to its poor
solubility dispersion coefficient, traditional emulsions have resulted
in only a tiny proportion of MEO being soluble in water.
Inhibitory activity of MNEO and MTEO against F. oxysporum
The antifungal effect of the two emulsions on the growth of F.
oxysporum was compared. At the same concentration (0.5 mg/mL), MNEO had
the most significant effect on the growth of F. oxysporum colonies
(Fig. [84]4A–B). The colony diameter did not change significantly
within five days, while the colonies treated with MTEO were less
affected and showed an increasing trend. The effects of the two
solvents on the colony development of F. oxysporum were determined to
test whether the selected solvents have toxic effects on colony growth.
There was no significant difference in the growth of colonies with two
solvent additions compared with the blank treatment group, indicating
that the two solvents had no toxic effect (Fig. [85]4A).
Figure 4.
[86]Figure 4
[87]Open in a new tab
Determination of the antifungal activity of two emulsions against F.
oxysporum. (A) Inhibitory effect of two emulsions on mycelial growth of
F. oxysporum. (B) Colony growth of F. oxysporum. (C) Germination rate
of spores. (D) The MIC of the two emulsions. CK1, An equal amount of
sterile water was added to the medium; CK2, Solvents with nanoemulsions
were added to the medium; CK3: Solvents of traditional emulsions were
added to the medium, MNEO: F. oxysporum grown under MNEO treatment,
MTEO: F. oxysporum grown under MTEO treatment. Data are presented as
the means ± SDs of five biological replicates performed in triplicate.
The effects of the two emulsions on the spore germination rate of F.
oxysporum were investigated at 0.5 mg/mL. After 15 h of culture, the
germination rate of untreated spores was 64.78%, and the germination
rate of MTEO-treated spores was 40.38%. At the same time, the spore
germination rate after MNEO treatment was as low as 24.37%
(Fig. [88]4C). The MICs of the two emulsions against F. oxysporum were
0.58 mg/mL and 3.51 mg/mL, respectively, as shown in Fig. [89]4D.
In conclusion, the antifungal activity of MNEO is greatly improved,
which can more effectively inhibit the growth of F. oxysporum and
improve its bioavailability.
Effects of gene expression in F. oxysporum following treatment with MNEO and
MTEO
Previous studies found that MNEO can inhibit the mycelial growth and
spore germination of F. oxysporum. To study the mechanism of action of
MNEO (0.5 mg/mL) on F. oxysporum, we evaluated the transcriptome
changes of F. oxysporum after treatment with two formulations of MEO.
PCA preliminarily verified the differences between CK, MNEO, and MTEO.
The trend of separation and distinction between groups was evident, and
the reproducibility of samples in the group was good (Fig. [90]5A).
Figure 5.
[91]Figure 5
[92]Open in a new tab
Transcriptomic analysis of F. oxysporum with MNEO and MTEO treatment.
(A) Principal component analysis (PCA) of transcriptome data. (B)
Upregulated and downregulated DEGs in the MNEO and MTEO treatment
groups. (C) Venn diagram showing shared and unique DEGs of the MNEO and
MTEO transcriptomes. Each treatment was the same as above.
Based on the differential expressed genes (DEGs) analysis of F.
oxysporum RNA-seq between MNEO treatment and control, 4196 DEGs were
identified, of which 1916 genes were upregulated and 2280 were
downregulated. Compared to the control, 2142 DEGs were identified in
the MTEO treatment group, of which 1250 genes were downregulated and 89
were upregulated (Fig. [93]5B). Under the same concentration treatment,
MNEO treatment had a greater impact on the transcriptome of F.
oxysporum (Fig. [94]5C). Then, KEGG enrichment analyse was performed on
the DEGs to explore the antifungal mechanism of the two emulsions and
further clarify the advantages of the MNEO treatment group at the
molecular level.
KEGG analysis between MNEO and MTEO treatment in transcriptomics
KEGG pathway enrichment analysis showed that many pathways were
significantly enriched in MNEO and MTEO compared to CK. In the early
stage of the experiment, we found that the two emulsions could inhibit
the growth and spore germination of F. oxysporum. Therefore, we further
analyzed the biological pathways involved in these phenotypes. By
analyzing the top 20 biological pathways, the meiosis,
glycolysis/gluconeogenesis, and starch and sucrose metabolism pathways
were directly related to fungal growth and development (Fig. [95]6A–B).
Figure 6.
[96]Figure 6
[97]Open in a new tab
KEGG pathway enrichment analysis of DEGs in F. oxysporum exposed to
MNEO (A) and MTEO (B). Upregulated and downregulated DEGs in 5
biological metabolic pathways shared by MNEO treatments (C) and MTEO
(D).
Most of the genes encoding major facilitator superfamily (MFS)
transporter, speckled protein (SP) family, sugar: H^+ symporter (HXT),
and guanine nucleotide-binding protein G(i) subunit α (GNAI) in the
meiosis pathway were significantly downregulated (Table [98]S1). In
addition, the gene difference was more significant in the MNEO
treatment group; GNAI and GTPase Kras (KRAS) were significantly
downregulated. Further analysis showed that the genes encoding
cyclin-dependent kinase (CDC28), meiosis-specific transcription factor
NDT80, and anaphase-promoting complex subunit 6 (APC6) were
significantly downregulated after MNEO treatment in the middle and late
stages of meiosis. In summary, this may be one of the reasons why MNEO
treatment has a more substantial inhibitory effect on F. oxysporum.
Most DEGs were downregulated in the glycolysis/gluconeogenesis and
starch and sucrose metabolism pathways (Fig. [99]6C–D). For example,
genes encoding active enzymes such as fructose-bisphosphate aldolase,
class II (ALDO), triosephosphate isomerase (TIM), phosphoglycerate
kinase (PGK), and 2,3-bisphosphoglycerate-independent phosphoglycerate
muta (gpmI) in the glycolysis/gluconeogenesis pathway are
downregulated. In addition, MNEO inhibited the expression of more genes
encoding aldehyde dehydrogenase NAD^+ (ALDH). Additionally, MNEO
inhibited the expression of genes encoding phosphoenolpyruvate
carboxykinase (pckA) and glyceraldehyde 3-phosphate dehydrogenase
(GAPDH), while there was no significant difference in the MTEO
treatment group (Table [100]S2). Similarly, the genes encoding 15
enzymes in starch and sucrose metabolism were significantly
downregulated, seven unique to the MNEO treatment group (Table
[101]S1).
Finally, ribosome and ribosome biogenesis in eukaryotes are pathways
for DEG enrichment only under MNEO stress. Most of the DEGs in the two
metabolic pathways were downregulated (Fig. [102]6C–D), indicating that
MNEO had a strong inhibitory effect on these pathways. In the ribosomal
pathway, MNEO mainly inhibited the 29 genes encoding S26e, S16, L26e,
and other ribosomal proteins (Fig. [103]6C, Table [104]S2). A total of
28 genes encoding enzymes were downregulated in ribosome biogenesis in
eukaryotes, such as GTPase1, RMP1, BMS1, POP4, and NOB1 (Fig. [105]6C,
Table [106]S2).
These results indicate that DEGs involved in the above pathways may be
more closely related to the response of F. oxysporum to MNEO and MTEO
stress. Furthermore, the stress effect of MNEO on F. oxysporum is more
substantial, which confirms that MNEO has a more potent antifungal
effect than MTEO.
Metabolic changes in F. oxysporum under the MNEO and MTEO treatment
The metabolomics of the two treatment groups was analyzed to understand
the antifungal mechanism of MNEO and MTEO on F. oxysporum. PCA results
showed that PC1 explained 42.46% of the total variance. PC2 explained
17.98% of the total variance, with good separation between the
treatment groups (Fig. [107]7A). There were 245 differentially
accumulated metabolites (DAMs) in the MNEO treatment group and 135 DAMs
in the MTEO treatment group. The intersection and specificity of
metabolites in different subgroups were analyzed using Wayne diagrams.
The number of DAMs shared in several differential subgroups was 111,
and the number of differential metabolites specific to each
differential subgroup was 134 and 24, respectively (Fig. [108]7B). The
numbers of upregulated and downregulated metabolites in the MNEO and
MTEO treatment groups were 138 and 107 and 97 and 38, respectively
(Fig. [109]7C).
Figure 7.
[110]Figure 7
[111]Open in a new tab
Extensive targeted metabolomic analysis of F. oxysporum following MNEO
and MTEO treatment. (A) Metabolite PCA plots. (B) Wayne plots of DAMs
between MNEO and MTEO treatment. (C) The number of upregulated and
downregulated DAMs under MNEO and MTEO stress. Metabolomics reveals the
enrichment pathway of MNEO (D) and MTEO (E) inhibition of F. oxysporum
growth. A DA score > 0 indicates upregulation of all identified
metabolites within the pathway, while a DA score < 0 indicates
downregulation.
The DAMs were categorized, with the MNEO treatment consisting of amino
acids and derivatives (18.37%), phenolic acid (16.73%), nucleotides and
organic derivatives (12.24%), organic acids (11.84%), and other types
of metabolites (Figure [112]S2A). The MTEO treatment was primarily
composed of phenolic acids (20.74%), amino acids and derivatives
(17.04%), organic acids (11.85%), and other types of metabolites
(Figure [113]S2B).
KEGG enrichment analysis of these metabolites revealed that DAMs in the
MNEO-treated group were in the starch and sucrose metabolism,
glycolysis/gluconeogenesis, and purine metabolism pathways. Starch and
sucrose metabolism and glycolysis/gluconeogenesis were also enriched in
the transcriptome; most of these differential metabolites were
downregulated (DA score < 0), and there were differences in 6 and 7
metabolites, respectively (Fig. [114]7D, Figure [115]S2C–D). In the
MTEO treatment group, DAMs were mainly enriched in phenylalanine
metabolism, sphingolipid metabolism, tryptophan metabolism, and other
pathways. Amino acid metabolism pathways accounted for a large
proportion, and most DAMs were differentially upregulated (DA
score > 0) (Fig. [116]7E). However, in the MTEO-treated group, no DAMs
were identified in the starch and sucrose metabolic pathways, and only
two DAMs, d-glycerate-3-phosphate (d-Glycerate-3P) and
d-fructose-1,6-bisphosphate (d-Fructose-1,6P[2]) were identified in the
glycolysis/gluconeogenesis pathway. These findings indicate that the
impact of MTEO on the accumulation of metabolites in these two pathways
was not statistically significant (Figure [117]S2C).
In summary, we found more differential metabolites in the MNEO
treatment group through metabolomics analysis, mainly manifested in the
downregulation of significantly enriched pathways (top 20). Starch and
sucrose metabolism and glycolysis/gluconeogenesis were also
significantly enriched, and the accumulation of multiple metabolites
decreased, but these two pathways were not enriched in the MTEO
treatment group. The DAMs enrichment pathways in the MTEO treatment
group were mainly upregulated and most related to fungal stress
resistance. This also shows that MNEO is more destructive to F.
oxysporum.
Integrated transcriptomics and metabolomics analyses
Through transcriptome and metabolomics analysis, we found that MNEO
treatment significantly suppressed glycolysis/gluconeogenesis and
starch and sucrose metabolism. In the presence of MNEO, 7 DAMs (6
downregulated) and 32 DEGs (11 upregulated and 21 downregulated) were
identified in the starch and sucrose metabolism of F oxysporum, while 6
DAMs (6 downregulated) and 29 DEGs (6 upregulated and 23 downregulated)
were identified in the glycolysis/gluconeogenesis pathway. However,
there was no significant enrichment of metabolites in these two
pathways after MTEO treatment. Therefore, a combined analysis of DEGs
and DAMs involved in glycolysis/gluconeogenesis and starch and sucrose
metabolism was conducted for the MNEO treatment group.
MNEO inhibits the metabolism of sucrose and UDP-glucose in F.
oxysporum, affecting subsequent glycogen synthesis and metabolism and
trehalose synthesis. UDP-glucose is synthesized through trehalose
6-phosphate phosphatase (otsA) and α, α-trehalose, in which the gene
encoding otsA is downregulated (Fig. [118]8). During the synthesis and
decomposition of glycogen, the accumulation of d-glucose-1-phosphate
(d-Glucose-1P), d-glucose-1,6-bisphosphate (d-Glucose-1,6P[2]), and
d-glucose-6-phosphate (d-Glucose-6P) decreased consistently with the
coding of glycogenin (GYG1). The related glycogen phosphorylase (PYG)
genes were consistent (Fig. [119]8A).
Figure 8.
[120]Figure 8
[121]Open in a new tab
Combined transcriptomic and metabolomic analysis of
glycolysis/gluconeogenesis and starch and sucrose metabolis (A) DEGs
and DAMs involved in glycolysis/gluconeogenesis and starch and sucrose
metabolism in the MNEO treatment group. (B) Combined transcriptomic and
metabolomic analysis of glycolysis/gluconeogenesis in the MTEO
treatment group. d-Glucose-1P: d-glucose-1-phosphate,
d-Glucose-1,6P[2]: d-glucose-1,6-bisphosphate, d-Glucose-6P:
d-glucose-6-phosphate, d-Fructose-6P: d-fructose-6-phosphate,
d-Fructose-1,6P[2]: d-fructose-1,6-bisphosphate, Glycerone-P: glycerone
phosphate, d-Glyceraldehyde-3P: d-glyceraldehyde-3-phosphate,
d-Glycerate-3P: d-glycerate-3-phosphate, SUS: sucrose synthase, otsA:
trehalose 6-phosphate synthase, TREH: α, α-trehalase, GYG1: glycogenin,
GYS: glycogen synthase, PYG: glycogen phosphorylase, PGM2L1:
glucose-1,6-bisphosphate synthase, pgm: phosphoglucomutase, GPI:
glucose-6-phosphate isomerase, INV: beta-fructofuranosidase, HK:
hexokinase, pfkc: ADP-dependent phosphofructokinase/glucokinase, FBA:
fructose-bisphosphate aldolase, class II, TPI: triosephosphate
isomerase, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, PGK:
phosphoglycerate kinase, gpmI: 2,3-bisphosphoglycerate-independent
phosphoglycerate mutase, ENO: enolase, pps: pyruvate, water dikinase.
After annotation, the DEGs and metabolites were presented as heatmaps
at the corresponding locations, with yellow (low) and red (high)
scales. (C) Coexpression network of DGEs and metabolites involved in
glycolysis/gluconeogenesis and starch and sucrose metabolism. The
higher the degree of connection, the deeper the color of the circle,
and the more remarkable.
The accumulation of d-Glucose-1P, d-Fructose-1,6P[2], and d-Glucose-6P
was reduced in the glycolysis/gluconeogenesis pathway (Fig. [122]8A).
d-glucose is catalyzed by hexokinase (HK), glucose-6-phosphate
isomerase (GPI), and ADP-dependent phosphofructokinase/glucokinase
(pfkc) to synthesize d-Fructose-1,6P[2], which provides the material
basis for the subsequent glycolysis process. As shown in the figure,
the gene encoding HK was downregulated, consistent with the decrease in
the content of the intermediate substances d-fructose-6-phosphate
(d-Fructose-6P) and d-Fructose-1,6P[2]. Under the action of
fructose-bisphosphate aldolase (FBA), triosephosphate isomerase (TPI),
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and phosphoglycerate
kinase (PGK), d-Fructose-1,6P[2] is decomposed into d-Glycerate-3P. The
genes encoding these enzymes were downregulated, and the metabolites
glycerone phosphate (Glycerone-P) and d-Glycerate-3P were also
downregulated. Finally, the upstream gene encoding
2,3-bisphosphoglycerate-independent phosphoglycerate mutase ((gpmI) and
enolase (ENO) of phosphoenolpyruvate was downregulated, consistent with
its content reduction. The key genes of glycolysis/gluconeogenesis and
starch and sucrose metabolism were found to be inhibited to varying
degrees in the MNEO and MTEO treatment groups. In contrast, the number
of DEGs in the MNEO treatment group was more affected. Moreover, the
differential metabolites of MTEO were only d-Glycerate-3P and
d-Fructose-1,6P[2] (Fig. [123]8B). This shows that the MNEO treatment
group has a more substantial inhibitory effect on this pathway, thus
having better antifungal activity.
A coexpression network showed that 5 DEGs highly contributed to starch
and sucrose metabolism and glycolysis/gluconeogenesis, including
FOYG_00893, FOYG_01354, FOYG_05053, FOYG_11832, and FOYG_11338, which
might highly contribute to carbohydrate metabolism under MNEO stress.
In addition, differential metabolites, including Glycerone-P
(Zmzn000078), d-Glucose-1P (mws1090), and d-Glucose-1,6P[2]
(Zmyn000083), were also key nodes in these pathways and should be
further studied (Fig. [124]8C).
Effectiveness of MNEO against root rot of P. notoginseng
MNEO exhibited strong inhibition against F. oxysporum in the early
stage of the experiment, and we assessed its impact on controlling root
rot in P. notoginseng caused by F. oxysporum. The roots inoculated with
F. oxysporum showed clear signs of decay, indicating its ability to
survive in P. notoginseng roots and cause root rot (Fig. [125]9A).
After infection with F. oxysporum, the incidence rate of P. notoginseng
roots treated with MNEO was 13.41%, representing a significant decrease
of 26.62% in incidence rate (Fig. [126]9C). The addition of the
nanoemulsion significantly inhibited the growth of the fungal masses
and reduced the infection rates and root rot occurrence.
Figure 9.
[127]Figure 9
[128]Open in a new tab
Effect of MNEO on controlling F. oxysporum infestation in P.
notoginseng seedlings and isolated roots. (A, C) Effect of MNEO on the
control of P. notoginseng isolated roots. (B, D) Effect of MNEO on the
control of root rot of P. notoginseng seedlings caused by F. oxysporum.
Data are presented as the means ± SDs of five biological replicates
performed in triplicate. ***, P < 0.01. **, 0.01 < P < 0.05.
The same results were found in the P. notoginseng seedlings
(Fig. [129]9B), where seedlings infected with only the conidial
solution of F. oxysporum had a high disease incidence of 78.63%. The
disease index and chlorophyll content were 42.93 and 2.39 mg/g,
respectively. After applying MNEO, the number of leaf discolorations
was significantly reduced. The disease incidence was reduced by 21.91%.
The disease index and chlorophyll content of P. notoginseng seedlings
were 28.61 and 3.53 mg/g, respectively (Fig. [130]9D). The results of
both experiments indicate that MNEO is effective in the suppression of
F. oxysporum and the control of root rot of P. notoginseng and has the
potential to be developed as a green pesticide.
Verification of the transcriptome reliability using RT-qPCR
In order to verify the accuracy and reproducibility of the
transcriptome analysis results, 9 genes related to sugar metabolism,
ribosomes, and meiosis metabolic pathway were randomly selected and
confirmed by RT-qPCR. For all 9 genes, RT-qPCR analysis revealed the
same expression trends as the RNA-Seq data (Figure [131]S2). It shows
that the expression data obtained by RNA-Seq in this study is reliable.
Discussion
The practical application of MEO as a fungal biocontrol agent for P.
notoginseng root rot is limited by its poor water solubility, low
bioavailability, and high volatility. Nanotechnology has been used to
address these challenges by creating nanoemulsions to encapsulate and
deliver EOs. Studies have shown that nanoemulsions can improve the
bioavailability of plant EOs by helping them enter microbial cells and
disrupting cell structure, thus enhancing their antifungal
activity^[132]30. In this study, the prepared MNEO using the ultrasonic
method has small homogeneous emulsion particle size (Fig. [133]2), good
water solubility, and stability under different conditions
(temperature, light, pH) without compromising its antifungal activity
(Figure [134]S2), consistent with previous reports^[135]31. In
addition, compared to MTEO, the MNEO is water-miscible and exhibits a
14.41-fold reduction in particle size (Fig. [136]2), along with a
6.05-fold decrease in MIC and a 16.01% reduction in spore germination
(Fig. [137]4). These results demonstrate that the nanoemulsion enhances
the dispersion efficiency and stability of MEO, thereby improving its
antifungal activity and bioavailability.
Plant EOs have complex chemical compositions influenced by a multitude
of factors, including plant species, growth conditions, collection
sites, and processing methods, which directly affect their antifungal
activity^[138]28. Previous studies using GC–MS analysis identified
l-Menthyl acetate and d-Limonene as the main components in MEO from M.
haplocalyx^[139]32, consistent with our GC–MS results (Fig. [140]3).
The relative proportion of these main components remains unchanged in
MNEO after conversion into nanoemulsion, suggesting excellent
inclusiveness that enhances the antifungal efficacy of MEO. l-menthol
has been shown to inhibit Candida albicans, Aspergillus niger, and
other fungi, while l-Menthol demonstrates strong inhibition against
Fusarium and Rhizobium^[141]33. The enhanced inhibition effect on F.
oxysporum observed upon conversion into nanoemulsion can be attributed
to higher content of l-Menthol, l-Menthone, and l-Menthyl acetate in
MNEO compared to MTEO, providing a theoretical basis for future
development of antifungal products derived from MEO.
The biosynthesis and metabolism of glycogen are crucial for starch and
sucrose metabolism, providing energy for vital life activities. GYG1 is
essential for glycogen biosynthesis, while PYG regulates its
decomposition by releasing d-Glucose-1P^[142]34. Inhibition of PYG
reduces cell proliferation^[143]35. Our study found that both MNEO and
MTEO suppress the gene expression of GYG1 and PYG, diminishing F.
oxysporum’s energy source. Additionally, MNEO inhibits the gene
expression of otsA, which synthesizes stress-protective trehalose using
UDP-glucose^[144]36. Metabolomics analyses revealed that MNEO led to a
significant decrease in the accumulation of seven metabolites
associated with starch and sucrose metabolism in F. oxysporum, thereby
disrupting energy metabolism processes and impeding the growth of the
fungus (Figure [145]S2). Notably, MNEO has stronger inhibitory effects
on gene expression and metabolite accumulation than MTEO in this
pathway (Fig. [146]6, Figure [147]S2).
The accumulation of d-Glycerate-3P and d-Fructose-1,6P[2] was mainly
affected in glycolysis/gluconeogenesis by MNEO and MTEO treatment,
leading to altered gene expression of FBA, TPI, PGK, and other
essential enzymes for this pathway (Fig. [148]8). The decreased
glycolytic synthesis of d-Glycerate-3P and reduced activity of the
glycerolipid/free fatty acid (GL/FFA) cycle hinder cell growth by
impeding lipid detoxification and critical metabolic intermediate
production necessary for maintaining cellular homeostasis^[149]37.
Lower levels of fungal spore formation and germination stages were
observed at low concentrations of d-Fructose-1,6P[2]. The enzyme
controlling the synthesis of d-Fructose-1,6P[2], FBA, was identified as
a potential inhibitory target for the natural fungicide iso butyryl
benzene analog that inhibits Magnaporthe grisea growth^[150]38,[151]39.
Furthermore, exclusive MNEO treatment led to a significant
downregulation of metabolites and genes such as d-Fructose-6P,
phosphoenolpyruvate, FOYG_01354, and FOYG_09920 (Figure [152]S2 Table
[153]S1), indicating a more effective inhibition of energy metabolism
in F. oxysporum by MNEO.
During meiosis, glucose is decomposed and converted through two main
pathways, with their products utilized in each meiotic cycle. One
pathway involves active transport through HXT into the membrane
followed by GNAI-mediated decomposition to provide energy and material
for subsequent meiotic processes^[154]40. The other pathway involves
stimulation of cAMP synthesis through interaction with the G
protein-coupled receptor (GPCR) Gpr1, GNAI, and glucose to generate
substances and energy^[155]41. In this study, it was observed that both
MNEO and MTEO inhibited the expression of genes encoding HXT and GNAI,
impacting the activity of these enzymes, which may result in inadequate
acquisition of actual energy, impeding normal meiosis and ultimately
leading to slowed or halted fungal growth (Table [156]S1). Furthermore,
MNEO also significantly inhibits genes encoding key enzymes such as
CDC28, NDT80, and APC6 (Table [157]S1). These regulators play a role in
meiosis, as well as in mitosis, DNA replication, and sexual
development^[158]42.
Eukaryotic ribosome biosynthesis is fundamental to cellular life and is
responsible for the regulation and coordination of many cellular
processes, including cell growth and division^[159]43,[160]44.
Additionally, inhibiting eukaryotic ribosome biosynthesis can block
mitochondrial respiration in Beauveria bassiana, disrupting energy
metabolism and inhibiting strain growth to prevent fungal
contamination^[161]45. In our study, following MNEO treatment, there
was a significant inhibition of ribosome and ribosome biogenesis,
leading to disruption of normal ribosomal function and impacting cell
division and energy metabolism. Conversely, after MTEO treatment, genes
associated with ribosome and ribosome biogenesis were not inhibited
(Fig. [162]6, Table [163]S2).
Conclusion
In this study, an MEO nanoemulsion was successfully prepared using the
ultrasonic method, effectively preserving key components of MEO such as
l-Menthol, l-Menthone, and l-Menthyl acetate, thus demonstrating
significant efficacy in inhibiting the growth of F. oxysporum in vitro.
Transcriptome and metabolome analyses revealed that MNEO inhibition of
F. oxysporum primarily impacts the expression and accumulation of genes
and metabolites in three major categories: energy metabolism (starch
and sucrose metabolism, glycolysis/gluconeogenesis), ribosomal function
(ribosome, ribosome biogenesis), and cell reproduction (meiosis),
including Glycerone-P, trehalose, UDP-glucose, and otsA, among others.
Additionally, MNEO modulates gene expression related to meiosis,
ribosome function, and biogenesis in eukaryotes to further inhibit the
growth of F. oxysporum (Fig. [164]10). In practical applications, MNEO
has shown promising outcomes. This study offers a theoretical framework
for the further advancement and application of MEO in the development
of eco-friendly pesticides and mitigation of root rot in medicinal
plants.
Figure 10.
[165]Figure 10
[166]Open in a new tab
The mechanism model of the inhibitory effect of MNEO on F. oxysporum
was analyzed from the perspective of multiomics. MNEO Stress indicates
that the metabolic pathways or biological processes of F. oxysporum are
inhibited after MNEO treatment.
Materials and methods
Fungus and plant material
The P. notoginseng root rot pathogen F. oxysporum was provided by the
Wu Kai research group at Yunnan Normal University (GenBank:
[167]OQ080022.1). The strains were cultured on potato dextrose agar
(PDA) medium and placed in a microbial incubator at 28 °C.
The seeds of P. notoginseng were purchased from Yunnan Wenshan Jinyuan
Agricultural Development Co., Ltd. They were sown in a matrix mixed
with nutrient soil: clay = 4:1 and cultured at a relative humidity of
65% ± 5% photoperiod for 14 h/d and a temperature of 20 °C until seed
germination. MEO was purchased from Anhui Huatian Perfume Co., Ltd.
Experimental research and field studies on the plants (either
cultivated or wild), including the collection of the plant material are
in compliance with relevant institutional, national, and international
guidelines and legislation.
Preparation of emulsion
Establishment of a nanoemulsion system for MEO
Surfactant (Tween 80) and cosurfactant (absolute ethanol) were
accurately weighed in a beaker at a particular mass ratio (Km = 1, 2,
3, and 4). The ultrasonic treatment was carried out in an ice bath for
20 min with an operating power of 300 W. Then, select the best Km
ratio. The total mass of the prepared mixed surfactant and MEO phase
was 5 g, and the mass ratios were 9:1, 8:2, 6:4, 5:5, 4:6, 3:7, 2:8,
and 1:9. Then, the mixture was allowed to stand at room temperature for
15 min, and distilled water was added dropwise with a 5 mL pipette,
with stirring while dropping, and repeated until the solution changed
from clear to turbid and back to clear. The mass fraction of the
components at the transition point was calculated. The ternary phase
diagram of the surfactant blend, MEO, and distilled water was drawn.
The area was calculated to determine the optimal Km of the EO
nanoemulsion system.
Preparation of MNEO
The surfactant (Tween 80) and the cosurfactant (anhydrous ethanol) were
accurately weighed at the optimal mass ratio (Km = 3) determined in
Sect. "[168]Establishment of a nanoemulsion system for MEO" (Figure
[169]S1), and thoroughly mixed to obtain surfactant I. The surfactant I
was then placed in a 50 mL beaker with the MEO at a ratio of 8:2,
stirred magnetically for 10 min, and treated by ultrasound in an ice
bath at 300 W for 20 min. Subsequently, deionised water was added in a
ratio of 5:4 (oil phase: water phase), and the oil and water phases
were stirred magnetically for 10 min. The resulting mixture was then
sonicated in an ice bath at 300 W for 30 min (Fig. [170]1A).
Preparation of MTEO
Referring to the literature with slight modifications^[171]46, MEO was
dissolved in surfactant II (aqueous solution of 2% DMSO and 0.1% Tween
80) according to the desired concentration, ultrasonicated in an ice
bath for 20 min and set aside at 4 °C (Fig. [172]1B).
Investigation of the appearance and stability of MNEO
Morphological observations and particle size analysis of the two emulsions
The two emulsions were diluted ten times with deionized water. For
particle morphology, samples were observed under a transmission
electron microscope (JEM-1011, JEOL, Co. Ltd., Tokyo, Japan). A laser
particle size meter (90Plus PALS) was used to measure the particle size
of the emulsions.
Centrifugation test
MNEO samples were prepared and centrifuged at 2000 r/min, 5000 r/min,
and 8000 r/min for 30 min to observe if they were stratified, using
distilled water as a blank control. The absorbance at 550 nm was
measured before and after centrifugation.
[MATH:
T=10-A :MATH]
T: Transmittance; A: Absorbance.
Salt stability
Five milliliters of MNEO were placed in 5 clean test tubes. Sodium
chloride was added to them to make the mass fractions of sodium
chloride 1%, 2%, 3%, 4%, and 5%, respectively, and the test tubes were
shaken to dissolve. The changes in the nanoemulsion were observed. And
use the same formula as above to calculate the transmittance.
Acid–base stability
Ten milliliters of MNEO were placed in a small beaker, and then 0.1 M
hydrochloric acid or sodium hydroxide solution was gradually added
dropwise to the cup. The pH was adjusted to acidic or alkaline, and the
system’s pH change was measured using a pH meter. The difference in the
nanoemulsion solution was observed, and use the same formula as above
to calculate the transmittance.
The effect of temperature on the stability of MNEO
The prepared MNEO was heated at − 20 °C, 4 °C, 20 °C, 30 °C, 40 °C and
50 °C for 30 min to observe whether the nanoemulsions were
delaminating; if not, the absorbance of the nanoemulsions before and
after heating was measured, and use the same formula as above to
calculate the transmittance.
Effect of light on antifungal activity of MNEO
The prepared MNEO was placed in a light incubator; the light intensity
was set to 0%, 30%, 60%, or 90%; and the constant temperature was 25 °C
for 48 h. After treatment, the antifungal effect was tested according
to the method detailed in Section “Fungal growth curves”.
Gas chromatography-mass spectrometry (GC‒MS) analysis
The prepared MTEO and MNEO were analyzed by GC‒MS using Agilent
Technologies 7890B-5977B gas chromatography. The compounds’ retention
times and mass spectra were compared with NIST 17.L database, and the
final chemical constituents were determined with relevant
literature^[173]47.
Comparison of antifungal activity between MNEO and MTEO
Fungal growth curves
The MNEO, MTEO, sterile water, nanoemulsion solvent, and traditional
emulsion solvent (the final emulsion concentration was 0.5 mg/mL) were
added to 20 mL of PDA medium for five replicates. The PDA medium was
inoculated with a 5 mm block of F. oxysporum and incubated at 28℃. The
colony diameter was measured at 24 h intervals until the seventh day,
and the growth curve of the two emulsions against F. oxysporum was
obtained^[174]48.
Fungal spore germination rate
MNEO and MTEO were added to a sterile EP tube containing PDA liquid
medium and mixed until the emulsion concentration was 0.5 mg/mL.
Finally, an appropriate spore suspension was added to a final
concentration of 1 × 10^6 spores/mL, and the control group was added to
sterile water. Each treatment was repeated three times^[175]49. The
total number of spores and the number of germinated spores were counted
by a blood cell counting plate under a light microscope.
[MATH: G=X1/X0×100%
:MATH]
G: spore germination rate; X[1]: number of spores that have germinated;
X[0]: total number of spores.
Determination of MIC
The MIC value test was carried out with 96-well plates according to the
reference with slight modification^[176]50. Fresh colonies were washed
with 1/3 PDA liquid medium, and spore suspensions were obtained by
filtration. The concentration was adjusted to 2 × 10^5 spores/mL. The
initial concentration of MNEO was 4 mg/mL, and MTEO was 25 mg/mL. Two
emulsions are diluted into eight concentration gradients by double
dilution. The 96-well plates were incubated at 28 °C for 20 h. A
microplate reader measured the absorbance of each well at a wavelength
of 595 nm, and the MIC value of the emulsion was calculated.
Transcriptome and metabolomics analysis
Sample preparation
Emulsion at the concentration of 0.5 mg/mL was prepared for 30 mL, and
then 1 g of F. oxysporum mycelium was added and treated at 28 °C for
24 h. The mycelium was harvested through filtration, rapidly frozen
with liquid nitrogen, and stored at − 80 °C for subsequent use.
RNA extraction and RNA-Seq
Total RNA was extracted from the samples using the RNAprep pure plant
kit (DP441, Tiangen, China). Illumina RNA-Seq was performed by Meisoft
Biotechnology Co., Ltd. (Wuhan, China). RNA quality was measured by a
nanophotometer spectrophotometer (IMPLEN, CA, USA), Qubit 2.0
fluorometer (Life Technologies, CA, USA) and Agilent Bioanalyzer 2100
system (Agilent Technologies, CA, USA). Poly (A) mRNA was enriched with
oligomer (dT) magnetic beads and randomly fragmented mRNA. The first
strand of cDNA was synthesized by the M-MuLV reverse transcriptase
system. Then, the RNA strand was degraded with RNase H, and cDNA was
synthesized with DNA polymerase. These double-stranded cDNAs were
connected to the sequencing adapter. The cDNA (~ 200 bp) was screened
with AMPure XP beads. After amplification and purification, the cDNA
library was obtained and sequenced using the Illumina Novseq 6000
system^[177]51.
Sequence data processing
The original sequence of the original image data was converted by base
recognition technology. To obtain high-quality data, the sequence
joints were cut, and fastp was used to remove low-quality reads
containing ≥ 5 uncertain bases or more than 50% Qphred ≤ 20 bases. The
GC content of the clean read was calculated. Fast QC also generated Q20
and Q30 values to evaluate the essential quality. Then, clean reads
were mapped to the F. oxysporum reference genome using HISAT2 with
default parameters. Gene expression levels were determined using the
RPKM (per million reads) method.
Extraction of metabolites
Biological samples were freeze-dried using a vacuum freeze dryer
(Scientz-100F). The freeze-dried samples were ground with a mixer mill
(MM 400, Retsch) and zirconia beads at 30 Hz for 90 s. Lyophilized
powder (100 mg) was dissolved in 1.2 mL of 70% methanol solution,
rotated for 30 s every 30 min six times, and placed in a refrigerator
at 4 °C overnight. After centrifugation at 12,000 rpm for 10 min, the
extract was subjected to UPLC‒MS/MS analysis (SCAA-104, 0.22 μm pore
size; an NPEL, Shanghai, China, [178]http://www.anpel.com.cn/).
Qualitative and quantitative analysis of metabolites
Metabolite data were log2 transformed for statistical analysis to
improve normality and normalization. Hierarchical clustering analysis
(HCA), principal component analysis (PCA), and orthogonal partial least
squares discriminant analysis (OPLS-DA) were performed on the
metabolites of 30 samples to study the specific accumulation of
metabolites. The p-value and fold change values were set to 0.05 and
2.0, respectively. The Venn diagram illustrates the number of
bidirectional metabolites. The Kyoto Encyclopedia of Genes and Genomes
(KEGG) database was used, with a p-value < 0.01, and the standard
database of F. oxysporum (String database species ID:5507) was
referenced. All data were plotted using GraphPad Prism 99v6.01
(GraphPad Software, La Jolla, CA, USA)^[179]51.
In vivo testing of the control effect
The roots of P. notoginseng with uniform size were selected and cut
into approximately 2 cm thick pieces after cleaning. After disinfection
with alcohol and sodium hypochlorite, the pieces were placed on a plate
covered with water agar. The 5 mm diameter of the fungal blocks was
positioned at the center of the P. notoginseng block and served as a
positive control, while 5 μL of MNEO at a concentration of 0.5 mg/mL
was added to the fungal block as a treatment. Following a seven-day
incubation period, the growth of fungal blocks on P.notoginseng was
quantified, and the incidence rate was calculated.
Uniformly grown annual P. notoginseng seedlings (10 cm high) were
selected, and 10 mL of F. oxysporum suspension at a concentration of
1 × 10^6 spores/mL was injected into the soil with a syringe around the
base of the plant stems. Forty plants were planted in each treatment.
MNEO at a 0.5 mg/mL concentration was prepared, and 10 mL was poured
into the base of each plant stem every seven days, while 10 mL of
sterile water was poured into the blank control. P. notoginseng was
incubated in a 12 h light/12 h dark incubation chamber at 24 °C to
observe disease development. The incidence and disease index of P
notoginseng were recorded on the 30th day after the initial application
of MNEO, and the chlorophyll content was determined using an
established method^[180]52,[181]53.
[MATH: Disease incidenceDI=number of diseased
plants/total
number of
plants×100%53<
/msup>. :MATH]
Disease severity was graded as follows.
* Grade 0: plants are healthy.
* Grade 1: spots on the leaves of the plants.
* Grade 2: wilting of the plant.
* Grade 3: plants are dead.
[MATH: Disease indexDi=100×∑<
mfenced close=")"
open="(">Dn×Dg/Tn×Mg :MATH]
where Dn indicates the number of plants with the same disease
level, Dg is the corresponding disease level, Tn is the total
number of plants, and Mg is the highest disease level.
RT‒qPCR analysis
The samples were taken from mycelia that were treated with different
EOs (CK, MNEO, MTEO) for 24 h and used for RNA extraction. Each sample
was reverse-transcribed into first strand cDNA using a cDNA reverse
transcription kit (TaKaRa PrimeScript RT Master Mix RR036B). The primer
list is shown in the supplementary table (Table [182]S3). RT‒qPCR was
performed using a ChamQ SYBR qPCR Master Mix (Vazyme) and a Bio-Rad CFX
Manager 3.1 thermal cycle system. Relative transcript levels were
calculated using the 2^−ΔΔCq method, using a housekeeping gene, QTUB,
as a reference. Three biological and technical replications were
performed.
Statistical analysis
All experiments in this study were repeated three times, and the data
shown are the mean ± SD (standard deviation). The results were
subjected to one-way ANOVA (Pvalue of ≤ 0.05), LSD (least-significant
difference), and Tukey’s multiple range test to determine significant
differences in mean comparisons. These data were analyzed using the
SPSS program version 19.0. The results were finally plotted using the
Origin program version 2019b, Adobe Illustrator 2022, and GraphPad
Prism 9.
Supplementary Information
[183]Supplementary Information.^ (23.6MB, doc)
Author contributions
Hongxin Liao: Methodology, Software, Writing—original draft. Jinrui Wen
and Hongyan Nie: Conceptualization, Methodology. Cuiqiong Ling:
Investigation. Liyan Zhang: Investigation. Furong Xu: Data curation.
Xian Dong: Writing—review & editing, Funding, Administration. All
authors read and approved the manuscript.
Funding
This work was funded by the National Natural Science Foundation of
China (82060683), Yunnan Provincial Science and Technology Plan-Basic
Research Project (202301AW070008), Wang Yuan Chao Expert Workstation in
Yunnan Province (202305AF150018), Yunnan Provincial Science and
Technology Department Applied Basic Research Joint Special Funds of
Yunnan University of Traditional Chinese Medicine (202101AZ070001-047),
Natural Science Foundation of Hubei Province (2023AFB982), Key
Laboratory Project of Sustainable Utilization of Yunnan Southern
Medicine (202105AG070012XS23010), and Yunnan Provincial Department of
Education Scientific Research Fund Project (2023Y0451).
Data availability
The Transcriptome sequencing data of this study were submitted and
deposited in the NCBI GenBank database under BioProject No.
PRJNA1076078. The datasets generated during and/or analysed during the
current study are available from the corresponding author on reasonable
request.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in
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Supplementary Information
The online version contains supplementary material available at
10.1038/s41598-024-67054-1.
References