Abstract
Plant-beneficial Pseudomonas and Bacillus have been extensively studied
and applied in biocontrol of plant diseases. However, there is less
known about their interaction within two-strain synthetic communities
(SynCom). Our study revealed that Pseudomonas protegens Pf-5 inhibits
the growth of several Bacillus species, including Bacillus velezensis.
We established a two-strain combination of Pf-5 and DMW1 to elucidate
the interaction. In this combination, pyoluteorin conferred the
competitive advantage of Pf-5. Noteworthy, pyoluteorin-deficient Pf-5
cooperated with DMW1 in biofilm formation, production of metabolites,
root colonization, tomato bacterial wilt disease control, as well as in
cooperation with beneficial bacteria in tomato rhizosphere, such as
Bacillus spp. RNA-seq analysis and RT-qPCR also proved the
pyoluteorin-deficient Pf-5 mutant improved cell motility and metabolite
production. This study suggests that the cooperative effect of
Bacillus–Pseudomonas consortia depends on the balance of pyoluteorin.
Our finding needs to be considered in developing efficient SynCom in
sustainable agriculture.
Subject terms: Biofilms, Microbiome, Antimicrobials, Soil microbiology,
Applied microbiology
Introduction
The quality of plant rhizosphere microbiomes is critical in
plant-disease suppression. Cooperative interactions between the
beneficial microbes can efficiently suppress the invasion of plant
pathogens, enhance efficient colonization, and positively affect
competition for niches in the plant rhizosphere^[52]1–[53]4. A diverse
and positively interacting microbial community structure is a
precondition in promoting plant growth, and the effective control of
plant diseases. Abundant evidence suggests that disease control can be
more efficient by the collective actions of the microbial community
rather than the individual contributions of specific bacterial or
fungal species^[54]5. In order to elevate the disease-controlling
effects of the natural microbiome, synthetic consortia, consisting of
two or more microbial strains, able to control plant pathogens
(multi-strain biocontrol agents, MSBCAs), have been employed to improve
the growth and harvest yield of crops^[55]5. In the reductionist
synthetic community approach, only a few well-characterized members of
the natural microbiome are assembled to form a defined synthetic
community (SynCom)^[56]6. However, in using this approach, the
interactions between different bacteria, which can directly determine
the type of microbial community structure, need to be carefully
considered^[57]7,[58]8.
Fluorescent pseudomonads and plant-associated bacilli occupy a
significant position in the microbial community structure of plant
rhizosphere^[59]9, and represent excellent models for beneficial
bacteria with biocontrol function^[60]9–[61]11, and might be promising
candidates for applying together within a SynCom. Fluorescent
pseudomonads, the most abundant bacteria in the plant rhizosphere, have
a dominant influence on plant growth and development and play a vital
role in plant-disease control^[62]12,[63]13. Pseudomonas putida strain
IsoF effectively eliminates both, plant pathogenic bacteria, and
bacterial competitors, by injecting toxic effectors into neighboring
bacterial cells utilizing a type IVB secretion system (T4BSS)^[64]14.
Among the group of fluorescent pseudomonads, Pseudomonas protegens has
obtained considerable attention in biocontrol research due to its
extraordinary antimicrobial properties, which are mainly due to their
secondary metabolites, such as pyoluteorin^[65]15, orfamide A^[66]16,
2,4-diacetylphloroglucinol (DAPG)^[67]7, pyrrolnitrin^[68]17, and
pyoverdine^[69]18. Also, volatile organic compounds (VOCs) enhance the
competitiveness of P. protegens within plant rhizosphere^[70]19.
Extensive documentation supports the notion that a gene cluster
involved in inositol degradation bestows Pseudomonas with exceptional
colonization abilities^[71]20.
Plant-associated bacilli are widely applied in the biocontrol of plant
diseases. Bacillus strains are advantageous, due to their ability to
form resistant endospores, and are well-suitable for large-scale
fermentation^[72]21. The plant-associated B. velezensis, previously B.
amyloliquefaciens subsp. plantarum, a member of the B. subtilis species
complex, is widely applied as a powerful BCA, and known to produce a
diverse array of antagonistic metabolites^[73]22. A representative of
this taxonomic group, B. velezensis DMW1, isolated from potato inner
tissues, is reported to synthesize non-ribosomal the lipopeptides
iturin, surfactin, fengycin, the polyketides difficidin, bacillaene,
macrolactin, and the dipeptide bacilysin. DMW1 possesses a remarkable
capacity to effectively suppress the growth of pathogenic bacteria,
fungi, and oomycetes^[74]23,[75]24.
We assumed that combining selected representatives of the two powerful
groups of gram-negative and gram-positive BCAs, P. protegens Pf-5 and
B. velezensis DMW1, within a two-strain SynCom might result in an
efficient BCA surpassing the efficiency of the corresponding single
strain BCAs. However, no mutualistic effect was registered. By
contrast, in this combination, P. protegens Pf-5 did strongly suppress
B. velezensis DMW1 leading to annulment of its biocontrol action. The
objective of this study was to elucidate the molecular interactions
between both strains and to identify the reason for suppressing DMW1 by
Pf-5 with the goal of constructing more efficient consortia from both
bacteria.
The coexistence in their natural environments between plant-associated
bacilli and pseudomonads was recently reviewed by Lyng and
Kovács^[76]9. However, in many cases the interactions observed in dual
cultures were negative. Amensalism (suppressive effects) against
Bacillus was often observed^[77]9. Secondary metabolites such as
lipopeptides produced by several pseudomonads can mediate inhibition of
B. velezensis during colonizing tomato roots^[78]25. Moreover, the
pseudomonad type VI secretion system (T6SS) and antibiotic
2,4-diacetylphloroglucinol can impact biofilm formation in B.
subtilis^[79]14. Recently, it was reported that antagonism between
bacilli and pseudomonads is shaped by competition for
iron^[80]26–[81]28. In order to design more compatible consortia, we
have identified in this study the key secondary metabolite responsible
for targeted inhibition of B. velezensis, and were able to design a
two-strain-SynCom, more efficient than the single strains. By contrast
to the wild type, the Pf-5 mutant lacking the hybrid polyketide
pyoluteorin can smoothly coexist with B. velezensis. The
two-strain-SynCom enhanced biofilm formation, metabolite production,
and tomato root colonization. This cooperative interaction improved the
efficacy of tomato bacterial wilt disease control and reshaped the
microbial community structure in the tomato rhizosphere. Our study
demonstrated that by targeted switching off of a functional metabolite
produced by a member of the synthetic consortia, their efficiency in
disease control can be markedly enhanced.
Results
P. protegens Pf-5 overcomes Bacillus strains including B. velezensis DMW1
P. protegens Pf-5, the model strain of biocontrol Pseudomonas, was
selected as one member of a two-member consortium consisting of
representatives of gram-negative and gram-positive biocontrol bacteria.
Several Bacillus strains, representing different species of the B.
subtilis species complex, were chosen as the second member of the
synthetic consortium. It was observed that P. protegens Pf-5 suppressed
the growth of B. velezensis DMW1, the model strain B. velezensis FZB42,
B. subtilis SYST2, B. paralicheniformis NMSW12, B. safensis GBSW22, B.
pumilus NMSW10, and B. halotolerans DGL6 (Fig. [82]1a). This indicated
that P. protegens Pf-5 contained an active factor or metabolite, which
could suppress the growth of all the Bacillus strains used in this
study. To explore the active factor(s), a bipartite consortium,
consisting of P. protegens Pf-5, and B. velezensis DMW1, chosen due to
its excellent biocontrol properties^[83]23, was established. Pf-5 and
DMW1 were differently labeled with either red fluorescence or green
fluorescence proteins. The competitive test on solid LB agar showed
that Pf-5 entered gradually into the growth region of DMW1, thus
inhibiting the growth of DMW1 during the time (Fig. [84]1b). The cell
number of DMW1 in the contact region with Pf-5 (near Pf-5, Pf-5(N)) was
found significantly reduced compared to the DMW1 region not contacted
by Pf-5 (far Pf-5, Pf-5(F)). Only less than 3‰ of the cell number,
detected in the unaffected control region, were registered after 48 h
growth (Fig. [85]1b, c). By contrast, the cell number of Pf-5 in the
DMW1 contact region (near DMW1, DMW1(N)) was not different compared to
the Pf-5 region without contact with DMW1 (far DMW1, DMW1(F)) (Fig.
[86]1d). B. velezensis DMW1 and its different metabolites (crude
extract, surfactin, iturin, and fengycin) did not much impair the
growth of P. protegens Pf-5 (Fig. [87]1e, f) suggesting that P.
protegens Pf-5 strongly suppressed DMW1, but is less inhibited by B.
velezensis and its metabolites.
Fig. 1. Antagonistic activities between P. protegens and Bacillus strains.
[88]Fig. 1
[89]Open in a new tab
a The inhibiting effect of P. protegens Pf-5 on the growth of different
Bacillus strains. b Criss-cross agar test for detecting antagonistic
activity against each other of P. protegens Pf-5 (red–mCherry) and B.
velezensis DMW1 (green–gfp). Images. Top: fluorescence image gfp
channel under a fluorescence microscope at the excitation/emission
wavelengths of 488/525 nm. Bottom: normal light (bright field) was
taken after 24 h, 48 h, and 72 h incubation. c Antagonistic effect of
Pf-5 on DMW1. Cell number of DMW1 close at Pf-5 or remote of the border
region affected by Pf-5. Pf-5(F) represents “far Pf-5” (yellow circle
in b); Pf-5(N) represents “near Pf-5” (green circle in b). d
Antagonistic effect of DMW1 on Pf-5. Cell number of Pf-5 growing in or
distant from the border region adjacent to DMW1. DMW1(F) represents
“far DMW1” (blue circle in b); DMW1(N) represents “near DMW1” (red
circle in b). Bars with different letters indicate significant
differences (two-sided, unpaired Student’s t-test, p < 0.0001 for c,
p > 0.9999 for d) (n = 9). e The inhibiting effect of DMW1 and the
crude extract on the growth of Pf-5. Sterile water (Control), bacterial
suspension, or crude extract was dripped onto a white circular paper
disc. f No effect of the lipopeptides of DMW1 on the growth of Pf-5.
Sterile water (Control) or a different substance dripped onto a white
circular paper disc.
The factor responsible for the superiority of P. protegens Pf-5 over B.
velezensis DMW1 is regulated by the GacS/GacA two-component system
To find out which factor is responsible for Pf-5’s superiority over
DMW1 within the two-member consortium, the GacS/GacA two-component
system, which globally regulates the synthesis of many secondary
metabolites, was considered. The mutant ΔgacA, impaired in expression
of the GacS/GacA two-component system, was constructed, and labeled by
red fluorescence. The competition experiments showed that ΔgacA lost
the ability to overcome DMW1 and displayed no antagonistic activity
against DMW1 in the criss-cross experiment (Fig. [90]2a). The cell
number of DMW1 in the region, adjacent to the ΔgacA mutant growth
region (near ΔgacA, ΔgacA(N)), was not significantly affected (Fig.
[91]2b). As expected, the cell number of the ΔgacA mutant growing in
the regions contacted (near DMW1, DMW1(N)) or not contacted (far DMW1,
DMW1(F)) by DMW1 was not significantly different (Fig. [92]2c).
Fig. 2. Growth superiority of P. protegens Pf-5 over B. velezensis DMW1 is
due to the expression of the secondary metabolites regulated by the GacS/GacA
two-component system.
[93]Fig. 2
[94]Open in a new tab
a Criss-cross agar test for detection of cross antagonistic activity of
ΔgacA (red–mCherry) and B. velezensis DMW1 (green–gfp). Images (top:
fluorescence image gfp channel, bottom: visual light). b Antagonistic
effect of ΔgacA on DMW1. Cell number of DMW1 growing in or distant from
the border region affected by ΔgacA. ΔgacA(F) represents “far ΔgacA”;
ΔgacA(N) represents “near ΔgacA”. c Antagonistic effect of DMW1 on
ΔgacA. Cell number of ΔgacA growing in or remote of the border region
adjacent to DMW1. DMW1(F) represents “far DMW1”; DMW1(N) represents
“near DMW1”. d The superiority of Pf-5 over DMW1 was completely
restored in the gacA mutant strains after complementation with the gacA
wild-type gene. Pf-5 gacA-C (red–mCherry) and B. velezensis DMW1
(green–gfp) growing on LB. Top: fluorescence image gfp channel; bottom:
bright field image. e Antagonistic effect of ΔgacA-C on DMW1. Cell
number of DMW1 growing in or distant from the border region adjacent to
ΔgacA-C. ΔgacA-C(F) represents “far ΔgacA-C”; ΔgacA-C(N) represents
“near ΔgacA-C”. f No antagonistic effect of DMW1 on ΔgacA-C. Cell
number of ΔgacA-C growing in or not in the border region adjacent to
DMW1. DMW1(F) represents “far DMW1”; DMW1(N) represents “near DMW1”. g
Effect of the culture, and of crude extract prepared from P. protegens
Pf-5, ΔgacA, ΔgacA-C on inhibition of B. velezensis DMW1. The ΔgacA
mutant has lost its inhibiting capability, whilst the mutant
complemented with the wild-type gacA gene has restored its ability to
inhibit DMW1. h Diameter of inhibition zones after inoculation of
Pseudomonas bacterial suspension in DMW1 lawn. i Diameter of inhibition
zones after inoculation of Pseudomonas crude extracts of secondary
metabolites in DMW1 lawn. Bars with different letters indicate
significant differences (two-sided, unpaired Student’s t-test,
p = 0.6806 for b, p = 0.9312 for c, p < 0.0001 for e, p = 0.1821 for f,
n=9; p < 0.0001 for h, p = 0.0.3559 for i, n=3).
After complementing the mutant gacA strain with the gacA^+ wild-type
gene, resulting in strain ΔgacA-C, the competitive superiority of
ΔgacA-C was completely restored (Fig. [95]2d, e). DMW1 had no apparent
effect on the cell number of ΔgacA-C (Fig. [96]2f). Agar-diffusion
tests performed with the culture, and the crude extract containing
secondary metabolites produced by the Pf-5 wild-type, the ΔgacA mutant,
and the complement ΔgacA-C strain corroborated that the GacA/GacS
system governs a metabolic factor responsible for the antagonistic
activity of Pf-5 exerted against DMW1 (Fig. [97]2g–i). The gacA mutant
lost the ability of the wild strain to suppress the growth of DMW1
completely, but this ability was restored by complementing the mutant
strain with the gacA^+ wild-type gene.
The secondary metabolite pyoluteorin is responsible for the growth
superiority of P. protegens Pf-5 in the two-member community
HPLC analysis of the crude extracts obtained from Pf-5 wild-type,
ΔgacA, and ΔgacA-C, corroborated earlier findings that synthesis of
secondary metabolites, such as pyoluterin, orfamide A, and
2,4-diacetylphloroglucinol (DAPG) is controlled by the GacS/GacA
system^[98]29. The gacA deficient mutant strain, impaired in the
synthesis of the GacA/GacS system, produced none of these metabolites
(Fig. [99]3a and Supplement Fig. [100]1). In order to identify the
compound(s) responsible for antagonistic activity of Pf-5 against DMW1,
the genes responsible for the synthesis of antimicrobial metabolites
were knocked out by triparental mating: ofaA (orfamide A), pltB
(pyoluteorin), phlA (2,4-diacetylphloroglucinol), prnA (pyrrolnitrin),
rzxB (rhizoxin), hcnABC (hydrogen cyanide), and pFL4656 (non-ribosomal
peptide). Only the ΔpltB mutant, unable to synthesize pyoluteorin, lost
its ability to antagonize DMW1 (Fig. [101]3b). In vitro experiments
performed with purified pyoluteorin confirmed that pyoluterin
efficiently inhibits the growth of DMW1 (Fig. [102]3c). Criss-cross
experiments demonstrated that, similar as in the ΔgacA mutant, ΔpltB
lost the growth advantage against DMW1 (Fig. [103]3d, e). Similar to
the ΔgacA mutant strain, the cell number in the ΔpltB mutant was not
affected by the presence of DMW1 (Fig. [104]3f). Taken together, these
results demonstrated that the polyketide pyoluteorin is the critical
factor, responsible for the growth advantage of P. protegens Pf-5 over
B. velezensis DMW1.
Fig. 3. The competitive advantage of Pf-5 over DMW1 is dependent on
pyoluteorin.
[105]Fig. 3
[106]Open in a new tab
a HPLC analysis of Pf-5, and of the ΔgacA and ΔgacA-C mutant strains.
After complementing the mutant strain with the gacA^+ wild-type gene,
the ability of strain ΔgacA-C to synthesize pyoluteorin was restored.
The pyoluteorin standard (50 µg/mL) is shown at the bottom. b
Antagonistic activity against DMW1 of Pf-5 gene mutants, impaired in
the biosynthesis of secondary metabolites, was only affected in the
ΔpltB mutant, impaired in the synthesis of pyoluteorin. The diameter of
inhibition zones was not significantly affected in all other mutants,
impaired in the synthesis of antimicrobial metabolites. Bars with
different letters indicate significant differences (p < 0.0001, one-way
ANOVA, Duncan’s multiple range test) (n = 3). c Effect of the crude
extract of P. protegens Pf-5, and of purified pyoluteorin (50 µg/mL) on
the B. velezensis DMW1 lawn. d Criss-cross test revealed no
antagonistic activities between ΔpltB (red–mCherry) and B. velezensis
DMW1 (green–gfp) on LB agar after 24 h, 48 h, and 72 h. Top:
fluorescence spectroscopy to distinguish green (DMW1) and red
fluorescence (ΔpltB); bottom: visual light image. e ΔpltB did not
affect the cell number of DMW1. Cell number of DMW1 in or remote of the
border region to ΔpltB. ΔpltB (F) represents “far ΔpltB”; ΔpltB (N)
represents “near ΔpltB”. f DMW1 did not affect the cell number in
ΔpltB. Cell number of ΔpltB in or remote of the border region to DMW1.
DMW1(F) represents “far DMW1”; DMW1(N) represents “near DMW1”. Bars
with different letters indicate significant differences (two-sided,
unpaired Student’s t-test, p = 0.2337 for e, p = 0.9034 for f) (n = 9).
In the pyoluteorin-deficient P. protegens Pf-5 mutant, thirty-one percent of
the differentially expressed genes were significantly upregulated, including
those associated with cell motility and metabolite production
To further explore the changes of DMW1 after treatment with Pf-5 or
ΔpltB, the transcriptome was analyzed. Gene Ontology (GO) term
enrichment among differentially expressed genes (DEGs) was performed to
comprehensively reveal the biological functions encoded by these genes.
This GO enrichment analysis not only highlighted the active roles of
DEGs across various biological dimensions but also provided deeper
insights into how these genes participate in and influence key
functional mechanisms within the bacterium (Fig. [107]4a, Table
[108]1). The results of the KEGG pathway enrichment analysis revealed
significant enrichments in multiple key metabolic and biosynthetic
pathways, including Carbon metabolism, Biosynthesis of amino acids,
Quorum sensing, Carbon fixation pathways in prokaryotes, Biosynthesis
of secondary metabolites, Non-ribosomal synthesized-peptides,
Propanoate metabolism, RNA degradation, and Valine, leucine, and
isoleucine biosynthesis (Fig. [109]4b). Based on the KEGG pathway
analysis, we focused on the differentially expressed genes (DEGs)
enriched under the two major categories of Cellular Processes and
Metabolism. In-depth analysis revealed that compared to Pf-5 treatment,
ΔpltB treatment significantly enriched DEGs closely related to
processes such as flagellin synthesis, amino acid metabolism, and
non-ribosomal peptide synthesis. These findings not only highlighted
the unique impact of ΔpltB treatment on cell motility, fundamental
metabolic activities, and non-ribosomal synthesis of antimicrobial
lipopeptides and polyketides but also provided important clues for
further exploring its potential biological effects and mechanisms
(Table [110]2). We analyzed the expression patterns of DEGs related to
flagellin synthesis, and non-ribosomal-peptide synthetases after
co-culturing Bacillus with the wild-type Pf-5 and the mutant ΔpltB,
respectively (Table [111]2). 21 out of 22 genes involved in flagellin
synthesis were upregulated after co-culturing with the mutant ΔpltB.
Furthermore, in screening for genes related to non-ribosomal peptide
synthetase, 7 out of 11 genes were found to be upregulated (Table
[112]2).
Fig. 4. Pseudomonas influences the metabolic process and cellular processes
of DMW1.
[113]Fig. 4
[114]Open in a new tab
a Gene Ontology (GO) annotation classification for differentially
expressed genes (DEG) (Top 15). Blue box: biological process, red box:
cellular component, green box: molecular function. b Bubble plot
showing results of KEGG enrichment (Top 15) analysis of repeated DEGs.
Transcriptional changes in genes related to the biological process,
cellular component, and molecular function of DMW1 after treatment with
wild-type Pf-5 and mutant ΔpltB are shown. The size of the circle
indicates the number of genes, orange indicates significant, and blue
indicates not significant. c Relative expression of the itu, fen, srf,
mln, dfn, and bae genes in DMW1 responsible for the synthesis of
iturin, fengycin, surfactin, macrolactin, difficidin and bacillaene,
flgD and fliR genes in DMW1 responsible for the synthesis of flagellum
respectively. The dotted line represents the average value measured in
the control.
Table 1.
Number of gene in transcriptome analyses
Process Num
Biological process
Cellular process 1333
Metabolic process 1131
Developmental process 283
Biological regulation 270
Localization 240
Response to stimulus 226
Regulation of the biological process 224
Cellular component organization or biogenesis 208
Multi-organism process 55
Negative regulation of the biological process 46
Locomotion 42
Signaling 35
Positive regulation of the biological process 23
Reproduction 12
Detoxification 6
Nitrogen utilization 5
Biological adhesion 3
Cell proliferation 3
Carbohydrate utilization 2
Reproductive process 2
Immune system process 1
Multicellular organismal process 1
Cellular_component
Cell 1393
Cell part 1393
Membrane 843
Membrane part 716
Protein-containing complex 146
Organelle 82
Extracellular region 64
Organelle part 39
Nucleoid 12
Extracellular region part 1
Molecular_function
Catalytic activity 1419
Binding 1127
Transporter activity 280
Transcription regulator activity 102
Structural molecule activity 47
Molecular transducer activity 22
Antioxidant activity 18
Molecular function regulator 9
Molecular carrier activity 4
Toxin activity 1
[115]Open in a new tab
Table 2.
Transcriptional changes in genes in DMW1 after treatment with
Pseudomonas
Pathway name log2(ΔpltB/Pf-5)
Flagellar assembly
Flagellar hook assembly protein FlgD 5.880498164
Flagellar biosynthesis chaperone FliJ 5.525115887
Flagellar type III secretion system protein FliR 4.722247044
Flagellar biosynthesis protein FlhB 4.657546554
Flagellar hook-length control protein FliK 4.515594176
Flagellar protein export ATPase FliI 4.458213445
Flagella biosynthesis regulatory protein FliZ 4.300241003
Flagellar hook-basal body complex protein FliE 4.132446628
Flagellar motor switch protein FliG 4.130199175
Flagellar assembly protein FliH 4.03836466
Flagellar basal body rod protein FlgG 3.99335507
Flagellar basal body rod protein FlgC 3.983929641
Flagellar type III secretion system pore protein FliP 3.925084142
Component of the flagellar export machinery 3.91961604
Flagellar M-ring protein FliF 3.85616435
Flagellar biosynthesis protein FlhA 3.820413385
Flagellar motor switch protein FliM 3.687212618
Flagellar motor switch phosphatase FliY 3.486289742
Flagellar basal body-associated protein FliL 3.388886511
Flagellar basal body rod protein FlgB 3.016592448
Flagellar motor protein MotB 2.212011148
Cystine ABC transporter substrate-binding lipoprotein TcyA
−4.085519801
Non-ribosomal peptide structures
Non-ribosomal peptide synthase 3.166922311
Non-ribosomal peptide synthetase 2.869741048
Non-ribosomal peptide synthetase 2.795045952
Non-ribosomal peptide synthetase 2.76320404
Non-ribosomal peptide synthetase 2.625956926
Non-ribosomal peptide synthetase 2.356591277
Non-ribosomal peptide synthetase 2.172344088
Non-ribosomal peptide synthetase 1.993738806
Non-ribosomal peptide synthase 0.267432218
Non-ribosomal peptide synthetase −1.46456935
Non-ribosomal peptide synthetase −1.482083249
[116]Open in a new tab
The data described above demonstrated that B. velezensis DMW1 growing
together with P. protegens Pf-5 was suppressed by the pyoluteorin
production of Pf-5. In order to evaluate whether the synthesis of
antimicrobial secondary metabolites produced by B. velezensis is
differently affected by Pf-5, and the ΔpltB mutant strain, unable to
secrete pyoluteorin, we analyzed the expression of their transcripts by
RT-qPCR. The results showed that the relative expression level of the
genes, responsible for the non-ribosomal peptide synthetase of iturin,
fengycin, surfactin, difficidin, macrolactin, and bacillaene, and the
genes involved in flagellum synthesis in B. velezensis DMW1, were
enhanced by 4.24–13.10 times when co-cultivated with the ΔpltB mutant
strain compared to the co-cultivation with the Pf-5 wild-type strain
(Fig. [117]4c).
The pyoluteorin-deficient P. protegens Pf-5 mutant promotes the production of
secondary metabolites in DMW1
HPLC analysis corroborated that synthesis of the main secondary
metabolites of DMW1 was significantly increased when co-cultivated with
ΔpltB (Fig. [118]5). Compared with the DMW1 treatment alone, the
relative abundance of fengycin, difficidin, and bacillaene in Pf-5
treatment decreased by 10.6%, 10.1%, and 37.3% respectively. Compared
with Pf-5 or DMW1 treatment, the relative abundance of the metabolites
in ΔpltB treatment significantly increased. Among the main metabolites,
the lipopeptides iturin, fengycin, and surfactin involved in
plant-induced systemic resistance and antifungal action increased by
1.37, 1.32, and 2.02 times compared to DMW1, while the antibacterial
polyketides macrolactin, difficidin, and bacillaene increased by 1.84,
1.21, and 1.39 times compared to DMW1 alone (Fig. [119]5). It has been
shown previously that these secondary metabolites are important for the
biocontrol exerted by DMW1 against fungal and bacterial plant
pathogens^[120]23. In conclusion, by using a ΔpltB mutant strain,
unable to synthesize pyoluteorin, together with the efficient DMW1
biocontrol strain in a bipartite synthetic consortium, it is possible
to enhance the efficiency of the consortium without negatively
affecting the growth of the gram-positive partner strain.
Fig. 5. Production of Bacillus metabolites upon addition of Pseudomonas Pf-5
or ΔpltB.
Fig. 5
[121]Open in a new tab
Fold increase in DMW1 bioactive secondary metabolite production upon
the addition of Pseudomonas Pf-5 or ΔpltB, compared to unsupplemented
DMW1 cultures (fold change = 1, dotted line). Graphs showed the
mean ± SD calculated from three biological replicates (n = 3). Bars
with different letters indicate significant differences as defined by
one-way ANOVA with Tukey’s HSD test.
Removal of pyoluteorin synthesis enables the P. protegens Pf-5/B. velezensis
DMW1 consortium to coexist harmoniously, and to be efficient in biofilm
formation, root colonization, and plant-disease control
Efficient biofilm formation is a precondition for root colonization by
plant-associated bacteria. DMW1 formed dense wrinkled biofilms when
cultivated solely. However, in the presence of Pf-5, this phenotype
disappeared, and the hybrid biofilm appeared thin and sparse, and was
characterized by an overwhelming number of Pf-5 cells. By contrast, the
replacement of the Pf-5 wild-type by the mutant ΔpltB, restored the
dense, wrinkled phenotype, which resembled the biofilm phenotype
produced in the absence of Pf-5. Fluorescence microscopy revealed that
the number of red-labeled Pseudomonas and green-labeled Bacillus cells
was nearly equal (Fig. [122]6a).
Fig. 6. DMW1 ability to colonize tomato roots was inhibited by pyoluteorin
produced by P. protegens Pf-5.
[123]Fig. 6
[124]Open in a new tab
a Effect of Pf-5 and ΔpltB on the biofilm formation of B. velezensis
DMW1 in liquid medium. b Effect of Pf-5 and ΔpltB on the gene
expression of the biofilm formation of B. velezensis DMW1 (n = 3) (fold
change = 1, dotted line). c Laser-scanning confocal microscopy images
of B. velezensis DMW1 (green–gfp) and P. protegens Pf-5 (red–mCherry)
co-colonizing tomato roots at 25 °C four days after inoculation. Scale
bar: 20 μm. d Boxplot of the number of DMW1 cells colonizing the tomato
rhizosphere solely, or together with Pf-5, or the ΔpltB mutant strain
(n = 6). e Boxplot of the relative number of Pseudomonas cells
(wild-type or ΔpltB) colonizing the tomato rhizosphere. Gene copies
were calculated according to the standard curve (see “Methods” section)
(n = 6). f 45-day-old tomato plants, infected with R. solanacearum, and
grown in normal soil either without bacteria or inoculated with DMW1,
Pf-5, ΔpltB, DMW1+Pf-5, DMW1+ΔpltB. g Disease control effect of the
different treatments is described in (f). Details of the disease assay
are described in “Methods” section. Bars with different letters
indicate significant differences as defined by one-way ANOVA with
Tukey’s HSD test (n = 3). DMW1 means only DMW1; +Pf-5 means DMW1+Pf-5;
+ΔpltB: means DMW1+ΔpltB.
The expression of the biofilm-related genes epsB, and matrix
components-related gene blsA, and the flagellar protein-related gene
fliC in DMW1 was, compared to the Pf-5 wild type, significantly
upregulated in coculture with the ΔpltB mutant (Fig. [125]6b). This
means that pyoluteorin produced by Pf-5 plays the key role in
inhibiting biofilm formation and suppressing of DMW1. Adding purified
pyoluteorin (10 µg/mL) had the same inhibiting effect on biofilm
formation as adding the Pf-5 wild-type strain (Supplementary Fig.
[126]2a).
If applied solely, the B. velezensis DMW1 efficiently colonized tomato
roots. Thereby, the meristem, and the elongation zone of the main root,
especially the junction between the epidermal cells, and the root
hairs, were preferentially colonized. But, when the P. protegens Pf-5
strain was co-applied with DMW1, both bacteria colonized different
sites of the tomato roots. By contrast, when the Pf-5 wild-type was
replaced by the ΔpltB mutant strain, B. velezensis and P. protegens
(ΔpltB) did nicely coexist within the same sites (Fig. [127]6c and
Supplementary Fig. [128]2b). This result is in accordance with our
previous finding that DMW1 and ΔpltB can form mixed biofilms consisting
of the members of both species (Fig. [129]6a).
The degree of colonization was quantified by qPCR followed by absolute
quantification of the number of gene copies^[130]30,[131]31, which
correspond to the colony forming units (CFU) colonizing the root
tissues according to the standard curves (Supplementary Fig. [132]3).
The degree of colonization by DMW1 was significantly declined when
co-applied with Pf-5 but was not significantly affected when co-applied
with ΔpltB (Fig. [133]6d). As expected DMW1 had no effect on the
colonization rate of Pf-5 (Fig. [134]6e). Our results demonstrated that
the inhibitory effect of Pf-5 on the root colonizing efficiency of DMW1
can be overcome when expression of pyoluteorin was turned off. There
was no statistical difference between the single DMW1 treatment and the
treatment of supplementary DMW1 with ΔpltB.
Both, P. protegens and B. velezensis could control when applied solely,
tomato bacterial wilt disease caused by R.
solanacearum^[135]19,[136]23. Here, we found that DMW1, Pf-5, and its
mutant ΔpltB efficiently controlled the tomato bacterial wilt disease
by 63.46%, 73.08%, and 72.12% (Fig. [137]6f, g). Notably, when Pf-5 or
ΔpltB were co-applied with DMW1, the control effect was significantly
enhanced. The best result was obtained with the bipartite consortium
consisting of DMW1 and the ΔpltB mutant strain yielding 91.35% and
there was no significant difference from those consisting of DMW1 and
Pf-5. These results indicated that in the absence of pyoluteorin
production, the bipartite consortium consisting of the P. protegens
Pf-5 mutant strain and B. velezensis DMW1 controls tomato bacterial
wilt disease in a very efficient manner.
P. protegens Pf-5 lacking pyoluteorin enhances the microbial diversity and
abundance of tomato rhizosphere
As demonstrated by the chord diagram, representatives of the
gamma-proteobacteria, flavobacteria, alpha-proteobacteria, and
beta-proteobacteria were the predominant components of the tomato
rhizosphere. The addition of P. protegens Pf-5 increased the diversity
of the rhizosphere microbial community. In the presence of Pf-5 or
ΔpltB, an increase of the members of the Bacteroidetes phylum, and of
the Proteobacteria compared to the water control treatment was
registered (Supplementary Fig. [138]4a). Network metrics values, such
as the number of edges, the clustering coefficient, the average degree,
and network density, indicated higher diversity in the microbiome after
ΔpltB treatment than after the Pf-5 treatment. Moreover, the network
obtained after ΔpltB treatment also featured an increase in the
occurrence of Bacillus spp. compared to the network obtained after Pf-5
treatment (ΔpltB/Pf-5 ratio of 0.4%/0.2%) (Fig. [139]7a). We observed
differences in the composition of the microbiome in Pseudomonas-treated
soil compared to natural soil (Fig. [140]7b and Supplementary Fig.
[141]4a). As expected, the microbiota developed after the ΔpltB
inoculation differed from that after adding of Pf-5 most likely due to
the absence of pyoluteorin (Fig. [142]7b and Supplementary Fig.
[143]4b). The unconstrained principal coordinate analysis (PCoA) of the
Bray–Curtis distance revealed different clustering of the uninoculated
rhizosphere soil microbiota (control), the microbiota after Pf-5
inoculation, and the microbiota after ΔpltB inoculation. These clusters
were found clearly separated along the second coordinate axis,
indicating significant variation (Fig. [144]7b). Measurement of the
α-diversity showed notable differences between the various treatments.
The introduction of the Pseudomonas strains did significantly enhance
the microbial diversity of tomato rhizosphere soil (Fig. [145]7c and
Supplementary Fig. [146]4c, d). By contrast to Pf-5, adding ΔpltB
significantly enriched the representatives of the Bacillus taxon (Fig.
[147]7d, e and Supplementary Fig. [148]4e). Remarkably, the abundance
of Bacillus spp. forced by ΔpltB treatment was notably higher than that
observed in the control without inoculation of additional strains and
in the Pf-5 treatment (Fig. [149]7f, g). There was a significant
positive correlation in the cell numbers between Bacillus and
Pseudomonas in the ΔpltB treatment (Fig. [150]7g). In summary, the
addition of P. protegens to the tomato rhizosphere microbiome
contributed to microbial diversity, which was found further enhanced in
the absence of pyoluteorin. Furthermore, in the absence of pyoluteorin
synthesis enrichment of beneficial bacteria such as Bacillus spp.
residing within the rhizosphere was observed.
Fig. 7. Assessment of Pseudomonas-associated bacterial communities.
[151]Fig. 7
[152]Open in a new tab
a Bacterial intra-kingdom co-occurrence networks^[153]32. The
intra-kingdom network exhibits a higher number of nodes and edges in
ΔpltB treatment rhizosphere soil compared to Pf-5 samples. Nodes are
colored according to bacterial lineages, and the size of the node
indicates the degree of association. Edges are colored to indicate
positive (pink) and negative (green) associations. b Unconstrained PCoA
(principal coordinates PCo1 and PCo2) using Bray–Curtis distance
reveals a distinct separation of the rhizosphere microbiota in the
first axis (p < 0.001, permutational multivariate analysis of variance
(PERMANOVA) by Adonis)^[154]30. c Microbiota community diversity among
the three treatments was implemented by analysis of richness indices.
Letters are used to indicate significant differences between groups.
Different letters denote distinct groups (p < 0.05, ANOVA, Tukey HSD
test)^[155]30. d Manhattan plots showing ASVs enriched or depleted in
the Pf-5/ΔpltB samples compared to Pf-5 treatment in tomato rhizosphere
soil microbiota. Each circle or triangle represents a single ASV. ASVs
enriched or depleted in the samples are represented by filled or empty
triangles, respectively (ASVs abundance >0.01%, p < 0.05)^[156]33. Red
arrows indicate an increased abundance of Bacillus sp. e Absolute
differences in the relative abundance of genera between Pf-5 and ΔpltB
(ANOVA with Tukey’s HSD test; p < 0.05). ASVs matched to Bacillus spp.
are marked in red. Error bars represent standard deviations. The right
side displays a STAMP diagram illustrating the differential
genera^[157]34, while the left side exhibits the respective proportions
of these genera in the two sample groups. On the right, the difference
between blue and yellow corresponds to high abundance colors, and the
error bars represent a 95% confidence interval. Correlations between
the abundance of Bacillus and Pseudomonas in the Pf-5 treatment
(p = 0.792) (f) and ΔpltB treatment (p < 0.0001) (g). One week or two
weeks samples were colored to blue or green.
Discussion
In this study, we explored the interaction within a synthetic
consortium formed by a selected gram-negative Pseudomonas and a
gram-positive Bacillus strain, both known for their excellent ability
to biocontrol pathogens in the plant rhizosphere.
The co-cultivation of P. protegens Pf-5 and B. velezensis DMW1 in a
solid medium led to the growth inhibition of B. velezensis DMW1, which
corresponds to earlier findings that fluorescent pseudomonads such as
P. protegens exert negative interactions with plant-associated
bacilli^[158]35. Here, the unknown inhibiting factor was shown to be
controlled by the GacS/GacA two-component system, known as a global
regulator of secondary metabolism. Further analyses revealed that the
chlorinated polyketide pyoluteorin, non-ribosomal synthesized by P.
protegens Pf-5^[159]16,[160]36, and regulated by the GacS/GacA system,
caused the antibiosis effect observed in DMW1. Pyoluteorin was shown to
inhibit plant pathogens such as Pantoea ananatis^[161]15,
Oomycetes^[162]37, Chladymonas reinhardtii^[163]38, and Heterobasidion
sp.^[164]39, Burkholderia glumae^[165]40, indicating a broad spectrum
of antimicrobial activity. We found that P. protegens Pf-5 and B.
velezensis DMW1 are incapable of forming together a robust biofilm. Due
to the presence of pyoluteorin, the growth of DMW1 became suppressed
within the consortium. P. protegens Pf-5 and B. velezensis DMW1
couldn’t coexist in the roots of tomato plants, and the ecological
niche was mainly occupied by P. protegens Pf-5. Notably, B. velezensis
DMW1 and P. protegens Pf-5 colonized two separate zones of the root
system, given that both zones are sufficiently distant from each other.
This observation might suggest that DMW1 can only colonize plant roots
when not negatively affected by pyoluteorin. Pyoluteorin was identified
as the key substance that hindered biofilm formation and affected the
expression of genes related to biofilm formation, but its targets and
inhibiting mechanisms in bacilli remain to be further studied.
Favorable interactions among members can enhance the inhibitory
activity of the SynCom against pathogenic bacteria, thereby boosting
its biocontrol potential^[166]7. In order to overcome the inhibiting
effect exerted by Pf-5 against B. velezensis DMW1, the pyoluteorin
knockout mutant ΔpltB of P. protegens Pf-5 was constructed.
Co-cultivating of the P. protegens ΔpltB mutant together with DMW1 in a
liquid medium resulted in increased synthesis of the main antimicrobial
metabolites. The increased production of Bacillus secondary metabolites
might contribute to enhanced biocontrol effects of the bipartite
consortium. The efficacy of the two-member DMW1/P. protegens ΔpltB
consortium in controlling tomato bacterial wilt disease surpassed the
efficacy of the treatment solely with Pf-5, suggesting that
target-directed-engineering of selected members of synthetic consortia
can overcome antagonistic interactions, and greatly enhance their
efficacy. It is known that interbacterial interaction can trigger the
activation of Bacillus biosynthetic gene clusters (BGCs) residing in
the root rhizosphere^[167]23.
Another way to overcome inhibition of biofilm formation of
plant-associated bacilli in the presence of fluorescent pseudomonads
has been reported. Mutations in negative regulators of biofilm
formation were generated during directed laboratory evolution resulting
in improved competitiveness of B. subtilis^[168]9. Through a
comprehensive comparative analysis of the effects of P. protegens Pf-5
and P. protegens ΔpltB on the microbial community structure in the
tomato rhizosphere, we made an intriguing discovery: both P. protegens
Pf-5 and P. protegens ΔpltB have the remarkable ability to
significantly augment the diversity of the microbial community
structure in the soil surrounding tomato roots, thus contributing to a
more robust and thriving ecosystem in the tomato rhizosphere. Applying
the strategy to overcome the limitation set by the kin boundary by
identifying and then eliminating the factor(s) hindering cooperative
behavior within synthetic consortia^[169]41,[170]42, we paved the way
for developing highly efficient synthetic consortia, e.g. in biocontrol
of phytopathogens. Interestingly, after introducing the ΔpltB mutation,
P. protegens supported the colonization of Bacillus in the tomato
rhizosphere and enhanced microbial biodiversity. This finding
highlights the unique contribution of P. protegens ΔpltB in enriching
Bacillus spp. (Fig. [171]7d–g). This provides further evidence that
positive interaction relationships are particularly beneficial for
promoting the diversity of microbial community structure.
As an alternative to the use of an engineered SynCom described here,
careful selection of beneficial species exhibiting mutualistic
interactions offers another way to obtain efficient SynComs. For
example, B. velezensis and P. stutzeri can achieve mutualistic
symbiosis through the cross-feeding of metabolites^[172]41.
Cross-feeding between bacteria is common and important^[173]39–[174]41.
Researchers utilized GutCP to predict numerous new cross-feeding
interactions in the human gut microbiota and revealed cross-feeding
interactions for nearly 65% of the microorganisms^[175]42. Leaf
bacteria play a significant role in cross-feeding interactions that
have functional relevance, and these interactions can be influenced by
the phyllosphere environment, indirectly contributing to the diversity
of bacterial populations^[176]33.
Mutual interactions among beneficial bacteria can lead to synergistic
effects. Here, we showed that by introducing the ΔpltB mutation, P.
protegens and B. velezensis developed a cooperative behavior, including
the capacity to co-form biofilms and to co-colonize tomato roots.
Furthermore, B. velezensis DMW1 enhanced colonization of tomato roots
in the presence of P. protegens ΔpltB, showcasing its beneficial
effects and biocontrol potential. The efficacy of the two-member
DMW1/P. protegens ΔpltB consortium in controlling tomato bacterial wilt
disease, surpassed the effectiveness of the treatment solely with Pf-5,
suggesting that target-directed-engineering of selected members of
synthetic consortia can overcome antagonistic interactions, and greatly
enhance their efficacy.
Taken together, the introduction of the targeted mutation connected
with the switch off of the pyoluteorin synthesis in the synthetic
consortium of two taxonomically very distant plant-growth-promoting
bacteria (PGPB), restores their beneficial action on plant growth due
to improved interactions on community-level, and enhanced ability to
colonize commonly plant roots. Our findings have implications for
designing and applying synthetic communities consisting of
remote-related PGPB.
Methods
Microorganisms and cultivation
The bacteria used in this study are listed in Table [177]3. Bacillus
strains, and Escherichia coli strains were cultured by inoculating a
single colony in 20 mL lysogeny broth (LB) (10 g L^−1 NaCl, 5 g L^−1
yeast extract, and 10 g L^−1 tryptone) medium, and incubated overnight
at 37 °C with shaking (200 rpm); P. protegens strains were cultured by
inoculating a single colony in 20 mL King’ B (KB) (20.0 g L^−1
tryptone, 10.0 g L^−1 NaCl, 1.5 g L^−1 MgSO[4]·7H[2]O, 1.5 g L^−1
K[2]HPO[4]), and incubated overnight at 30 °C with shaking (200 rpm).
Plasmid cloning was performed in E. coli DH5α (Table [178]4). The
cultured bacteria are regarded as cultures. Primers used for cloning
and verification are found in Table [179]5.
Table 3.
Strains and plasmids used in this study
Strains Relevant genotype and description Sources
Pseudomonas protegens
Pf-5 Wild type; Amp^R Joyce E. Loper
Pf-5ΔgacA Pf-5 deleted the gacA gene; Amp^R This study
Pf-5ΔgacA/pBBR-gacA Mutant complement; Amp^R, Gm^R This study
Pf-5ΔpltB Pf-5 deleted the pltB gene; Amp^R This study
Pf-5ΔphlA Pf-5 deleted the phlA gene; Amp^R This study
Pf-5Δprna Pf-5 deleted the prna gene; Amp^R This study
Pf-5ΔrzxB Pf-5 deleted the rzxB gene; Amp^R This study
Pf-5ΔhcnABC Pf-5 deleted the hcnABC gene; Amp^R This study
Pf-5ΔpFL4656 Pf-5 deleted the pFL4656 gene; Amp^R This study
Pf-5/pBBR-mCherry Pf-5 with vector pBBR-mCherry; Amp^R, Gm^R
Laboratory stock
Pf-5ΔgacA/pBBR-mCherry ΔgacA with vector pBBR-mCherry; Amp^R, Gm^R
This study
ΔgacA/pBBR-gacA-pUCP26-mCherry ΔgacA/pBBR-gacA with vector
pUCP26-mCherry; Amp^R, Gm^R, Tc^R This study
Pf-5ΔofaA/pBBR-mCherry ΔofaA with vector pBBR-mCherry; Amp^R, Gm^R
This study
Pf-5ΔpltB/pBBR-mCherry ΔpltB with vector pBBR-mCherry; Amp^R, Gm^R
This study
Bacillus velezensis
DMW1 Wild type Laboratory stock
DMW1/pAD43-25 DMW1 with vector pAD43-25; Cm^R Laboratory stock
Escherichia coli Top10 TaKaRa Company
[180]Open in a new tab
Antibiotics resistance abbreviations: Amp^R (Ampicillin), Gm^R
(Gentamicin), Tc^R (Tetracycline), Cm^R (Chloramphenicol)
Table 4.
Strains and plasmids used in this study
Plasmid Relevant genotype and description Sources
pK18mobsacB Vector; Km^R Laboratory stock
pRK-2013 Vector; Km^R Laboratory stock
pBBR Vector; Gm^R Laboratory stock
pBBR-gacA The gacA gene was cloned into the BamH I and Hind III sites
of pBBR; Gm^R This study
pBBR-mCherry The mCherry gene was cloned into the Hind III and EcoR I
sites of pBBR; Gm^R Laboratory stock
pUCP26 Vector; Tc^R Laboratory stock
pUCP26-mCherry The mCherry gene was cloned into the EcoR I and Hind III
sites of pUCP26; Tc^R This study
pK18-ofaA Knockout of ofaA gene; Km^R This study
pK18-pltB Knockout of pltB gene; Km^R This study
pK18-phlA Knockout of phlA gene; Km^R This study
pK18-prna Knockout of prna gene; Km^R This study
pK18-rzxB Knockout of rzxB gene; Km^R This study
pK18-hcnABC Knockout of hcnABC gene; Km^R This study
pK18-pFL4656 Knockout of pFL4656 gene; Km^R This study
[181]Open in a new tab
Antibiotics resistance abbreviations: Km^R (Kanamycin), Gm^R
(Gentamicin), Tc^R (Tetracycline)
Table 5.
DNA primers used in this study
Primer Primer sequence (5′→3′)
Construct mutants
ΔgacA F1 ACGCGTCGACCGGGCATAGTTCAAAACC (Sal I)
ΔgacA R1 TGACGAACCGCCAATAGCGCAGACACCTCGCGATAT
ΔgacA F2 ATATCGCGAGGTGTCTGCGCTATTGGCGGTTCGTCA
ΔgacA R2 CCCAAGCTTCGTAGGGGTAGGACTTATC (Hind III)
ΔgacA out F TTTGCCTTTCTTGCGGCCT
ΔgacA out R TTCTGCAACAGGCTGAGG
ΔofaA F1 ACGCGTCGACTGGGTCAAGCCCTTGCGA (Sal I)
ΔofaA R1 TGACATGGGAAGCCGGGAGGCGGAAAAATGCGTCAT
ΔofaA F2 ATGACGCATTTTTCCGCCTCCCGGCTTCCCATGTCA
ΔofaA R2 CCCAAGCTTAGCAGGTCCTCCTGTTCC (Hind III)
ΔofaA out F AAATCGCTGATCGATGCGC
ΔofaA out R TCCGGGGTGATCCTGAAGG
ΔpltB F1 CTAGTCTAGATCGACTACATCTTCATTCACT (Xba I)
ΔpltB R1 ATCAGCTCGGCCCGTGACAATCCACTCCCGATGATTGCAA
ΔpltB F2 TTGCAATCATCGGGAGTGGATTGTCACGGGCCGAGCTGAT
ΔpltB R2 CCCAAGCTTGTGCAGGGCCACCAGGGAT (Hind III)
ΔpltB out F AAGGCCAAGATGGTGGTGG
ΔpltB out R CGCTTGAGCGCAAGGATCA
ΔphlA F1 CTAGTCTAGAGGAAGTGAGAATGGCTTTA (Xba I)
ΔphlA R1 CTTGCGTAGACAGGCGTAAGTTCATTTTCCTCTTGATTCC
ΔphlA F2 GGAATCAAGAGGAAAATGAACTTACGCCTGTCTACGCAAG
ΔphlA R2 CCCAAGCTTATAGCCGAACTTCTGGAAG (Hind III)
ΔphlA out F TAAACCTCGGCGATCAACG
ΔphlA out R CGCTGTTCTTCAACACTTCC
Δprna F1 CTAGTCTAGACTGGCCCATGACGGCCCG (Xba I)
Δprna R1 TTCCTGAGCCGCGAGCGTGCGGTGCCGCCGCCCACG
Δprna F2 CGTGGGCGGCGGCACCGCACGCTCGCGGCTCAGGAA
Δprna R2 CCCAAGCTTTTCTCGATAAGCTGGGTCGTCC (Hind III)
Δprna out F GCTTCGTGGCCATGGAAAT
Δprna out R CGCAGCAGGACCTTGAAGA
ΔrzxB F1 CTAGTCTAGACGTTGTCTTGCAGCAAGCG (Xba I)
ΔrzxB R1 GCGCTGGTGAAGGACATCAGAGCTCGCGCAGGTAGTGG
ΔrzxB F2 CCACTACCTGCGCGAGCTCTGATGTCCTTCACCAGCGC
ΔrzxB R2 CCCAAGCTTCGGACAAACCTTCATCCCACT (Hind III)
ΔrzxB out F TTGCCATTGGCAATTTTTCTG
ΔrzxB out R CTCGATCAGCACCTGATTG
ΔhcnABC F1 CTAGTCTAGAGCCGTGGTCTGGCTTGAAA (Xba I)
ΔhcnABC R1 GCCAGCGCCTACAGAGCGGTGTGGGTTCATCCGTGAAAA
ΔhcnABC F2 TTTTCACGGATGAACCCACACCGCTCTGTAGGCGCTGGC
ΔhcnABC R2 CCCAAGCTTCATCGACTACCTGGAAACCC (Hind III)
ΔhcnABC out F TGGAAGAAGCCAAGCAGGC
ΔhcnABC out R CTGCCCTTCGAGCATCACT
ΔpFL4656 F1 CTAGTCTAGACCATTTTTCAGCAACCCGCA (Xba I)
ΔpFL4656 R1 GGCGTTCATGGAGGTTTCTGATATCCCTGCCTTGTTGTT
ΔpFL4656 F2 AACAACAAGGCAGGGATATCAGAAACCTCCATGAACGCC
ΔpFL4656 R2 CCCAAGCTTAGGTCTTCTGGATGTGCTCC (Hind III)
ΔpFL4656 out F CTGATCATCGAGCCGATCCA
ΔpFL4656 out R TACAGCAGGATGCCGAGAT
Construct plasmids
gacA F (pBBR) CCCAAGCTTGTTGATAAGGGTGCTAGTAGT (Hind III)
gacA R (pBBR) CGCGGATCCTCAGAGGCTGGCATCAACC (BamH I)
mCherry F (pUCP26) TATGACCATGATTACGAATTCATGGTGAGCAAGGGCGAGG (EcoR I)
mCherry R (pUCP26) ACGACGGCCAGTGCCAAGCTTTTACTTGTACAGCTCGTCCATGCC (Hind
III)
pUCP26 JD F GCGCAACGCAATTAATGTGAG
pUCP26 JD R AACTGTTGGGAAGGGCGATC
Quantify gene expression levels related to cell motility and metabolite
production using qPCR
itu F AGCCGCAATCCTTTATTCG
itu R CAGTCCGTGATAGTTGTC
srf F TCGGCGTTCATTTGGAAG
srf R TCTCTTTATGCTCAGGCG
fen F CTATCTCGGTGCGTTTGA
fen R CCGGTGCAAATACATCGGTA
bae F GAAAAGAGCTTTATCCCG
bae R GCCGCTTATTTAATGCAG
mln F GAGATAATGAGAAAGACGGC
mln R TCCTGCGGTTAATTCTCC
dfn F ATGATTTCCTCAGCGGCAAG
dfn R TTGGCCTTTCGTCCGGATAT
epsB F GGGACTGGCTCAAATATCCG
epsB R ATTTTCCTTCTCCCGGAACG
blsA F GCCTGGTTTTGCTTCTTTCT
blsA R GTATCTTTTGTTGTAGCCGC
fliC F ATCCATCTCTTTCTGTAACTCGTC
fliC R AACGCTCAAGACGGAATCTC
rpsU F GCTCTTCGTCGCTTCAAACGC
rpsU R TTCGCGCTTTCTTGCTTCTTGC
FlgD F TCAGCAATTCGTTTCCAATCTTG
FlgD R CAGCTTTGTCTTCAAGTGCG
FliR F TGACCAGGCATTTCCGTC
FliR R ATTTTCAGAGGAAGCCCGAC
Quantify Pseudomonas gene copy number using qPCR
fPs16S F ACTGACACTGAGGTGCGAAAGCG
rPs23S R ACCGTATGCGCTTCTTCACTTGACC
Quantify Bacillus gene copy number using qPCR
Bacillus F ATGTTAGCGGCGGACGGGTGAG
Bacillus R AAGTTCCCCAGTTTCCAATGACC
Amplification of 16S rRNA gene fragments for amplicon
515 F GTGCCAGCMGCCGCGGTAA
806 R GGACTACHVGGGTWTCTAAT
[182]Open in a new tab
Underlined sites indicate restriction enzyme cutting sites added for
cloning. Letters in boldface denote the annealing regions for overlap
PCR.
Tagging Pseudomonas and Bacillus with fluorescent proteins
To visualize the development of bacterial strains co-cultivated on a
solid agar surface, Pf-5 and its mutants were tagged with mCherry red
fluorescent protein. The pBBR-mCherry plasmid was transformed into E.
coli Top10, and the recombinant Pf-5/pBBR-mCherry, ΔpltB/pBBR-mCherry,
ΔgacA/pBBR-mCherry strains were generated by triparental mating of
Top10/pBBR-mCherry (donor), HB101/pRK-2013 (helper), and the Pf-5,
ΔpltB and ΔgacA strains of interest (recipient) ^[183]15,[184]43. In
the same way, the mCherry fragment was amplified from the pBBR-mCherry
plasmid to construct pUCP26-mCherry. This plasmid was then transformed
into E. coli Top10 and the recombinant ΔgacA-C/pUCP26-mCherry
(ΔgacA-C-mcherry) strain was generated according to the method
described above. DMW1 was labeled with green fluorescence protein gfp
by transfer of the pAD43-25 plasmid^[185]23.
Detection of competition ability
The effect of P. protegens on bioactivity toward Bacillus spp. was
determined in an inhibition assay on a solid medium. Cultures of
Bacillus spp., as well as P. protegens Pf-5, were adjusted to an OD[600
nm] = 1.0. Five μL of the P. protegens Pf-5 culture were placed onto
1.0% LB agar supplemented with 1.0% (v/v) of Bacillus sp. culture. The
activity of P. protegens Pf-5 toward Bacillus spp. was determined as
described above. The LB agar plates were incubated at 30 °C for 48 h,
followed by measuring the size of inhibition zones.
For bacterial co-cultivations, 1 mL cultures of P. protegens Pf-5 or
its mutants labeled with mCherry, and B. velezensis DMW1 labeled with
gfp were washed twice in sterilized water by centrifuging at 5000 rpm
for 4 min, followed by discarding the supernatant and resuspending the
pellet in sterilized water without the antibiotic. Subsequently, the
optical density at 600 nm (OD [600 nm]) was adjusted to 1.0 (Bacillus)
and 0.1 (Pseudomonas). For co-cultivations on agar surfaces, lines were
drawn by streaking out P. protegens Pf-5 or its mutants labeled with
mCherry, crossed by B. velezensis DMW1 labeled with gfp. The incubation
time varied from 24 to 72 h, at 30 °C. Fluorescent colonies were imaged
by fluorescent stereo microscopy (Nikon SMZ25, Nikon, Japan). Due to
the excitation wavelength for red fluorescent protein being close to
that of green protein, strong excitation (at 488 nm) can simultaneously
excite both types of fluorescence.
To determine the colony number of the bacteria, labeled with different
fluorescent proteins, after contacting each other, the bacterial disks
were taken at the midpoint of the segment formed by the intersection of
two perpendicular lines after 48 h. Three beads (φ = 3 mm) were put in
each tube, which was then filled with 1 mL of sterilized water. The
bacterial disks in the sterilized water were subjected to grinding for
30 sec at 60 Hz using tissuelyser-64 (Shanghai Jingxin Technology). The
grinding steps were repeated twice, and then the bacteria in the medium
were released and diluted in sterilized water. According to the
gradient dilution method, the bacterial suspensions were then spread on
KB medium plates containing ampicillin (100 µg/mL) and gentamicin
(25 µg/mL) for Pf-5, ΔgacA and ΔpltB labeled with mCherry, or
ampicillin (100 µg/mL) and tetracycline (25 µg/mL) for ΔgacA-C labeled
with mCherry. LB medium plates containing chloramphenicol (5 µg/mL)
were used for B. velezensis DMW1 labeled with gfp. The plates were
incubated at 30 °C (Pseudomonas), or 37 °C (Bacillus) for 12 h, and
then the number of colonies was counted and statistically analyzed.
Crude extract preparation
To extract secondary metabolites of B. velezensis or P. protegens
strains, bacteria were cultured by inoculating a single colony in 20 mL
LB medium and incubated overnight at 37 °C under shaking (200 rpm).
Then, the bacteria were further cultivated in 200 mL of LB medium for
24 h. Afterward, the pre-treated XAD16 resin was added and adjusted to
2.5%, and the mixture was incubated for another 24 h. Centrifuge at
8000 rpm for 15 min at room temperature, discard the supernatant,
resuspend the mixed resin precipitate with 30–40 mL of methanol, and
place it at 37 °C at 200 rpm for 4 h, then centrifuge at 8000 rpm at
room temperature for 15 min. The supernatant was dissolved in methanol,
and the crude extract was obtained.
Purification of iturin, surfactin, and fengycin
For the purification of iturin, surfactin, and fengycin, crude extract
sample was dissolved in a methanol solution and subjected to
purification through preparative high-performance liquid chromatography
(HPLC) using a C18 column (5 µm, 100.0 mm × 21.2 mm; flow rate:
10 mL/min; gradient: 20–100% MeOH/H[2]O in 30 min, followed by 100%
MeOH in 10 min) to generate sub-fractions. The distinctive peaks in the
DMW1 sub-fraction underwent semipreparative HPLC (C18 column, 5 µm;
250.0 mm × 10.0 mm; flow rate: 3 mL/min; with 0.1% formic acid in 54%
MeOH/H[2]O in 53 min, 65% MeOH/H[2]O in 53–70 min) to obtain a pure
compound. NMR spectra were recorded in methanol-d4 on a Bruker AVANCE
II 400 MHz instrument, and high-resolution mass spectra were acquired
using an Agilent 6530 Accurate-Mass Q-TOF LC/MS coupled to an Agilent
1260 HPLC.
Inhibition assay
The effect of B. velezensis DMW1, and its secondary metabolites
extracts on P. protegens Pf-5 was determined in an inhibition assay on
a solid medium. Cultures of B. velezensis DMW1, as well as P. protegens
Pf-5 were normalized to an optical density at 600 nm (OD[600 nm]) of
1.0. Five μL of the culture of DMW1 or its secondary metabolites
extracts were applied to a paper disk (φ = 5 mm) and then placed onto
LB agar containing 1.0% (v/v) of P. protegens Pf-5 culture. The
activity of P. protegens Pf-5 or its secondary metabolites extracts
towards B. velezensis DMW1 was determined using the same method. Plates
were incubated at 30 °C for 48 h, followed by measuring the size of
inhibition zones.
Construction of deletion mutants in Pf-5 genes involved in the synthesis and
regulation of antimicrobial metabolites
To explore the effect of secondary metabolites of P. protegens Pf-5 on
the activity of B. velezensis DMW1, several Pseudomonas deletion
mutants were generated by triparental mating according to the
description above^[186]15,[187]43. Mutants of P. protegens Pf-5 were
created by the triparental mating. Briefly, the upstream (770 bp) and
downstream (770 bp) fragments were amplified by PCR, fused via overlap
PCR, and introduced into the pK18mobsacB vector. The recombinant
plasmid was transferred from E. coli Top10 to P. protegens Pf-5, with
the help of E. coli Top10 (pRK-2013)^[188]15,[189]43. The following
mutations were introduced: gacA (secondary metabolite regulatory
system), ofa (orfamide A biosynthetic gene), pltB (pyoluteorin
biosynthetic gene), phlA (2,4-diacetylphloroglucinol biosynthetic
gene), prnA (pyrrolnitrin biosynthetic gene), rzxB (rhizoxin
biosynthetic gene), hcnABC (VOC hydrogen cyanide biosynthetic gene),
pFL4656 (non-ribosomal peptide biosynthetic gene).
Purification of pyoluteorin
For the purification of pyoluteorin, high-performance liquid
chromatography (HPLC, Waters), equipped with a reverse-phase column
(ZORBAX SB-C18) was performed. The running program was a gradient
elution from 5% solvent A (HPLC-grade acetonitrile containing 0.1%
trifluoroacetic acid), 95% solvent B (Milli-Q water containing 0.1%
trifluoroacetic acid) to 95% A, 5% B for 20 min. A concentration of 95%
solvent A and 5% solvent B were then held for 5 min. A gradient elution
from 95% solvent A, 5% solvent B to 5% A, 95% B for 1 min; A
concentration of 95% solvent A and 5% solvent B were then held for
2 min. For the purification of pyoluteorin, the semipreparative HPLC
was used with a flow rate of 4 mL/min and an injection volume of 1 mL.
Pyoluteorin was done under UV absorption at 310 nm. In the last step,
purified compounds were identified via ultraperformance liquid
chromatography-mass spectrometry (UPLC-MS).
RNA-seq
B. velezensis DMW1 was grown as a single culture or co-cultured with P.
protegens Pf-5 or ΔpltB, respectively. Two mL of the culture,
containing either the single strain or a mix of B. velezensis DMW1
(OD[600 nm] = 1.0) and P. protegens (OD[600 nm] = 0.1) (volume
ratio = 19:1), were used for inoculation of 200 mL medium as described
previously^[190]26. The cultures were grown at 37 °C under shaking
(200 rpm) for 12 h. The bacteria were collected for total RNA
extraction and RNA-seq by the DNBSEQ platform (BGI Genomics Co., Ltd.,
China). The reads were mapped to the DMW1 genome v1.1
([191]https://www.ncbi.nlm.nih.gov/nuccore/NZ_CP114180.1) using HISAT2.
Utilizing the SOAPnuke tool, the expression levels of individual DMW1
genes were quantified through normalization to the “Fragments Per
Kilobase of exon per Million mapped reads (FPKM)” metric, enabling the
identification of differentially expressed genes (DEGs). The genes with
at least two-fold change and p-value ≤ 0.05 were considered DGEs
between the two samples. Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway enrichment analysis of DEGs was performed using the “KEGG
enrichment analysis” tool embedded in the BGI platform
([192]https://report.bgi.com/ps/mrna/index.html).
RNA isolation and RT-qPCR
To investigate srf, itu, fen, mln, bae, dfn, flgD, and fliR gene
expression in Bacillus, firstly RNA extraction was carried out using
the Bacteria Total RNA Kit (ZP403) (Zoman Biotechnology Co., Ltd;
Beijing) following the Gram-positive manufacturer’s protocol. RNA
quality and quantity were performed with a Thermo Scientific NanoDrop
2000 UV-vis Spectrophotometer. Primer 3 program available online was
used for primer design and primers were synthesized by GenScript. The
primers used for this purpose are listed in Table [193]5.
Reverse transcription-polymerase chain reaction (RT-qPCR) was performed
to quantify gene expression of itu (iturin synthesis), fen (fengycin
synthesis), srf (surfactin synthesis), bae (bacillaene synthesis), mln
(marcolactin synthesis), dfn (difficidin synthesis), flgD (flagellar
hook assembly protein FlgD) and fliR (flagellar type III secretion
system protein FliR)^[194]24. Reverse transcriptase and RT-qPCR
reactions were conducted using the HiScript III 1st strand cDNA
Synthesis Kit (+gDNA) (Vazyme, China). The qPCR reaction was performed
in a total volume of 20 μL: 10 μL of ChamQ Universal SYBR qPCR Master
Mix, 0.4 μL of each primer (5 μM), 2 μL of cDNA (100 ng), 7.2 µL of
Nuclease-free water. The thermal cycling program applied on the ABI
StepOne was: preheating at 95 °C for 30 s, (95 °C for 5 s and 60 °C for
30 s) × 40 cycles, and the specificity of the PCR product amplification
was verified based on the Tm value for 15 s at 95 °C and 1 min at
60 °C. Finally, the qPCR amplification was run on the ABI StepOne qPCR
instrument (Applied Biosystems) with software version 2.3. The relative
gene expression analysis was conducted using the 2^–ΔΔCT method with
the rpsU gene as a housekeeping gene to normalize mRNA levels between
different samples^[195]44.
Analysis of secondary metabolites with HPLC
Using a C18 reversed-phase column (ZORBAX SB-C18), we performed a
gradient elution starting from 30% solvent A (HPLC-grade acetonitrile
with 0.1% trifluoroacetic acid) to 70% solvent B (Milli-Q water with
0.1% trifluoroacetic acid) over a period of 30 min. This was followed
by a gradient change to 95% solution A and 5% solution B, maintained
for 10 min. The flow rate was set at 1 mL/min. Secondary metabolites
were detected at UV wavelengths of 210 nm and 280 nm, respectively, and
the peak area represented the production.
Biofilm in analysis Bacillus–Pseudomonas interactions
For the biofilm assay, 200 μL overnight culture was transferred into a
well containing 20 mL liquid LB medium. After incubation at
30 °C/37 °C, 200 rpm for OD[600 nm] = 1.0, 10 μL B. velezensis DMW1
(OD[600 nm] = 1.0) and 10 μL P. protegens Pf-5/ΔpltB (OD[600
nm] = 0.25) culture or sterilized water was added into 2 mL fresh LBGM
medium (LB supplemented with 1% glycerol and 100 μM MnSO[4]) in a flat
bottom 12-well microplate^[196]45. To visualize the distribution of two
bacteria in the cultures, the culture was examined with a Zeiss LSM 700
microscope, equipped with a 20× objective. GFP fluorescence was excited
at 488 nm and detected between 500 and 530 nm; for mCherry, excitation
was at 561 nm with emission detection from 580 to 620 nm after
incubation at 30 °C for 24 h without shaking. Biofilm-related gene
expression level among the different treatments was analyzed at the
transcriptional level by RT-qPCR.
Construction of standard curve for qPCR
After ligating the target fragment into the pMD-18T vector, the
resulting construct was transformed into DH5α competent cells. Plasmids
from the transformants were extracted and then diluted in a 10-fold
gradient. These diluted plasmids were subsequently used as templates
for fluorescence quantitative PCR amplification. The logarithm of the
initial copy number of the template was plotted on the x-axis, while
the number of cycles reaching the threshold (Threshold cycle, Ct) was
plotted on the y-axis, thus generating a standard curve. The CT number
was introduced into the standard curve equation to calculate the
initial gene copy number of the sample to reveal the gene copy number
per gram of dry soil^[197]31.
Root colonization assay
To determine the colony number of tomato root colonizing B. velezensis
DMW1 and P. protegens Pf-5/ΔpltB in the rhizosphere soil,
thirty-day-old tomato-plant seedling roots were dipped into the
bacterial suspension containing 5 × 10^7 cells/mL Bacillus and 3 × 10^8
cells/mL Pseudomonas for 40 min. Afterward, all seedlings were
transplanted into pots (10 cm in diameter) containing 200 g sterilized
peat soil from Danish Pindstrup company (located in the Kingdom of
Denmark), and incubated in the greenhouse (30 °C, 75% relative
humidity, 14 h light/10 h darkness). Rhizosphere soil samples from
these treatments were collected 7 d after transplanting. Five tomato
roots were collected from the points of each plot and vigorously shaken
to remove excess soil. Then, the soil adhering to the roots
(rhizosphere soil) was suspended in phosphate-buffered saline (PBS).
The rhizosphere soil suspensions were centrifuged at 10,000 × g for
10 min, and the sediments were stored at −80 °C for later DNA
extraction. Soil samples from the pot experiments were chosen for
subsequent DNA extraction using Power Soil DNA Isolation Kits (MoBio
Laboratories Inc., Carlsbad, CA, USA) following the manufacturer’s
protocol. The concentration and quality of the DNA were determined
using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington,
NC, USA). The abundance of Pseudomonas spp. was quantified by
quantitative polymerase chain reaction (qPCR) with specific
primers^[198]46. The abundance of Bacillus spp. was quantified by qPCR
with specific primers^[199]47.
For imaging of B. velezensis DMW1/pAD43-25-gfp, and P. protegens Pf-5
or ΔpltB/pBBR-mCherry colonization in the tomato root,
surface-sterilized tomato seeds with roots 10 mm long (3 d) were
inoculated with suspensions of B. velezensis DMW1 (5 × 10^7 cells), and
P. protegens Pf-5/ΔpltB (3 × 10^8 cells). After incubation for 30 min
at 25 °C and shaking with 100 rpm, seedlings were transferred onto
square Petri dishes (12 × 12 cm) containing 0.5× Murashige and Skoog
(MS) semisolid agar medium (0.7% agar) without sucrose^[200]48. The
square plates were kept in a vertical position during the incubation
time of seven days at 25 °C under long daylight conditions (16 h
light/8 h darkness) in a plant-growth chamber. To determine the spatial
pattern of root colonization by DMW1/pAD43-25-gfp and Pf-5/pBBR-mCherry
or ΔpltB/pBBR-mCherry, the root system (ca. 10 mm long) was imaged
using a Zeiss LSM 700. GFP fluorescence was excited at 488 nm and
detected between 500 and 530 nm; for mCherry, excitation was at 561 nm
with emission detection from 580 to 620 nm. To determine the root
colonization by B. velezensis DMW1 and P. protegens Pf-5/ΔpltB, the
root system (ca. 10 mm long) was subjected to grind for 30 sec at 60
Hertz in a 2 mL tube containing 1 mL of distilled water and three beads
(φ = 3 mm) using tissuelyser-64 (Shanghai Jingxin Technology). Using
the gradient dilution method, the diluted bacterial suspensions were
placed onto LB plates. The plates were incubated at 30 °C/37 °C for
12 h to count the number of colonies.
Disease control
Thirty-day-old tomato seedlings (variety cultivar ‘Mao fen’) roots were
inoculated with either: 10 mL 1 × 10^7 CFU/mL DMW1, or 10 mL 1 × 10^8
CFU/mL Pf-5, or 10 mL 1 × 10^8 CFU/mL ΔpltB culture, or a mix of DMW1
and Pf-5 or ΔpltB. Then, 10 mL 5 × 10^7 CFU/mL R. solanacearum
suspension was added. Each treatment was repeated in triplicate, and
every repeat contained 15 seedlings. All seedlings were maintained in
the greenhouse (30 °C, 75% relative humidity, 14 h light/10 h
darkness), and inspected periodically until disease symptoms appeared.
The disease severity was evaluated according to an empirical scale:
Level 0 = tomato plants without visible symptoms; Level 1 = striped
necrosis on stems occasionally or less than half of the leaves wilted
on unilateral stems; Level 2 = black streaks less than half the height
of the stem or between half to two-thirds of the leaves wilted on
unilateral stems; Level 3 = more than two-thirds of the leaves wilted
on unilateral stems; Level 4 = the plant is dead^[201]3. The disease
severity index was calculated according to the following formula:
[MATH: Diseaseseverityindex(DSI)=[Σ(x×y)/
(z×4)
]×100 :MATH]
1
[MATH: Diseasecontroleffect(%)=(DSITreatment−
DSIControl)
/DSIControl×100% :MATH]
2
Where: x = number of different degrees infected plants in the
treatments; y = relative degree value; and z = number of total plants
in the treatments.
Inoculation of tomatoes and sample collection
Tomato seedlings (variety cultivar ‘Mao fen’) grown in natural soil
were inoculated with 5% (v/DW) P. protegens in order to explore the
effect of Pseudomonas on the microbial community structure. The sample
of rhizosphere-associated microbiota was taken four weeks after the
transplantation of tomato seedlings, ensuring that the root microbiota
had been well established during this period^[202]49. From each
treatment, five root samples from five representative plants were
harvested. Rhizosphere soil samples and the corresponding DNA extracts
were investigated by using PowerSoil Soil DNA Isolation Kits (MoBio
Laboratories Inc., Carlsbad, CA, USA) following the manufacturer’s
protocol.
Illumina MiSeq sequencing
We generated the bacterial community profiles for each sample via PCR
amplification of the 16S ribosomal RNA (rRNA) gene targeting regions V4
region using primers 515F and 806R (listed in Table [203]5), followed
by Illumina sequencing at Personal MAGIAENE Co. Ltd, Guangdong, China.
The raw sequence data was processed using the QIIME2 pipeline, and the
operational taxonomic unit (OTU) table was constructed with the UPARSE
pipeline^[204]50. Briefly, reads were truncated at 200 bp and were
quality-filtered. After discarding replicates and singletons, the
remaining reads were assigned to OTUs at a 97% identity threshold. We
obtained 2,219,565 high-quality sequences from 15 samples. High-quality
reads were then analyzed with USEARCH, where chimeric and organelle
sequences were removed.
Statistical analysis
For statistical analyses, the software GraphPad PRISM 9.0 with an
unpaired T-test was performed. Further, the RStudio 4.3 statistical
software environment (R language version 4.3) was used for multiple
comparisons. For the drawing of the HPLC peak diagram, Origin 2022 was
used. Network analysis was carried out using Gephi 0.10. The screening
for differential ASV was performed by using the STAMP software.
Supplementary information
[205]Supplementary Material^ (522KB, pdf)
Acknowledgements