Content uploaded by Maedeh Kamali
Author content
All content in this area was uploaded by Maedeh Kamali on Jan 14, 2022
Content may be subject to copyright.
Citation: Kamali, M.; Guo, D.;
Naeimi, S.; Ahmadi, J. Perception of
Biocontrol Potential of Bacillus
inaquosorum KR2-7 against Tomato
Fusarium Wilt through Merging
Genome Mining with Chemical
Analysis. Biology 2022,11, 137.
https://doi.org/10.3390/biology
11010137
Academic Editor: Bernard R. Glick
Received: 6 December 2021
Accepted: 10 January 2022
Published: 14 January 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biology
Article
Perception of Biocontrol Potential of Bacillus inaquosorum
KR2-7 against Tomato Fusarium Wilt through Merging
Genome Mining with Chemical Analysis
Maedeh Kamali 1, Dianjing Guo 2, *, Shahram Naeimi 3and Jafar Ahmadi 4
1College of Veterinary Medicine and Life Sciences, City University of Hong Kong,
Hong Kong, China; mkamali@cityu.edu.hk
2State Key Laboratory of Agrobiotechnology and School of Life Sciences,
Chinese University of Hong Kong, Hong Kong, China
3Department of Biological Control Research, Iranian Research Institute of Plant Protection,
Agricultural Research, Education and Extension Organization (AREEO),
Tehran 19858-13111, Iran; sh.naeimi@areeo.ac.ir
4Department of Genetics and Plant Breeding, Imam Khomeini International University,
Qazvin 34149-16818, Iran; j.ahmadi@eng.ikiu.ac.ir
*Correspondence: djguo@cuhk.edu.hk; Tel.: +852-3943-6298
Simple Summary:
Bacillus is a bacterial genus that is widely used as a promising alternative to
chemical pesticides due to its protective activity toward economically important plant pathogens.
Fusarium wilt of tomato is a serious fungal disease limiting tomato production worldwide. Recently,
the newly isolated B. inaquosorum strain KR2-7 considerably suppressed Fusarium wilt of tomato
plants. The present study was performed to perceive potential direct and indirect biocontrol mech-
anisms implemented by KR2-7 against this disease through genome and chemical analysis. The
potential direct biocontrol mechanisms of KR2-7 were determined through the identification of genes
involved in the synthesis of antibiotically active compounds suppressing tomato Fusarium wilt.
Furthermore, the indirect mechanisms of this bacterium were perceived through recognizing genes
that contributed to the resource acquisition or modulation of plant hormone levels. This is the first
study that aimed at the modes of actions of B. inaquosorum against Fusarium wilt of tomatoes and
the results strongly indicate that strain KR2-7 could be a good candidate for microbial biopesticide
formulations to be used for biological control of plant diseases and plant growth promotion.
Abstract:
Tomato Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (Fol), is a destructive
disease that threatens the agricultural production of tomatoes. In the present study, the biocon-
trol potential of strain KR2-7 against Fol was investigated through integrated genome mining and
chemical analysis. Strain KR2-7 was identified as B. inaquosorum based on phylogenetic analysis.
Through the genome mining of strain KR2-7, we identified nine antifungal and antibacterial com-
pound biosynthetic gene clusters (BGCs) including fengycin, surfactin and Bacillomycin F, bacillaene,
macrolactin, sporulation killing factor (skf), subtilosin A, bacilysin, and bacillibactin. The correspond-
ing compounds were confirmed through MALDI-TOF-MS chemical analysis. The gene/gene clusters
involved in plant colonization, plant growth promotion, and induced systemic resistance were also
identified in the KR2-7 genome, and their related secondary metabolites were detected. In light of
these results, the biocontrol potential of strain KR2-7 against tomato Fusarium wilt was identified.
This study highlights the potential to use strain KR2-7 as a plant-growth promotion agent.
Keywords:
Fusarium wilt; biocontrol; B. inaquosorum KR2-7; genome mining; gene clusters; MALDI-
TOF-MS; secondary metabolites
Biology 2022,11, 137. https://doi.org/10.3390/biology11010137 https://www.mdpi.com/journal/biology
Biology 2022,11, 137 2 of 26
1. Introduction
Tomato Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici (Fol), is one of
the most destructive diseases, causing a considerable loss in the production of both field
and greenhouse tomatoes worldwide [
1
]. Since Fusarium wilt is a difficult disease to con-
trol [
2
–
4
], control strategies including physical and cultural methods, chemical fungicides
treatment, and the cultivation of resistance tomato cultivars [
5
] achieved limited efficacy [
6
].
In addition, excessive usage of agrochemicals imposed serious negative impacts on the
environment, causing the pollution of soil and groundwater reservoirs, an accumulation of
chemical residues in the food chain, an emergence of pesticide-resistance pathogens, and
health hazards [
7
]. As a result, biocontrol microbes have been suggested as a promising
alternative to agrochemicals in plant disease control. Numerous biocontrol microbes, espe-
cially Bacillus strains, have been commercially developed as biopesticides and biofertilizers
worldwide [7].
Biocontrol microbes protect the crops from an invasion of phytopathogens via (1) direct
modes of action, e.g., the antibiosis and production of antimicrobial secondary metabo-
lites [
8
,
9
]; and (2), the indirect modes of action, including induced systemic resistance
(ISR) and the competition for nutrients and space [
10
,
11
]. The investigation of biocontrol
microbes through conventional genetic and biochemical approaches could not unveil the
full potential of these microbes due to the absence of appropriate natural triggers or stress
signals under laboratory conditions [
12
]. With the development of high-throughput DNA
sequencing technologies and genome mining, along with MS-based analytical methods (e.g.,
GC/LC-MS, LC-ESI-MS, and MALDI-TOF-MS), more potential biocontrol microbes can
be revealed. For instance, the Bacillus amyloliquefaciens FZB42 genome contains nine giant
gene clusters synthesizing secondary metabolites which are involved in the suppression of
soil-born plant pathogens. Several gene/gene clusters are implicated in swarming motility,
plant colonization, biofilm formation, and the synthesis of plant growth-promoting volatile
compounds and hormones [
13
]. A wide range of extracellular proteins and phytase in the
FZB42 secretome were detected through two-dimensional electrophoresis, MALDI-TOF-
MS, and the proteomics approach, indicating this strain can grow on the plant’s surface and
supply phosphorus for the plant under phosphorus starvation. Additionally, four members
of the macrolactin family were identified in an FZB42 culture filtrate by combining mass
spectrometric and ultraviolet-visible data which perfectly agree with the overall structure
of the macrolactin gene cluster found in the FZB42 genome [
13
]. Recently, the genome
analysis of plant-protecting bacterium B. velezensis 9D-6 demonstrated that this strain can
synthesize 13 secondary metabolites, of which surfacin B and surfactin C were detected
as antimicrobial compounds against Clavibacter michiganensis through LC-MS/MS [
14
].
Furthermore, the genome mining of B. inaquosorum strain HU Biol-II revealed that this
bacterial genome contains eight bioactive metabolite clusters and the production of seven
metabolites was confirmed through HPLC MS/MS [15].
In our previous study, the B. inaquosorum strain KR2-7 was isolated from the rhizo-
sphere soil of the tomato (Solanum lycopersicum) and was introduced as a highly effective
biocontrol agent against Fol with a biocontrol efficiency of 80% under greenhouse condi-
tions [
16
]. To better understand the biocontrol mechanisms of strain KR2-7 against Fol,
whole-genome sequencing was conducted to identify putative gene clusters for secondary
metabolites biosynthesis and to characterize gene/gene clusters involved in plant coloniza-
tion, plant growth promotion, and induced systemic resistance (ISR). Moreover, secondary
metabolites and other compounds related to identified BGCs and gene/gene clusters were
detected using MALDI-TOF-MS analysis to confirm the results of genome mining.
2. Materials and Methods
2.1. Strains and Culture Conditions
The fungal pathogen Fol strain Fo-To-S-V-1 used in this study was obtained from
the culture collection from the Iranian Research Institute of Plant Protection. The fungus
Biology 2022,11, 137 3 of 26
was maintained on a potato dextrose agar (PDA, Merck, Germany) slant at 4
◦
C and was
sub-cultured onto a fresh PDA plate at 27 ◦C for 7 days for further tests.
Strain KR2-7 was maintained on nutrient agar (NA, Merck, Germany; with a 0.3% beef
extract, 0.5% peptone, and 1.5% agar) plate with a periodic transfer to a fresh medium. For
long-term storage, it was kept at
−
80
◦
C in lysogeny broth (LB, Merck, Germany) with
20% glycerol (v/v).
2.2. Dual Culture Assay
In order to investigate the antagonism efficiency of strain KR2-7 against various
tomato pathogens, five destructive fungal pathogens, including Alternaria alternata f. sp.
lycopersici,Athelia rolfsii,Botrytis cinerea,Rhizoctonia solani, and Verticillium albo-atrum, were
selected. The antifungal activity of strain KR2-7 against each pathogen was evaluated
through a dual culture assay in three replications. In the dual culture assay, strain KR2-7
was simultaneously cultured 3cm apart from the 5-mm plug of a pathogen in a 9 cm PDA
plate. The control plate was inoculated only with the pathogen. Plates were incubated at
27
◦
C. The fungal growth was checked daily by measuring the diameter of the colony for a
period of three days. The percentage of fungal growth inhibition (PFGI) was calculated
by the formula (1) developed by Skidmore and Dickinson [17], where R1 is the maximum
radius of the growing fungal colony in the control plate, and R2 is the radius of the fungal
colony that grew in the presence of strain KR2-7:
PFGI = ((R1−R2)/R1)×100 (1)
2.3. MALDI-TOF-MS Analysis of KR2-7 Secondary Metabolites
The secondary metabolite analysis was performed from the whole-cell surface extract
of bacterium obtained during the dual culture of KR2-7 and Fol. The bacterial surface
extract was prepared according to the methodology described by Vater et al. [
18
]. The Dual
culture was done on potato dextrose agar (PDA) instead of Landy agar. Strain KR2-7 was
streaked on one side of the plate and a 5 mm plug of Fol was placed on the opposite side
simultaneously and incubated at 27
◦
C. After 24 h, two loops of bacterial cells from the
interface of the bacterium-fungus in the inhibition zone were suspended in 500 µL of 70%
acetonitrile with 0.1% trifluoroacetic acid for 2 min. The suspension was gently vortexed to
produce a homogenized suspension. The bacterial cells were pelleted by centrifuging at
5000 rpm for 10 min. The cell-free supernatant was transferred to a new microcentrifuge
tube and stored at 4
◦
C for further analysis. One microliter of supernatant liquid was
spotted onto the target of the mass spectrometer with an equal volume of
α
-cyano-4-
hydroxycinnamic acid (CHCA) matrix and was air-dried. The sample mass fingerprints
were obtained using an ultrafleXtreme MALDI-TOF/TOF-MS (Bruker, Billerica, MA, USA)
within a mass range of 100–3000 Da. The MALDI-TOF-MS analysis was performed at
the school of life sciences, Chinese University of Hong Kong (CUHK), Hong Kong. The
whole-cell surface extract of strain KR2-7 grown on a potato dextrose agar was used as a
control.
2.4. Genome Sequencing, Assembly, and Annotation
The genomic DNA of strain KR2-7 was extracted using a commercial DNA extraction
kit (Thermo Fisher Scientific, Waltham, MA, USA). The whole-genome sequencing was
performed using the Illumina HiSeq 4000 and PacBio RSII platforms (BGI, Shenzhen, China).
The quality control of raw sequences was performed by FastQC v0.11.9 and the de novo
assembly was done using SPAdes v3.14.1. The genome was annotated using the NCBI
Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP), and Bacterial Annotation
System (BASys) webserver. The proteome of KR2-7 was subjected to BLASTP against
the Cluster of Orthologous Group (COGs) database at E-value < 1
×
10
−5
to identify the
Cluster of Orthologous Groups (COGs) [19].
Biology 2022,11, 137 4 of 26
2.5. Genome Phylogeny
In this study, 32 Bacillus strains belonging to various species were selected among
those recorded in the NCBI GenBank database. For all the selected strains, the nucleotide
and the corresponding amino acid sequences were retrieved from the GenBank database.
Whole-genome alignments were performed using REALPHY (http://realphy.unibas.ch
(accessed on 22 December 2021); [
20
]) and the phylogenetic tree was constructed using
the MEGA v. 7 [
21
] by maximum likelihood method [
22
], with evolutionary distances
computed using the general time-reversible model [
23
]. Branch validity was evaluated by
the bootstrap test with 1000 replications. The average nucleotide identity (ANI) values
of selected Bacillus strains were calculated using the server EzBioCloud (http://www.
ezbiocloud.net/tools/ani (accessed on 21 August 2021); [24]). According to the algorithm
developed by
Goris et al. [25]
, 95
∼
96% cut-off value was used for the species boundary [
26
].
The web-based DSMZ service (http://ggdc.dsmz.de (accessed on 7 January 2022); [
27
])
with 70% species and sub-species cut-off was used to estimate the in silico genome-to-
genome distance values for the selected strains.
2.6. Pathway Analysis
The annotated genome was analyzed using KEGG (Kyoto Encyclopedia of Genes
and Genomes) to determine the existing pathways, which were then manually validated
through matching the assigned gene functions to the corresponding KEGG pathway.
2.7. Genome-Wide Identification of Secondary Metabolite Biosynthesis Gene Clusters
The antibiotics and secondary metabolite analysis shell (antiSMASH) is a compre-
hensive resource that allows the automatic genome-wide identification and analysis of
secondary metabolite biosynthesis gene clusters in bacterial and fungal
genomes [28,29]
.
Thereby, the KR2-7 genome was submitted to the antiSMASH web server (https://antismash.
secondarymetabolites.org) (accessed on 22 December 2021) to detect the putative BGCs for
secondary metabolites. Each identified BGC in the KR2-7 genome was aligned against
corresponding BGC in B. subtilis strain 168 and B. amyloliquefacience stain FZB42 using
Geneious Prime v.2021.2.2. to find out the BGCs similarity between KR2-7, 168 and, FZB42.
3. Results
3.1. General Genomic Features of Strain KR2-7
The assembled genome of B. inaquosorum KR2-7 contained 4 contigs, with an N50
of 2,144,057 bp and 700X sequence coverage. The KR2-7 genome was obtained with a
length of 4,248,657 bp, the G+C content of 43.1%, and 4265 predicted genes consisting
of 4017 protein-coding genes, 50 rRNA genes, and 83 tRNA genes. Interestingly, strain
KR2-7 possesses the larger number of genes contributing to amino acid transport and
metabolism (322 genes), carbohydrate transport and metabolism (278 genes), inorganic ion
transport and metabolism (200 genes), and secondary metabolites biosynthesis, transport
and catabolism (76) compared to the reputable biocontrol agent B. velezensis strain FZB42
(Figure S1; Table S1). Therefore, the genome content of KR2-7 indicates that the strain has
considerable potential as a biocontrol agent. The genome sequence of B. inaquosorum KR2-7
was deposited in NCBI GenBank under the accession number QZDE00000000.2.
3.2. Genome Phylogeny
The genomes of 31 Bacillus strains were selected for aligning with the KR2-7 genome
and phylogenomic analysis. The selected strains and their corresponding genome sequence
accession numbers were presented in Table 1. Selected Bacillus strains were accurately
distributed on branches of the maximum likelihood phylogenomic tree (Figure 1).
Biology 2022,11, 137 5 of 26
Table 1.
Average nucleotide identity (ANI) and Genome-to-Genome Distance Calculation (GGDC)
values between each selected Bacillus strain and strain KR2-7.
Species Strain Accession Number ANI (%) GGDC
Bacillus altitudinis P-10 NZ_CP024204.1 71.39 0.2368
Bacillus amyloliquefaciens
B15 NZ_CP014783.1 77.31 0.2087
CC178 NC_022653.1 77.35 0.2082
FZB42 NC_009725.1 77.65 0.2081
L-H15 NZ_CP010556.1 77.38 0.2104
L-S60 NZ_CP011278.1 77.37 0.2101
S499 NZ_CP014700.1 77.36 0.2097
Y2 NC_017912.1 77.43 0.2086
Bacillus atrophaeus GQJK17 NZ_CP022653.1 80.4 0.1895
UCMB-5137 NZ_CP011802.1 80.3 0.192
Bacillus cellulasensis GLB197 NZ_CP018574.1 71.45 0.2368
Bacillus flexus KLBMP 4941 NZ_CP016790.1 68.97 0.1585
Bacillus megaterium YC4-R4 NZ_CP026736.1 68.87 0.1619
Bacillus muralis G25-68 NZ_CP017080.1 68.82 0.1484
Bacillus mycoides Gnyt1 NZ_CP020743.1 68.46 0.1317
Bacillus oceanisediminis 2691 NZ_CP015506.1 69.26 0.1506
Bacillus paralicheniformis MDJK30 NZ_CP020352.1 73.09 0.2242
Bacillus pumilus SAFR-032 NC_009848.4 71.3 0.2341
Bacillus pumilus TUAT1 NZ_AP014928.1 71.2 0.2366
Bacillus sp. B25 (2016b) CP016285.1 68.41 0.177
WP8 NZ_CP010075.1 71.11 0.2347
Bacillus subtilis
BSn5 NC_014976.1 93.06 0.0706
HJ5 NZ_CP007173.1 93.1 0.0703
XF-1 NC_020244.1 93.01 0.0707
Bacillus subtilis subsp. inaquosorum KCTC 13429 NZ_CP029465.1 99.26 0.0075
Bacillus subtilis subsp. inaquosorum DE111 NZ_CP013984.1 98.8 0.01222
Bacillus subtilis subsp. spizizenii W23 NC_014479.1 94.18 0.0585
Bacillus subtilis subsp. subtilis 168 NC_000964.3 93.03 0.0707
Bacillus thuringiensis subsp. kurstaki HD-1 NZ_CP004870.1 68.83 0.1575
Bacillus vallismortis NBIF-001 NZ_CP020893.1 77.57 0.2081
Bacillus velezensis SQR9 NZ_CP006890.1 77.38 0.2095
Bacillus weihenstephanensis KBAB4 NC_010184.1 68.46 0.1386
Biology 2022,11, 137 6 of 26
Figure 1. Maximum Likelihood phylogenomic tree of strain KR2-7 and selected Bacillus strains based on REALPY.
Numbers at nodes represent the percentages of occurrence of nodes in 1000 bootstrap trials. The Listeria
monocytogenes strain HCC23 (CP001175.1) was served as outgroup.
Figure 2. Antifungal activity of strain KR2-7 towards various phytopathogenic fungi. (A1–E1): a 5-mm agar plug
of each phytopathogenic fungi including Alternaria alternata, Athelia roflsii, Botrytis cinerea, Rhizoctonia solani, and
Verticillium albo-atrum was cultured on the center of the PDA plate for 6 days at 28 °C, respectively. (A2–E2): strain
KR2-7 was simultaneously cultured 3cm apart from the plug of (A2): Alternaria alternata, (B2): Athelia roflsii, (C2):
Botrytis cinerea, (D2): Rhizoctonia solani, (E2): Verticillium albo-atrum.
A1
B1
C1
D1
E1
A2
B2
C2
D2
E2
Figure 1.
Maximum Likelihood phylogenomic tree of strain KR2-7 and selected Bacillus strains based
on REALPY. Numbers at nodes represent the percentages of occurrence of nodes in 1000 bootstrap
trials. The Listeria monocytogenes strain HCC23 (CP001175.1) was served as an outgroup.
Moreover, closely related Bacillus species such as B. amyloliquefaciens and B. velezensis
were distributed on the same branch (Figure 1). The genome-based phylogeny approaches
well recognized B. methylotrophicus,B. amyloliquefaciens subsp. plantarum, and B. oryzicola
as the heterotypic synonyms of B. velezensis [
30
]. Recently, three subspecies of Bacillus
subtilis, including B. subtilis subsp. inaquosorum,B. subtilis subsp. Spizizenii, and B. subtilis
subsp. stercoris were promoted to species status through comparative genomics. Each
subspecies encompasses unique bioactive secondary metabolite genes which cause the
unique phenotypes [
31
]. According to REALPHY results, strain KR2-7 was identified as B.
inaquosorum, owing to being placed within the B. subtilis branch close to B. subtilis subsp.
inaquosorum strain KCTC 13429 and strain DE111 in the phylogenomic tree (Figure 1).
Notably, the results of ANI and GGDC analysis were consistent with REALPHY results
as the KR2-7 genome displayed the highest ANI values (99.26%) and the lowest GGDC
values (0.0075) with respect to the genome of strain KCTC 13429 (Table 1). Interestingly,
the phylogeny analysis of several B. amyloliquefaciens strains based on core-genome was
consistent with the ANI and GGDC values [
32
]. Altogether, the aforesaid genome-based
phylogeny approaches identified strain KR2-7 as Bacillus inaquosorum.
Biology 2022,11, 137 7 of 26
3.3. Secondary Metabolites Biosynthetic Gene Clusters in the KR2-7 Genome
Genome mining of the strain KR2-7 revealed that more than 700 kb (i.e., nearly 17% of
the genome) is devoted to 13 putative BGCs. Of the 13 found BGCs, nine were identified
to contain one polyketide synthase (PKS) for macrolactin; five non-ribosomal peptide
synthetases (NRPSs) for bacillibactin, bacillomycin F, bacilysin, fengycin, and surfactin;
one PKS-NRPS hybrid synthetases (PKS-NRPS) for bacillaene; one thiopeptide synthase
for subtilosin A, and one head-to-tail cyclised peptide for the sporulation killing factor.
The nine annotated BGCs encode secondary metabolites which contribute to plant growth
promotion through the fungal/bacterial pathogen suppression, ISR, nutrient uptake, and
plant colonization (Table 2) [
33
–
36
]. The distribution of identified BGCs within the KR2-7
genome underlies its vigorous potential in plant disease biocontrol application [
16
]. The
coding genes of secondary metabolites in KR2-7 were different from those in B. velezensis
FZB42, while these genes showed more similarity with those in B. subtilis 168 (Table 2).
Interestingly, the BGC of bacillomycin F in KR2-7 was absent in B. subtilis 168 and B.
velezensis FZB42 (Table 2) as this gene cluster conserved in B. inaquosorum [
31
]. Moreover,
the KR2-7 genome contains four unannotated BGCs (data not shown) which showed less
similarity to compounds listed in the MIBiG database.
Table 2.
The comparison of secondary metabolites biosynthetic gene clusters in B. inaquosorum strains
KR2-7, B. subtilis 168 and B.velezensis strain FZB42.
Metabolite Synthetase
Type
Gene Cluster
Function
Gene Similarity
with Strain
KR2-7
B. inaquosorum
KR2-7 B. subtilis 168 B. velezensis
FZB42 168 FZB42
Bacillaene PKS-NRPS pksABCDE,acpK,
pksFGHIJLMNRS
pksABCDE,acpk,
pksFGHIJLMNRS
baeBCDE,acpK,
baeGHIJLMNRS Antibacterial 89.63% 75.25%
Bacillibactin NRPS dhbABCEF dhbABCEF dhbABCEF Nutrient uptake 92.25% 73.07%
Bacilysin NRPS
bacABCDE,ywfAG
bacABCDEFG,
ywfA
bacABCDE,ywfAG
Antibacterial 93.50% 80.67%
Fengycin NRPS ppsABCDE ppsABCDE fenEABCD Antifungal 92.01% 72.05%
Macrolactin PKS pksJL,baeE -pks2ABCDEFGHI Antibacterial - 74.12%
Bacillomycin F NRPS ituD, ituABC - - Antifungal, ISR - -
Sporulation
killing factor
Head-to-tail
cyclised
peptide
skfABCEFGH skfABCEFGH - Antibacterial 96.08% -
Subtilosin A Thiopeptide sboA,
albABCDEFG
sboA,
albABCDEFG - Antibacterial 91.86% -
Surfactin NRPS
srfAA,AB,AC,AD srfAA,AB,AC,AD srfAA,AB,AC,AD
Antifungal,
Antibacterial,
Colonization, ISR
92.20% 74.65%
3.4. Antifungal Secondary Metabolites Production in Strain KR2-7
The KR2-7 genome mining showed that this strain harbors three BGCs with anti-
fungal function, including fengycin, surfacing, and bacillomycin F (a variant of iturin)
belonging to Bacillus cyclic-lipopeptides (CLPs). Bacillus CLPs represented the power-
ful fungitoxicity properties by interfering with cell membrane integrity, permeabilizing
the cell membrane, and perturbing membrane osmotic balance due to the formation of
ion-conducting pores [37].
Strain KR2-7 not only suppressed the Fusarium wilt of tomato caused by Fusarium
oxysporum f. sp. lycopersici [
16
] but also showed a broad-spectrum antifungal activity
towards various phytopathogenic fungi including Alternaria alternata f. sp. lycopersici,
Athelia rolfsii,Botrytis cinerea,Rhizoctonia solani, and Verticillium albo-atrum (Figure 2).
Biology 2022,11, 137 8 of 26
Figure 1. Maximum Likelihood phylogenomic tree of strain KR2-7 and selected Bacillus strains based on REALPY.
Numbers at nodes represent the percentages of occurrence of nodes in 1000 bootstrap trials. The Listeria
monocytogenes strain HCC23 (CP001175.1) was served as outgroup.
Figure 2. Antifungal activity of strain KR2-7 towards various phytopathogenic fungi. (A1–E1): a 5-mm agar plug
of each phytopathogenic fungi including Alternaria alternata, Athelia roflsii, Botrytis cinerea, Rhizoctonia solani, and
Verticillium albo-atrum was cultured on the center of the PDA plate for 6 days at 28 °C, respectively. (A2–E2): strain
KR2-7 was simultaneously cultured 3cm apart from the plug of (A2): Alternaria alternata, (B2): Athelia roflsii, (C2):
Botrytis cinerea, (D2): Rhizoctonia solani, (E2): Verticillium albo-atrum.
A1
B1
C1
D1
E1
A2
B2
C2
D2
E2
Figure 2.
Antifungal activity of strain KR2-7 towards various phytopathogenic fungi. (
A1
–
E1
): a
5-mm agar plug of each phytopathogenic fungi including Alternaria alternata,Athelia roflsii,Botrytis
cinerea,Rhizoctonia solani, and Verticillium albo-atrum was cultured on the center of the PDA plate for
6 days at 28
◦
C, respectively. (
A2
–
E2
): strain KR2-7 was simultaneously cultured 3cm apart from
the plug of (
A2
): Alternaria alternata, (
B2
): Athelia roflsii, (
C2
): Botrytis cinerea, (
D2
): Rhizoctonia solani,
(E2): Verticillium albo-atrum.
Fengycin (plipastatin), the powerful fungitoxic compound—especially against fila-
mentous fungi [
37
]—is synthesized by NRPS and encoded by a 39302 bp gene cluster with
five genes including ppsA-E in KR2-7, which showed a 92% and 72.05% similarity to the
fengycin gene cluster of B. subtilis 168 and B.amyloliquefacience FZB42, respectively (Table 2).
The first three genes (ppsABC) each encode two amino acid modules. The fourth gene
(ppsD) encodes three amino acid modules, and the last gene (ppsE) encodes one amino acid
module (Figure 3). Ions of m/z values 1471.8, 1485.7, 1487.9, 1499.9, 1501.9, 1513.8, 1515.9,
1527.8 1529.9 and, 1543.8 were observed in a whole-cell surface extract of KR2-7 grown
on a dual culture plate (thereafter, dual culture cell extract) and assigned to C15 to C18
fengycin homologues while in the whole-cell surface extract of KR2-7 grown on the control
plate (thereafter, control cell extract) only four aforesaid peaks (m/z 1501.9, 1515.9, 1529.9
and, 1543.8) were detected (Table 3, Figure S2). The result indicated that the KR2-7 strain
secreted various fengycin homologues to inhibit the growth of Fol.
Figure 3. The biosynthetic gene cluster of fengycin in strain KR2-7.
Figure 4. The biosynthetic gene cluster of bacillomycin F in strain KR2-7.
Figure 5. The biosynthetic gene cluster of surfactin in strain KR2-7.
ppsE
Contig QZDE02000001.1
Contig QZDE02000002.1
ppsA
ppsB
ppsC
ppsD
ppsD
2113791
2144057
1
5259
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
E
Te
P
C
P
A
Glu
Orn
Tyr
Thr
Tyr
Glu
Val
Pro
Glu
lle
ppsA
ppsB
ppsC
ppsD
ppsE
ituD, ituA, ituB
Asn
C
P
C
P
C
A
P
C
P
C
Tyr
Asn
Pro
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
Te
ituB, ituC
Thr
Asn
ituD
ituA
ituB
ituC
25611
62841
E
Contig QZDE02000002.1
1717326
sfp
srfAA
srfAB
srfAC
srfAD
yczE
srfAA
srfAB
srfAC
srfAD
Leu
Glu
Leu
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
Te
P
C
P
Leu
Leu
Val
Asp
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
Te/
At
Contig QZDE02000002.1
1743473
Figure 3. The biosynthetic gene cluster of fengycin in strain KR2-7.
Biology 2022,11, 137 9 of 26
Table 3.
Assignments of all fengycin mass peaks obtained by MALDI-TOF mass spectrometry of
whole cells of strain KR2-7 grown on control and dual culture plates.
Mass Peak (m/z) Assignment Reference
On PDA control
1501.9 Ala-6-C16 fengycin [M + H, Na, K]+[38]
1515.9 Ala-6-C17 fengycin [M + H, Na, K]+[38]
1529.9 Val-6-C16 fengycin [M + H, Na, K]+[38]
1543.8 Val-6-C17 fengycin [M + H, Na, K]+[38]
On PDA dual culture
1471.9 Ala-6-C15 fengycin [M + H, Na, K]+[38]
1485.7 C16-Fengycin A [M + Na] [39]
1487.9 Ala-6-C15 fengycin [M + H, Na, K]+[38]
1499.9 Ala-6-C17 fengycin [M + H, Na, K]+[38]
1501.9 Ala-6-C16 fengycin [M + H, Na, K]+[38]
1513.8 C18-fengycin A [M + Na] [39]
1515.9 Ala-6-C17 fengycin [M + H, Na, K]+[38]
1527.8 Val-6-C17 fengycin [M + H, Na, K]+[38]
1529.9 Val-6-C16 fengycin [M + H, Na, K]+[38]
1543.8 Val-6-C17 fengycin [M + H, Na, K]+[38]
1501.9 Ala-6-C16 fengycin [M + H, Na, K]+[38]
1515.9 Ala-6-C17 fengycin [M + H, Na, K]+[38]
1527.8 Val-6-C16 fengycin [M + H, Na, K]+[38]
1529.9 Val-6-C17 fengycin [M + H, Na, K]+[38]
1543.8 Ala-6-C15 fengycin [M + H, Na, K]+[38]
More strikingly, a 37074 bp gene cluster encoding bacillomycin F was also identified
immediately downstream of the fengycin gene cluster of KR2-7 (Figure 4). The bacillomycin
F is one of seven main variants within the iturin family [
40
] encoded by a gene cluster
consisting of four genes designated ituD, ituA, ituB and, ituC. The gene cluster code a cyclic
heptapeptide in which the first three amino acids are shared among iturin family members,
whereas the remaining four amino acids are conserved in B. inaquosorum [
31
]. Furthermore,
iturins are characterized by a heptapeptide of
α
-amino acids attached to a
β
-amino fatty acid
chain with a length of 14 to 17 carbons [
37
]. They possess potent antifungal activity against
a wide variety of fungi and yeast, but bounded antibacterial and no antiviral actions [
41
–
43
].
Furthermore, these molecules also showed strong haemolytic activity, which limits their
clinical use [
44
]. The antifungal mechanism of iturins launches by their interaction with the
target cell membrane and osmotic perturbation of the membrane, owing to the formation
of ion-conducting pores. Subsequently, the change in the permeability of a membrane is
conducive to the release of biomolecules, such as proteins, nucleotides, and lipids from
cells, which ultimately causes cell death [
44
,
45
]. In the dual culture cell extract of KR2-7, six
mass peaks assigned to C16, C18 and C19 forms of iturin were observed while they were
absent in the control cell extract of KR2-7 (Table 4, Figure S3). This result indicated that
strain KR2-7 produced different variants of iturins to limit the growth of Fol hyphae.
Figure 3. The biosynthetic gene cluster of fengycin in strain KR2-7.
Figure 4. The biosynthetic gene cluster of bacillomycin F in strain KR2-7.
Figure 5. The biosynthetic gene cluster of surfactin in strain KR2-7.
ppsE
Contig QZDE02000001.1
Contig QZDE02000002.1
ppsA
ppsB
ppsC
ppsD
ppsD
2113791
2144057
1
5259
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
E
Te
P
C
P
A
Glu
Orn
Tyr
Thr
Tyr
Glu
Val
Pro
Glu
lle
ppsA
ppsB
ppsC
ppsD
ppsE
ituD, ituA, ituB
Asn
C
P
C
P
C
A
P
C
P
C
Tyr
Asn
Pro
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
Te
ituB, ituC
Thr
Asn
ituD
ituA
ituB
ituC
25611
62841
E
Contig QZDE02000002.1
1717326
sfp
srfAA
srfAB
srfAC
srfAD
yczE
srfAA
srfAB
srfAC
srfAD
Leu
Glu
Leu
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
Te
P
C
P
Leu
Leu
Val
Asp
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
Te/
At
Contig QZDE02000002.1
1743473
Figure 4. The biosynthetic gene cluster of bacillomycin F in strain KR2-7.
Biology 2022,11, 137 10 of 26
Table 4.
Assignments of iturin mass peaks obtained by MALDI-TOF mass spectrometry of whole
cells of strain KR2-7 grown on control and dual culture plates.
Mass Peak (m/z) Assignment Reference
On PDA control
1106.6 C17-iturin [M + Na]+[18]
1122.6 C17-iturin [M + K]+[18]
1134.6 C19-iturin [M + Na]+[18]
1136.6 C18-iturin [M + K]+[18]
On PDA dual culture
1092.6 C16-iturin [M + Na]+[18]
1098.6 C16-iturin [M + H]+[18]
1106.6 C17-iturin [M + Na]+[18]
1112.6 C19-iturin [M + H]+[18]
1120.6 C18-iturin [M + Na]+[18]
1122.6 C17-iturin [M + K]+[18]
1134.6 C19-iturin [M + Na]+[18]
1136.6 C18-iturin [M + K]+[18]
1150.6 C19-iturin [M + K]+[18]
Similar to fengycin, surfactin was synthesized by NRPS and encoded by a srf gene
cluster that spans 26073 bp in the KR2-7 genome. The gene cluster harbors four genes
(srf AA-AD) and showed a 92.20% and 74.65% similarity to those of B. subtilis 168 and
B.amyloliquefacience FZB42, respectively. The product of the srf gene cluster is a linear
array of seven modules, six of which are encoded by srf AA and srf AB genes and the
last module is encoded by a srf AC gene (Figure 5). The fourth gene (srf AD) encodes
thioesterase/acyltransferase (Te/At-domain) which triggers surfactin biosynthesis [
37
].
Hence, the sfp gene encodes an essential enzyme (phosphopantetheinyl transferase) for the
non-ribosomal synthesis of lipopeptides and the synthesis of polyketides. The regulatory
gene yczE encoding an integral membrane protein was detected within the KR2-7 genome
(Figure 5). Surfactin enables bacteria cells to interact with plant cells as a bacterial elicitor
for stimulating ISR [
37
], especially through the activation of jasmonate- and salicylic
acid-dependent signaling pathways [
46
]. Several studies indicated the ISR-elicitor role of
surfactin against phytopathogens in various host plants, e.g., tomato [
47
], wheat [
48
], citrus
fruit [
49
], lettuce [
50
], and grapevine [
51
]. Comparing the MALDI-TOF mass spectra of
KR2-7 grown on a PDA control and dual culture revealed that surfactin contributed to the
suppression of Fol as eight mass peaks assigned to C13, C14 and C15 surfactin homologs
were detected in dual culture cell extracts, while only four of which were observed in the
control cell extract (Table 5, Figure S3). Furthermore, the bacterium produced more surfactin
to suppress Fol. Additionally, C14 and C15 surfactin tend to stimulate stronger ISR rather
than those with shorter chain lengths [
47
]. Moreover, suppression of taxonomically diverse
fungal pathogens including Fusarium oxysporum,F. moniliforme,F. solani,F. verticillioides,
Magnaporthe grisea,Saccharicola bicolor,Cochliobolus hawaiiensis, and Alternaria alternata by
the surfactin family demonstrated that surfactins are strong fungitoxic compounds [
52
–
55
].
Figure 3. The biosynthetic gene cluster of fengycin in strain KR2-7.
Figure 4. The biosynthetic gene cluster of bacillomycin F in strain KR2-7.
Figure 5. The biosynthetic gene cluster of surfactin in strain KR2-7.
ppsE
Contig QZDE02000001.1
Contig QZDE02000002.1
ppsA
ppsB
ppsC
ppsD
ppsD
2113791
2144057
1
5259
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
E
Te
P
C
P
A
Glu
Orn
Tyr
Thr
Tyr
Glu
Val
Pro
Glu
lle
ppsA
ppsB
ppsC
ppsD
ppsE
ituD, ituA, ituB
Asn
C
P
C
P
C
A
P
C
P
C
Tyr
Asn
Pro
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
Te
ituB, ituC
Thr
Asn
ituD
ituA
ituB
ituC
25611
62841
E
Contig QZDE02000002.1
1717326
sfp
srfAA
srfAB
srfAC
srfAD
yczE
srfAA
srfAB
srfAC
srfAD
Leu
Glu
Leu
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
C
A
Te
P
C
P
Leu
Leu
Val
Asp
C
A
P
C
P
C
A
P
C
P
E
C
A
P
C
P
Te/
At
Contig QZDE02000002.1
1743473
Figure 5. The biosynthetic gene cluster of surfactin in strain KR2-7.
Biology 2022,11, 137 11 of 26
Table 5.
Assignments of surfactin mass peaks obtained by MALDI-TOF mass spectrometry of whole
cells of strain KR2-7 grown on control and dual culture plates.
Mass Peak (m/z) Assignment Reference
On PDA control
1044.6 C14-surfactin [M + Na, K]+[18]
1046.6 C13-surfactin [M + K]+[18]
1058.6 C15-surfactin [M + Na]+[18]
1060.5 C14-surfactin [M + Na, K]+[18]
1074.6 C15-surfactin [M + Na, K]+[18]
On PDA dual culture
1030.6 C13-surfactin [M + Na]+[18]
1032.7 C13-surfactin [M + K]+[18]
1044.6 C14-surfactin [M + Na, K]+[18]
1046.6 C13-surfactin [M + K]+[18]
1058.6 C15-surfactin [M + Na]+[18]
1060.5 C14-surfactin [M + Na, K]+[18]
1074.6 C15-surfactin [M + Na, K]+[18]
3.5. Antibacterial Secondary Metabolites Production in Strain KR2-7
The KR2-7 genome contained six BGCs coding for antibacterial compounds including
bacillaene and macrolactin, sporulation killing factor (skf), subtilosin A, bacilysin, and
surfactin. Several studies on surfactin and its isoforms proved that these metabolites played
a major role in combating bacterial plant diseases, such as fruit bloch caused by Acidovorax
citrulli in melon [
56
], tomato wilt caused by Ralstonia solanacearum [
57
], and root infection
by Pseudomonas syringae in Arabidopsis [
58
]. Moreover, surfactin produced by B. subtilis R14
exhibited pronounced antagonistic efficacy against several multidrug-resistance bacterial
strains of Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus, and Enteroccoccus
faecalis [59].
Bacillaene is a polyketide known as a selective bacteriostatic agent that inhibits
prokaryotic, not eukaryotic growth by disrupting protein synthesis [
60
]. Its antimicro-
bial efficacy against various bacteria (Myxococcus xanthus and Staphylococcus aureus) and
fungi (Fusarium spp) have been reported [
60
–
62
]. In the KR2-7 genome, bacillaene was
synthesized by a PKS/NRPS hybrid pathway and encoded by a giant pks gene cluster
(76.355 Kbp) containing 16 genes (pksA-S and acpK) showing an 89.63% and 75.25% sim-
ilarity to those of B. subtlis 168 and B. velezensis FZB42, respectively (Table 2, Figure 6B).
Another polyketide, macrolactin, can be encoded by a 54.225 kbp gene cluster in strain
KR2-7 and showed a 74.12% similarity to the mln cluster of B. velezensis FZB42 (Table 2,
Figure 6A). Macrolactins are a large class of macrolide antibiotics that inhibited the growth
of several bacteria, including Ralstonia solanacearum,Staphylococcus aureus, and Burkholderia
epacian [
63
,
64
]. In the dual culture cell extract of KR2-7, one ion corresponding to 7-o-
succinyl macrolactin A ([M + Na]
+
= 525.4), and another ion corresponding to bacillaene
A ([M + H]
+
= 583.5) were detected (Figure 6C,D), while they were not observed in the
control cell extract of KR2-7.
Biology 2022,11, 137 12 of 26
1
(A)(B)
(C)(D)
Figure 6.
The biosynthetic gene clusters of (
A
) macrolactin and (
B
) bacillaene in strain KR2-7 and
MALDI-TOF MS analysis of antibacterial secondary metabolites produced by strain KR2-7 grown on
a dual culture plate. (
C
) m/z 525.4: 7-o-succinyl macrolactin A [M + Na]+; (
D
) m/z 583.5: Bacillaene
A [M + H]+.
Bacilysin (also known as tetaine) is a dipeptide suppressing a wide variety of de-
structive phytopathogenic bacteria, e.g., Erwinia amylovora,Xanthomonas oryzae pv. oryzae,
X. oryzae pv. Oryzicola, and Clavibacter michiganense subsp. sepedonicum [
65
–
67
]. This bac-
tericidal property is due to the inhibition of glucosamine-6- phosphate synthase by the
anticapsin moiety of bacilysin. Its inhibition represses the biosynthesis of peptidoglycans,
the essential constituents of the bacterial cell wall [
68
,
69
]. In the KR2-7 genome, bacilysin
can be encoded by a 7128 bp bac gene cluster consisting of seven genes (bacA-E, ywf AG),
and display high gene similarity to those of B. subtilis 168 (Table 2, Figure 7). This metabolite
and its derivatives were detected neither in the KR2-7 control cell extract nor dual culture
cell extract, likely due to culture conditions or the assay method.
1
Figure 7. The biosynthetic gene cluster of bacilysin antibacterial metabolite in strain KR2-7.
Furthermore, the KR2-7 genome encompassed two distinct gene clusters encoding
bacteriocins, including subtilosin A and sporulation killing factors (SKFs). Subtilosin A
is a macrocyclic anionic antimicrobial peptide originally obtained from wild-type strain
B. subtilis 168 [
70
] but is also produced by B. amyloliquefaciens and B. atrophaeus [
71
,
72
]. This
bacteriocin displayed a bactericidal effect on a broad spectrum of bacteria, including Gram-
positive and Gram-negative bacteria and both aerobes and anaerobes [
73
], possibly through
an interaction with membrane-associated receptors, or binding to the outer cell membrane,
and is conducive to membrane permeabilization [
73
–
75
]. Subtilosin A is ribosomally
synthesized by an alb gene cluster containing eight genes (albA-G, sboA) spanning 6.8 kbp
in the KR2-7 genome (Figure 8). The sboA gene encodes presubtilosin, and albA-G genes
encode proteins whose functions are presubtilosin processing and subtilosin export [
76
].
Biology 2022,11, 137 13 of 26
The mass peaks corresponding to subtilosin A and its homologs appeared neither in the
KR2-7control cell extract nor the dual culture cell extract. These peaks are detectable by
altering the culture condition and/or evaluating the procedure.
1
Figure 8. The biosynthetic gene cluster of subtilosin A antibacterial metabolite in strain KR2-7.
The KR2-7 genome also harbored a 5976 bp skf gene cluster encompassing skf ABCEFGH,
and involves the production and release of killing factors during sporulation (Figure 9).
During the early stages of sporulation, sporulating cells of B. subtilis exude extracellular
killing factors to kill the nonsporulating sister cells whose immunity to these toxins was not
developed. As a result, the nutrient from the dead cells are released and then used by the
sporulating cells to resume their growth. This phenomenon is termed “cannibalism” and
causes a delay in sporulation [
77
,
78
]. The SKF bacteriocin produced by the sporulating cells
can destroy other soil-inhabiting bacteria. Similarly, the expression of skf genes in B. subtilis
inhibits the growth of X. orzae pv. oryzae, the causative agent of rice bacterial blight [79].
1
Figure 9.
The biosynthetic gene cluster of sporulation killing factor antibacterial metabolite in
strain KR2-7.
3.6. Plant Colonization by Strain KR2-7
The most crucial step for a PGPR (Plant Growth Promoting Rhizobacteria) agent to
survive, enhance plant growth, and suppress plant disease is the efficient colonization of
plant tissues. The plant colonization process comprises two steps. In the first step, PGPR
agents reach the surface of plant tissue either by passive movement in water flow or by
flagellar movement. The second step is to establish the plant-bacterium interaction reliant
on bacterial biofilm formation [36,80].
The KR2-7 genome harbored the gene clusters for flagellar assembly (flg cluster, flh
cluster and, fli cluster) and bacterial chemotaxis (che cluster) together with other genes
known to be necessary for swarming motility, including hag, two stator elements (motAB),
as well as regulatory genes swrAA, swrAB, swrB and, swrC (Table 6). In the step of efficient
colonization, the PGPR agent forms bacterial biofilm and not only strengthens the plant-
bacterium interaction but protects the plant root system as a bio-barrier against pathogen
attacks [
80
]. The main component of bacterial biofilm is the extracellular polymeric sub-
stances (EPS) with a chemical composition including proteins, neutral polysaccharides,
charged polymers, and amphiphilic molecules [
80
]. The eps cluster (epsC-O) encoding
exopolysaccharide of biofilm and its regulatory genes sinR and arbA (repressors) and, sinI
(antirepressor), the yqxM-sipW-tasA gene cluster encoding amyloid fiber (TasA protein
of biofilm) and pgcA encoding phosphoglucomutase were found in the KR2-7 genome
(Table 6). Moreover, the involvement of surfactin in cell adhesion and biofilm formation
due to its 3D topology and amphiphilic nature has been illustrated [
81
,
82
]. Baise et al. [
58
]
declared that deleting surfactin gene expression in B. subtilis strain 6051 led to disability to
form robust biofilm on Arabidopsis root surface, and reduced the suppression of disease
caused by Pseudomonas syringae. Besides, the deficiency in surfactin production in B. sub-
tilis, strain UMAF6614 resulted in a biofilm formation defect on melon phylloplane and
partially reduced the suppression of bacterial soft root rot, bacterial leaf spot, and cucurbit
powdery-mildew by the biocontrol stain [83].
Biology 2022,11, 137 14 of 26
Table 6. Genes and gene clusters involved in plant-bacterium interaction in the genome of KR2-7.
Bioactivity Gene/Gene
Cluster From To Product Remark
Root
colonization
yfiQ 1181545 1180457 Putative membrane-bound
acyltransferase YfiQ Involved in surface adhesion [13,84]
sacB 2852367 2850946 Levan sucrase Levan contributed to the aggregation
of wheat root-adhering soil [85]
Swarming
motility
swrB 319026 318514 Swarming motility protein swrB Essential for swarming motility [86]
swrC 1348113 1344916 Swarming motility protein SwrC Self-resistance to surfactin [86]
sfp 1712320 1712994
4’-phosphopantetheinyl transferase sfp
Necessary for lipopeptide and
polyketide synthesis which is
essential for surface motility and
biofilm formation [13,86]
swrAA 2770348 2770776 Swarming motility protein swrAA Essential for swarming motility [87]
swrAB 2770855 2772051 Swarming motility protein swrAB Essential for swarming motility [87]
efp 3829945 3830526 Elongation factor P Essential for swarming motility [88]
flhA 328577 326544 Flagellar biosynthesis protein flhA
Flagellar assembly
flhB 329692 328610 Flagellar biosynthetic protein flhB
fliR 330489 329692 Flagellar biosynthetic protein fliR
fliQ 330757 330479 Flagellar biosynthetic protein FliQ
fliP 331428 330763 Flagellar biosynthetic protein fliP
fliY 333625 332483 Flagellar motor switch phosphatase
FliY
fliM 334613 333606 Flagellar motor switch protein FliM
fliL 335060 334638 Flagellar protein FliL
flgG 336163 335312 Flagellar basal-body rod protein flgG
fliK 338009 336546 Probable flagellar hook-length control
protein
fliJ 339085 338642 Flagellar FliJ protein
fliI 340404 339088 Flagellum-specific ATP synthase
fliH 341153 340401 Probable flagellar assembly protein
fliH
fliG 342162 341146 Flagellar motor switch protein FliG
fliF 343785 342175 Flagellar M-ring protein
fliE 344151 343831 Flagellar hook-basal body complex
protein FliE
flgC 344618 344163 Flagellar basal-body rod protein flgC
flgB 345010 344615 Flagellar basal-body rod protein flgB
motA 654985 655887 Motility protein A
motB 655835 656650 Motility protein B
Swarming
motility
flhO 2629891 2630742 Flagellar hook-basal body complex
protein flhO
Flagellar assembly
flhP 2630949 2631584 Flagellar hook-basal body complex
protein flhP
flgM 2751851 2752117 Negative regulator of flagellin
synthesis
flgK 2752634 2754151 Flagellar hook-associated protein 1
flgL 2754161 2755057 Flagellar hook-associated protein 3
hag 2756494 2757405 Flagellin
fliD 2757987 2759483 Flagellar hook-associated protein 2
fliS 2759505 2759906 Flagellar protein fliS
fliT 2759903 2760244 Flagellar protein FliT
cheD 320333 319833 Chemoreceptor glutamine deamidase
CheD
Bacterial chemotaxi
cheC 320959 320330 CheY-P phosphatase CheC
cheW 321457 320978 Chemotaxis protein CheW
cheA 323488 321470 Chemotaxis protein CheA
cheB 324555 323485 Chemotaxis response regulator
protein-glutamate methylesterase
cheY 332463 332095 Chemotaxis protein CheY
cheV 616969 616058 Chemotaxis protein CheV
cheR 3987238 3988155 Chemotaxis protein methyltransferase
Biology 2022,11, 137 15 of 26
Table 6. Cont.
Bioactivity Gene/Gene
Cluster From To Product Remark
Biofilm
formation
pgsA 273607 273026
CDP-diacylglycerol-glycerol-3-
phosphate3-Phosphatidyl
transferase
Member of pgsB-pgsC-pgsA-pgsE gene
cluster encoding PGA which is
contributed to robustness and
complex morphology of the colony
biofilms [89]
pgcA 1083817 1082072 Phosphoglucomutase
Phosphoglucomutase plays an
important role in biofilm
formation [90]
ybdK 1903365 1902379 Sensor histidine kinase ybdK Transcriptional regulation of biofilm
formation [91,92]
sigW 1928876 1928313 RNA polymerase sigma factor sigW Transcriptional regulation of biofilm
formation [91,92]
sigH 2011463 2010807 RNA polymerase sigma-H factor Involves in the initial stage of biofilm
formation [93]
abrB 2082858 2083148 Transition state regulatory protein
AbrB
Transcriptional regulation of biofilm
formation [94]
epsC-O 2731363 2874940 Gene cluster for capsular
poly-saccharide biosynthesis
Encoding exopolysaccharide which is
essential for biofilm formation [91]
lytS 3426013 3427803 Sensor protein lytS Transcriptional regulation of biofilm
formation [91,92]
yqxM 3813390 3814151 Protein yqxM
Belongs to yqxM-sipW-tasA gene
cluster that is necessary for biofilm
formation [95]
tasA 3814771 3815556 Spore coat-associated protein N
Required for development of complex
colony architecture [94]
sinR 3816019 3815651 HTH-type transcriptional regulator
sinR
Transcriptional regulation of biofilm
formation [91,92]
sinI 3816289 3816020 Protein sinI Transcriptional regulation of biofilm
formation [91,92]
Biofilm
formation
spo0A 3849786 3850625 Stage 0 sporulation protein A Involved in the initial stage of biofilm
formation [93]
resE 3951273 3953042 Sensor histidine kinase resE Transcriptional regulation of biofilm
formation [91,92]
ymcA 261856 261425 Protein ymcA These genes are involved in the
development of multicellular
communities [91]
ylbF 467892 467434 Regulatory protein ylbF
yqeK 3725618 3726187 Protein yqeK
sipW 3814123 3814707 Signal peptidase I W
Mineral
assimilation
moaD 594413 594180 Molybdopterin synthase sulfur carrier
subunit
Nitrogen assimilation
moaE 594879 594406 Molybdopterin synthase catalytic
subunit
moaC 1469542 1469000 Molybdenum cofactor biosynthesis
protein C
moaA 2592667 2593692 Molybdenum cofactor biosynthesis
protein A
moaB 3369200 3369751 Molybdenum cofactor biosynthesis
protein B
nasA-F 1758586 1768809 Gene cluster for Nitrate transport and
reduction
Nitrogen assimilation
narK 2532129 2533373 Nitrite extrusion protein
fnr 2533450 2534190 Anaerobic regulatory protein
arf M 2534994 2535563 Probable transcription regulator arfM
narG-J 2535783 2542171 Gene cluster for Nitrate reductase
nrgB 2619035 2618670 Nitrogen regulatory PII-like protein
nrgA 2620246 2619032 Ammonium transporter nrgA
mgtE 694316 692961 Magnesium transporter mgtE Magnesium assimilation
corA 3806910 3807869 Magnesium transport protein CorA
mntH 1626982 1628259 Manganese transport protein mntH
Manganese assimilation
mntA-D 3237914 3241819 Gene cluster for Manganese
binding/transport protein
mntR 3824735 3825202 Transcriptional regulator mntR
Biology 2022,11, 137 16 of 26
Table 6. Cont.
Bioactivity Gene/Gene
Cluster From To Product Remark
ktrC 573312 572647 Ktr system potassium uptake protein C
Potassium assimilation
ykqA 574212 573469 Putative gamma-glutamylcyclo
transferase ykqA
ktrD 674275 672926 Ktr system potassium uptake protein D
yugO 3174241 3173258
Putative potassium channel protein yugO
ktrB 3203187 3201850 Ktr system potassium uptake protein B
ktrA 3203862 3203194 Ktr system potassium uptake protein A
Mineral
assimilation
yclQ 1683673 1682711 Ferrichrome ABC transporter
Iron assimilation
yvrC 2985220 2986197 Putative iron binding lipoprotein yvrC
yusV 3009672 3010583 Putative iron (III) ABC transport ATPase
component
dhbABCEF 3101063 3112861 Gene cluster encoding Bacillibactin
tuaA-H 2733487 2742546 Gene cluster for teichuronic acid
biosynthesis Bivalent cations assimilation
yvdK 2839830 2842166 Glycosyl hydrolase yvdK Ferrochrome assimilation
Plant growth
promotion/ISR
alsR 2672398 2671511 HTH-type transcriptional regulator alsR These genes encode enzymes of the
biosynthetic pathway from pyruvate
to 3-hydroxy-2-butanone
alsS 2672549 2674270 Acetolactate synthase
alsD 2674320 2675099 Alpha-acetolactate decarboxylase
bdhA 1448068 1449108 (R, R)-butanediol dehydrogenase
This gene encodes enzyme to
acatlyse 3-hydroxy-2-butanone to
2,3-butanediol
Plant growth
promotion
yhcX 1092936 1091395 Carbon-nitrogen hydrolase These genes are involved in indole
acetic acid biosynthesis
ysnE 3489604 3489101 N-acetyltransferase
dhaS 4136609 4135263 Putative aldehyde dehydrogenase dhaS
phy 4084788 4085936 3-phytase
Phytase hormones biosynthesis gene
treR 1237083 1236367 Trehalose gene cluster transcriptional
repressor
These genes are involved in trehalose
biosynthesis
treA 1238792 1237104 Trehalose-6-phosphate hydrolase
treP 1240275 1238863 PTS system trehalose-specific EIIBC
component
ilvH 3492606 3493124 Acetolactate synthase small subunit These genes are parts of leucine,
valine, and isoleucine biosynthesis
pathway
ilvB 3490858 3492609 Acetolactate synthase large subunit
ilvC 3493148 3494176 Ketol-acid reductoisomerase
speA 501879 503375 Arginine decarboxylase These genes may transform amino
acids to plant growth-promoting
substances [80]
speE 2512298 2513128 Spermidine synthase
speB 2513189 2514061 Agmatinase
3.7. Genes Involved in Bacterium-Plant Interactions
Quite apart from the antagonistic mechanisms of bacterial biocontrol strains, these
bacterial microorganisms are also involved in plant growth augmentation through making
nutrients available for host plants, production of plant growth-promotion hormones, and
the induction of systemic resistance within the plant by specific metabolite secretion [
96
,
97
].
Similar to other biocontrol microorganisms, the KR2-7 genome contains the genes/gene
clusters related to plant growth promotion (Table 6).
The KR2-7 genome contained moaA-E genes encoding molybdenum cofactor and
may be a relic of a nitrogen-fixing gene cluster or a cofactor for nitrogen assimilation [
80
].
Moreover, the genes for nitrate reduction (narG-J), nitrate transport (narK), probable tran-
scription regulator genes (arf M), regulatory protein (fnr), an ammonium acid transporter
(nrgA), and its regulator gene (nrgB), along with the nas gene cluster (nasA-F), were also
identified in the KR2-7 genome. The nas gene cluster is involved in nitrite transport and
reduction (Table 6).
In addition to nitrogen assimilation, the KR2-7 genome encompassed potassium trans-
porting genes, including ktr system potassium uptake proteins (ktrA-D), a putative potas-
sium channel protein (yugO), and putative gamma-glutamyl cyclotransferase (ykqA) [
80
,
98
].
Furthermore, the presence of genes for transportation of magnesium (mgtE, corA), fer-
Biology 2022,11, 137 17 of 26
rochrome (yvdK), manganese (mntH), and a gene cluster for manganese binding/transport
(mntA-D), along with the transcription regulator protein (mntR), were identified in the
KR2-7 genome. These genes uptake the nutrients or detoxify the heavy metal ions for
both the bacteria and host plants [
80
]. An 11.7 kb dhb gene cluster (dhbABDEF) encoding
siderophore bacillibactin was identified in the KR2-7 genome.
Furthermore, ions of m/z values 883.4 and 905.2 were detected in the KR2-7dual
culture cell extract and were identified as bacillibactin [M + H]
+
and bacillibactin [M + Na]
+
(Figure 10) by comparison with previously reported data [
99
,
100
]. Notably, the molecular
ion peaks corresponding to bacillibactin were not observed in the control cell extract of
KR2-7
. Siderophores are low-molecular-weight molecules with a high affinity for ferric
iron that solubilize iron from minerals and organic compounds under iron limitation condi-
tions [
101
]. Siderophore-producing bacterial strains impact plant health by supplying iron
to the host plants [
102
,
103
], depriving fungal pathogens of iron [
104
], and suppressing fun-
gal phytopathogens, including F. oxysporum f. sp. capsici [
105
] and Phytophthora capsici [
106
].
Furthermore, it was reported that siderophores mitigate heavy metal contamination of soil
through the formation of a stable complex with environmental toxic metals such as Al, Cd,
Cu, Ga, In, Pb, and Zn [101].
(A)
(B)
Figure 10. MALDI-TOF MS analysis of bacillibactin produced by strain KR2-7 grown on PDA dual culture. (A)
m/z 883.4: bacillibactin [M + H]+; (B) m/z 905.2: bacillibactin [M + Na]+
Figure 10.
MALDI-TOF MS analysis of bacillibactin produced by strain KR2-7 grown on a PDA dual
culture. (A) m/z 883.4: bacillibactin [M + H]+; (B) m/z 905.2: bacillibactin [M + Na]+.
Volatile organic compounds (VOCs) produced by PGPR agents play a significant role
in promoting plant growth through the regulation of synthesis or metabolism of phytohor-
mones [
107
], the induction of systemic disease resistance [
108
,
109
], and the control of plant
pathogens [
110
]. A 2,3-butanediol and 3-hydroxy-2-butanone (acetoin) are the best-known
growth-promoting VOCs that produced B. subtilis and B. amyloliquefaciens. The genome of
the KR2-7 harbored als gene cluster (alsR, alsS, alsD), along with the bdhA gene is together
required for the biosynthesis pathway of 2,3-butanediol from pyruvate. In this pathway,
alsS encodes the acetolactate synthase enzyme, which catalyzes the condensation of two
pyruvate molecules into acetolactate. Then, acetolactate decarboxylase, encoded by alsD,
converts decarboxylate acetolactate into acetoin. The alsR regulates two aforesaid steps.
Finally, the bdhA encoded (R, R)-butanediol dehydrogenase enzyme catalyzes 3-hydroxy-2-
butanone (acetoin) to 2,3-butanediol [
111
]. In addition, the KR2-7 genome contained ilvH,
ilvB, ilvC genes and a leu gene cluster (leuABCD) which are required for the biosynthesis
pathway of three branched-chain amino acids (BCAA), including leucine, isoleucine, and
valine. Acetolactate is a central metabolite between 2,3-butanediol and BCAA biosynthesis
and can involve in both anabolism and catabolism by acetolactate decarboxylase. It was
reported that acetolactate decarboxylase is an enzyme with a dual role that can direct
acetolactate flux to catabolism in favour of valine and leucine biosynthesis or can catalyze
the second step of the 2, 3-butanediol anabolism pathway [
112
]. Bacillus spp. can enhance
plant growth through the synthesis of plant growth-promoting hormones, such as auxin,
indole-3-acetic acid (IAA), and gibberellic acid. The genome of KR2-7 may encompass
Biology 2022,11, 137 18 of 26
genes/gene clusters responsible for the biosynthesis of indole acetic acid, phytase, and
trehalose (Table 6). Moreover, a large variety of PGPRs produce polyamines, such as pu-
trescine, spermine, spermidine, and cadaverine, and are known to be involved in promoting
plant growth and improving abiotic stress tolerance in plants [
113
]. The genes coding for
arginine decarboxylase (SpeA), agmatinase (SpeB), and spermidine synthase (SpeE), which
direct polyamines biosynthesis, were also found in the KR2-7 genome (Table 6).
4. Discussion
Previously, the Bacillus subtilis species complex was composed of four close subspecies,
i.e., subspecies subtilis,spizizenii,inaquosorum, and stercoris, which were differentiated
through a phylogenetic analysis of multiple protein-coding genes and genome-based
comparative analysis [
114
,
115
]. B. subtilis subsp. Inaquosorum was deemed as a distinctive
taxon encompassing strains KCTC 13429 and NRRL B-14697 [
116
]. Recent phylogenomic
studies clearly distinguished subspecies inaquosorum from subspecies spizizenii, as the
estimated ANI among them was smaller than the defined ANI for species delineation
(95% ANI) [
117
]. In addition to a low ANI value (<95%), the BGC of subtilin exclusively
presents in the genomes of subspecies spizizenii, but was not characterized in subspecies
inaquosorum genome [
115
]. Accordingly, B. inaquosorum KR2-7 was clearly differentiated
from B. subtilis subsp. spizizenii W23 because of the low ANI value among them (94.18%)
and the lack of subtilin gene cluster in the genome content of strain KR2-7. In addition,
it was reported that B. inaquosorum is the only species to produce bacillomycin F. It was
approved by detecting a unique MALDI-TOF-MS biomarker at m/z 1120 in the MALDI-
TOF-MS spectra of B. inaquosorum that cannot be produced by other species [
114
]. Since
this unique biomarker (m/z value 1120.6) was observed in the MALDI-TOF-MS spectra of
strain KR2-7, it can be concluded that this strain is a B. inaquosorum. Recently, the ability of
B. inaquosorum strain HU Biol-II in producing bacillomycin F was confirmed through HPLC
MS/MS [
15
]. Most recently, subspecies spizizenii,inaquosorum, and stercoris were promoted
to species status through a comparative genome study [
118
]. This study determined that
each subspecies had unique secondary metabolite genes encoding unique phenotypes,
thereby each subspecies can be promoted to species. According to the aforesaid results,
strain KR2-7 was identified as a B. inaquosorum.
The genome-driven data highlighted the plant-beneficial functions of strain KR2-7.
This strain can efficiently colonize the plant root surface, relying on its swarming motility
and biofilm formation abilities. Efficient root colonization of biocontrol bacteria is necessary
for suppressing phytopathogens, and biofilm formation is an essential prerequisite for
persistent root colonization [
119
,
120
]. The biofilm-deficient mutant of B. pumilus HR10
produced weakened biofilms with reduced contents of extracellular polysaccharides and
proteins, and thereby could not efficiently control pine seedling damping-off disease [
121
].
Hence, the suppression of tomato Fusarium wilt by strain KR2-7 [
16
] may contribute to
efficient tomato root colonization of this strain.
In addition to efficient root colonization, strain KR2-7 is able to directly suppress soil-
dwelling phytopathogens through producing eight antimicrobial secondary metabolites,
e.g., fengycin, surfactin, bacillomycin F, macrolactin, bacillaene, bacilysin, subtilosin A, and
sporulation killing factor. The combination of obtained data via MALDI-TOF-MS with our
previous observations [
16
] confirmed that strain KR2-7 produced at least four bioactive
metabolites (including fengycin, surfactin, macrolactin, and bacillaene) to directly protect
the tomato plant from the invasion and penetration of Fol. The cyclic lipodecapeptide
fengycin exhibits strong fungitoxic properties by inhibiting phospholipase A2 and aro-
matase functions [
122
], disruption of biological membrane integrity [
123
], deformation and
permeabilization of hyphae [
124
,
125
], and induction of ISR [
126
]. In this context, the strong
antifungal activity of B. inaquosorum strain HU Biol-II against a diverse group of fungi
highly pertained to the fengycin produced by this strain. Interestingly, 97.47% of the ppsA-E
gene cluster in KR2-7 was similar to the fengycin gene cluster in strain HU Biol-II [
15
].
Fengycin produced by B. subtilis SQR9 and B. amyloliquefaciens NJN-6 significantly inhibited
Biology 2022,11, 137 19 of 26
the growth of F. oxysporum [
127
,
128
]. Moreover, fengycin BS155 isolated from B. subtilis
BS155 destroyed Magnaporthe grisea through damaging the plasma membrane and cell
wall, disruption of mitochondrial membrane potential (MMP), chromatin condensation,
and the induction of reactive oxygen species (ROS) [
129
]. In addition to fengycin, the
contribution of other secondary metabolites in the biocontrol of various pathogens has
been reported. The supernatant of B. subtilis GLB191, consisting of surfactin and fengycin,
highly controlled grapevine downy mildew caused by Plasmopara viticola by means of
direct antagonistic activity and the stimulation of plant defence [
51
]. Furthermore, the
strong antifungal effect of B. velezensis strains Y6 and F7 against Ralstonia solanacearum and
F. oxysporum was attributed to the production of fengycin, iturin, and surfactin, among
which iturin played a key role in the suppression of F. oxysporum [
130
]. The biocontrol
mechanism of B. amyloliquefaciens DH-4 against Penicillium digitatum, the causal agent
of citrus green mold, was secreting a cocktail of antimicrobial compounds consisting of
macrolactin, bacillaene, iturins, fengycin, and surfactin [100].
Additionally, strain KR2-7 can exert hormones, such as IAA, phytase, and trehalose for
root uptake and rebalance hormones in the host plant to boost growth and stress response.
Phytate (inositol hexa- and penta-phosphates) is the predominant form of soil organic
phosphorus, which is unavailable for plant uptake due to the rapid immobilization of
phosphorus and the lack of adequate phytase levels in plants [
131
]. Phytase is a phosphatase
enzyme responsible for the transformation of organic phytate to inorganic phosphate, which
is acquirable for plant roots. Similarly, phytase-producing Bacillus strains can effectively
enhance plant growth through the liberation of reactive phosphorus from phytate and make
this element available for plant uptake. In the presence of phytate, the comparison of the
culture filtrate of B. amyloliquefaciens strain FZB45 with those of a phytase-deficient mutant
provided evidence that the phytase activity of strain FZB45 enhanced the growth of corn
seedling [
132
]. The bacterization of Brassica juncea with Bacillus sp. PB-13 considerably
boost phosphorus content and growth parameters in 30-day-old seedlings [
133
]. More
recently, the soil inoculation of Bacillus strain SD01N-014 resulted in the enhancement of
soil phosphorus content and the promotion of maize seedling growth [
134
]. Accordingly,
extracellular phytase activity of strain KR2-7 mediated with phy gene can be expectable.
In addition, the presence of genes involved in the biosynthesis of IAA and trehalose in
the KR2-7 genome (Table 6) is an indication of this strain’s potential in the mitigation
of salt toxic stress on plants. Inoculation of tomato plants subjected to salt stress with
OxtreS (trehalose over-expressing strain) mutant of Pseudomonas sp. UW4 considerably
boosted the dry weight, root and shoot length, and chlorophyll content of the tomato
plant [
135
]. Moreover, canola seedlings treated with over-expressed IAA transformant
of UW4 represented longer primary root with an increased number of root hairs than
seedlings treated with wild-type UW4 [
136
]. The growth promotion of root hairs by IAA
improves the assimilation of water and nutrients from the soil, which in turn raises plant
biomass [
136
]. Similarly, Japanese cypress seedlings inoculated with B. velezensis CE 100
showed significant increases in growth parameters and biomass due to the production of
indole-3-acetic acid (IAA) by CE 100 strain [137].
5. Conclusions
According to genome-driven data, along with chemical analysis results, strain KR2-7
most likely exploits four possible modes of action to control tomato Fusarium wilt, as
shown in Figure 11:
(1) Inhibition of the pathogen growth through the diffusion of antifungal and antibacterial
secondary metabolites and biofilm formation;
(2)
Stimulation of ISR in tomato via the production of surfactin and volatile organic
compounds;
(3)
Promotion of plant health and growth by producing plant growth promotion hor-
mones and polyamines, supplying iron for tomato, depriving the pathogen of iron,
Biology 2022,11, 137 20 of 26
and relieving heavy metal stress in the soil as a result of siderophore bacillibactin
activity;
(4)
Efficient colonization of plant roots.
The described modes of action were highly based on the identified gene clusters
encoding secondary metabolites and characterized gene/gene clusters involved in plant
colonization, plant growth promotion, and ISR. Furthermore, future studies using inte-
grated omics approaches and the mutagenesis of strain KR2-7 are required to approve the
aforesaid possible modes of action of strain KR2-7 and exact functions of the putative genes
and gene clusters in the suppression of fungal pathogen Fol.
Figure 11. Schematic presentation of putative biocontrol mechanism of strain KR2-7 against Fol. (A) An untreated
tomato plant in which Fol (yellow 16-point star) penetrated root tissue, colonized and blocked vascular system to
prevent water and nutrients from being transferred to plant organs. It caused yellowing began with bottom leaves,
followed by wilting, browning, and defoliation. Growth is typically stunted, and little or no fruit develops. (B)
Strain KR2-7 (blue rod) reaches to tomato root and colonizes on the root surface through its motility potential and
biofilm formation. As a result of root colonization, strain KR2-7 diffuses a wide variety of antifungal and
antibacterial secondary metabolites to establish a protective zone (green dash line semicircular) in tomato
rhizosphere. Strain KR2-7 directly limits the invasion of Fol fungal pathogen through diffused antifungal
secondary metabolites and also control the bacterial pathogens of tomato by means of produced antibacterial
secondary metabolites. Meanwhile, volatile organic compounds and surfactin stimulate tomato systemic resistance
to provide ISR-mediated protection (yellow dash line arrow) against phytopathogens. Moreover, the tomato
growth is enhanced assisted by growth-promoting hormones, polyamines, and siderophore bacillibactin.
A
B
Figure 11.
Schematic presentation of putative biocontrol mechanism of strain KR2-7 against Fol.
(
A
) An untreated tomato plant in which Fol (yellow 16-point star) penetrated root tissue, colonized
and blocked the vascular system to prevent water and nutrients from being transferred to plant
organs. It caused yellowing began with bottom leaves, followed by wilting, browning, and defoliation.
Growth is typically stunted, and little or no fruit develops. (
B
) Strain KR2-7 (blue rod) reaches to
tomato root and colonizes on the root surface through its motility potential and biofilm formation.
As a result of root colonization, strain KR2-7 diffuses a wide variety of antifungal and antibacterial
secondary metabolites to establish a protective zone (green dash line semicircular) in the tomato
rhizosphere. Strain KR2-7 directly limits the invasion of Fol fungal pathogen through diffused
antifungal secondary metabolites and also control the bacterial pathogens of tomato by means of
produced antibacterial secondary metabolites. Meanwhile, volatile organic compounds and surfactin
stimulate tomato systemic resistance to provide ISR-mediated protection (yellow dash line arrow)
against phytopathogens. Moreover, tomato growth is enhanced assisted by growth-promoting
hormones, polyamines, and siderophore bacillibactin.
Biology 2022,11, 137 21 of 26
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10
.3390/biology11010137/s1, Figure S1. Circular map of the B. inaquosorum KR2-7 genome. Outermost
circle (1st): all genes are color-coded according to their functions (see top right); 2nd circle: GC content
(black); 3rd circle: GC skew+ (green); 4th circle: GC skew
−
(violet); 5th circle: scale (bps). GC views
were prepared using CGView Server V1.0 (http://wishart.biology.ualberta.ca/cgview/), Table S1:
The comparison of COG functional categories between B. subtilis KR2-7 and B. velezensis FZB42,
Figure S2: The fengycin mass peaks detected by MALDI-TOF mass spectrometry in (A) control cell
extract of KR2-7 and (B) dual culture cell extract of KR2-7, Figure S3: The iturin, mycosubtilin, and
surfactin mass peaks detected by MALDI-TOF mass spectrometry in (A) control cell extract of KR2-7
and (B) dual culture cell extract of KR2-7.
Author Contributions:
M.K.; Conceptualization, Methodology, Formal analysis, Investigation, Data
curation, Writing–original draft and supplementary materials, Visualization. D.G.; Supervision,
review & editing, Funding acquisition. S.N.; Supervision, Methodology, review & editing. J.A.;
Supervision, review & editing. All authors have read and agreed to the published version of the
manuscript.
Funding:
This research was funded by Guangdong Science and Technology Department (project ID:
2020A0505090009), and partially supported by a fund from State Key Laboratory of Agrobiotechnol-
ogy (project ID: 8300052).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The genome sequence of B. inaquosorum KR2-7 was deposited in
NCBI GenBank under the accession number QZDE00000000.2.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Agrios, G.N. Plant Pathology, 5th ed.; Elsevier Academic Press: London, UK, 2005; pp. 522–534.
2.
Borrero, C.; Ordovas, J.; Trillas, M.I.; Aviles, M. Tomato Fusarium wilt suppressiveness. The relationship between the organic plant
growth media and their microbial communities as characterised by Biolog. Soil. Biol. Biochem. 2006,38, 1631–1637. [CrossRef]
3.
Elmer, W.H. Effects of acibenzolar-S-methyl on the suppression of Fusarium wilt of cyclamen. Crop Prot.
2006
,25, 671–676.
[CrossRef]
4.
L’Haridon, F.; Aimé, S.; Duplessis, S.; Alabouvette, C.; Steinberg, C.; Olivain, C. Isolation of differentially expressed genes during
interactions between tomato cells and a protective or a non-protective strain of Fusarium oxysporum.Physiol. Mol. Plant Pathol.
2011,76, 9–19. [CrossRef]
5.
Ajilogba, C.F.; Babalola, O.O. Integrated Management Strategies for Tomato Fusarium Wilt. Biocontrol. Sci.
2013
,18, 117–127.
[CrossRef] [PubMed]
6.
Arie, T. Fusarium diseases of cultivated plants, control, diagnosis, and molecular and genetic studies. J. Pestic. Sci.
2019
,44,
275–281. [CrossRef] [PubMed]
7.
Cawoy, H.; Bettiol, W.; Fickers, P.; Ongena, M. Bacillus-based biological control of plant diseases. In Pesticides in the Modern
World-Pesticides Use and Management, 1st ed.; Stoytcheva, M., Ed.; IntechOpen Press: London, UK, 2011; pp. 273–302. Available
online: https://www.intechopen.com/chapters/21989 (accessed on 21 May 2021).
8.
Raaijmakers, J.M.; Mazzola, M. Diversity and natural functions of antibiotics produced by beneficial and plant pathogenic
bacteria. Annu. Rev. Phytopathol. 2012,50, 403–424. [CrossRef]
9.
Kohl, J.; Kolnaar, R.; Ravensberg, W.J. Mode of action of microbial biological control agents against plant diseases: Relevance
beyond efficacy. Front. Plant Sci.
2019
,10, 845. Available online: https://www.frontiersin.org/article/10.3389/fpls.2019.00845
(accessed on 21 May 2021). [CrossRef]
10.
Conrath, U.; Beckers, G.J.M.; Langenbach, C.J.G.; Jaskiewicz, M.R. Priming for enhanced defense. Annu. Rev. Phytopathol.
2015
,
53, 97–119. [CrossRef] [PubMed]
11.
Spadaro, D.; Droby, S. Development of biocontrol products for postharvest diseases of fruit: The importance of elucidating the
mechanisms of action of yeast antagonists. Trends Food Sci. Technol. 2016,47, 39–49. [CrossRef]
12.
Paterson, J.; Jahanshah, G.; Li, Y.; Wang, Q.; Mehnaz, S.; Gross, H. The contribution of genome mining strategies to the
understanding of active principles of PGPR strains. FEMS Microbiol. Ecol. 2017,93, fiw249. [CrossRef] [PubMed]
13.
Chen, X.; Koumoutsi, A.; Scholz, R.; Eisenreich, A.; Schneider, K.; Heinemeyer, I.; Morgenstern, B.; Voss, B.; Hess, W.R.; Reva, O.;
et al. Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens
FZB42. Nat. Biotechnol. 2007,25, 1007–1014. [CrossRef]
Biology 2022,11, 137 22 of 26
14.
Grady, E.N.; MacDonald, J.; Ho, M.T.; Weselowski, B.; McDowell, T.; Solomon, O.; Renaud, J.; Yuan, Z.C. Characterization and
complete genome analysis of the surfactin-producing, plant-protecting bacterium Bacillus velezensis 9D-6. BMC Microbiol.
2019
,19,
5. [CrossRef]
15.
Knight, C.A.; Bowman, M.J.; Frederick, L.; Day, A.; Lee, C.; Dunlap, C.A. The first report of antifungal lipopeptide production by
a Bacillus subtilis subsp. inaquosorum strain. Microbiol. Res. 2018,216, 40–46. [CrossRef] [PubMed]
16.
Kamali, M.; Ahmadi, J.; Naeimi, S.; Guo, D. Characterization of Bacillus isolates from the rhizosphere of tomato suppressing
Fusarium wilt disease. Acta Phytopathol. Entomol. Hung. 2019,54, 53–68. [CrossRef]
17.
Skidmore, A.M.; Dickinson, C.H. Colony interactions and hyphal interference between Septoria Nodorumand phylloplane fungi.
Trans. Brit. Mycol. Soc. 1976,66, 57–64. [CrossRef]
18.
Vater, J.; Gao, X.; Hitzeroth, G.; Wilde, C.; Franke, P. “Whole cell”-matrix assisted laser desorption ionization-time of flight mass
spectrometry, an emerging technique for efficient screening of biocombinatorial libraries of natural compounds- present state of
research. Comb. Chem. High Throughput Screen. 2003,6, 557–567. [CrossRef] [PubMed]
19.
Tatusov, R.L.; Fedorova, N.D.; Jackson, J.D.; Jacobs, A.R.; Kiryutin, B.; Koonin, E.V.; Krylov, D.M.; Mazumder, R.; Mekhedov, S.L.;
Nikolskaya, A.N.; et al. The COG database: An updated version includes eukaryotes. BMC Bioinform. 2003,4, 41. [CrossRef]
20.
Bertels, F.; Silander, O.K.; Pachkov, M.; Rainey, P.B.; van Nimwegen, E. Automated reconstruction of whole-genome phylogenies
from short- sequence reads. Mol. Biol. Evol. 2014,31, 1077–1088. [CrossRef]
21.
Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol.
Biol. Evol. 2013,30, 2725–2729. [CrossRef]
22.
Felsenstein, J. Evolutionary trees from DNA sequences: A maximum likelihood approach. J. Mol. Evol.
1981
,17, 368–376.
[CrossRef]
23. Tavare, S. Some probabilistic and statistical problems in the analysis of DNA sequences. Am. Math. Soc. 1986,17, 57–86.
24.
Yoon, S.H.; Ha, S.M.; Kwon, S.; Lim, J.; Kim, Y.; Seo, H.; Chun, J. Introducing EzBioCloud: A taxonomically united database of
16S rRNA and whole genome assemblies. Int. J. Syst. Evol. Microbiol. 2017,67, 1613–1617. [CrossRef] [PubMed]
25.
Goris, J.; Konstantinidis, K.T.; Klappenbach, J.A.; Coenye, T.; Vandamme, P.; Tiedje, J.M. DNA-DNA hybridization values and
their relationship to whole-genome sequence similarities. Int. J. Syst. Evol. Microbiol. 2007,57, 81–91. [CrossRef] [PubMed]
26.
Richter, M.; Rosselló-Móra, R. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl. Acad. Sci.
USA 2009,106, 19126–19131. [CrossRef] [PubMed]
27.
Meier-Kolthoff, J.P.; Auch, A.F.; Klenk, H.P.; Göker, M. Genome sequence-based species delimitation with confidence intervals
and improved distance functions. BMC Bioinf. 2013,14, 60. [CrossRef]
28.
Medema, M.H.; Blin, K.; Cimermancic, P.; de Jager, V.; Zakrzewski, P.; Fischbach, M.A.; Weber, T.; Takano, E.; Breitling, R.
antiSMASH: Rapid identification, annotation, and analysis of secondary metabolite biosynthesis gene clusters in bacterial and
fungal genome sequences. Nucleic Acids Res. 2011,39, W339–W346. [CrossRef] [PubMed]
29.
Weber, T.; Blin, K.; Duddela, S.; Krug, D.; Kim, H.U.; Bruccoleri, R.; Lee, S.Y.; Fischbach, M.A.; Müller, R.; Wohlleben, W.; et al.
antiSMASH 3.0- a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res.
2015
,43,
W237–W243. [CrossRef]
30.
Dunlap, C.A.; Kim, S.J.; Kwon, S.W.; Rooney, A.P. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens;
Bacillus methylotrophicus,Bacillus amyloliquefaciens subsp. plantarum and “Bacillus oryzicola” are later heterotypic synonyms of
Bacillus velezensis based on phylogenomics. Int. J. Syst. Evol. Microbiol. 2016,66, 1212–1217. [CrossRef]
31.
Dunlap, C.A.; Bowman, M.J.; Rooney, A.P. Iturinic Lipopeptide diversity in the Bacillus subtilis species group–important
antifungals for plant disease biocontrol applications. Front. Microbiol. 2019,10, 1794. [CrossRef] [PubMed]
32.
Belbahri, L.; Chenari Bouket, A.; Rekik, I.; Alenezi, F.N.; Vallat, A.; Luptakova, L.; Petrovova, E.; Oszako, T.; Cherrad, S.; Vacher,
S.; et al. Comparative genomics of Bacillus amyloliquefaciens strains reveals a core genome with traits for habitat adaptation and a
secondary metabolites rich accessory genome. Front. Microbiol. 2017,8, 1438. [CrossRef]
33.
Stein, T. Bacillus subtilis antibiotics: Structures, syntheses and specific functions. Mol. Microbiol.
2005
,56, 845–857. [CrossRef]
[PubMed]
34.
Schneider, K.; Chen, X.; Vater, J.; Franke, P.; Nicholson, G.; Borriss, R.; Süssmuth, R.D. Macrolactin is the polyketide biosynthesis
product of the pks2 cluster of Bacillus amyloliquefaciens FZB42. J. Nat. Prod. 2007,70, 1417–1423. [CrossRef] [PubMed]
35.
Ongena, M.; Henry, G.; Thornart, P. The roles of cyclic lipopeptides in the biocontrol activity of Bacillus subtilis. In Recent Develop-
ments in Management of Plant Diseases, 1st ed.; Gisi, U., Chet, I., Gullino, M., Eds.; Springer Press:
Dordrecht, The Netherlands
,
2009; Volume 1, pp. 59–69. [CrossRef]
36.
Chen, L.; Heng, J.; Qin, S.; Bian, K. A comprehensive understanding of the biocontrol potential of Bacillus velezensis LM2303
against Fusarium head blight. PLoS ONE 2018,13, e0198560. [CrossRef]
37.
Ongena, M.; Jacques, P. Bacillus lipopeptides: Versatile weapons for plant disease biocontrol. Trends Microbiol.
2008
,16, 115–125.
[CrossRef] [PubMed]
38.
Koumoutsi, A.; Chen, X.H.; Henne, A.; Liesegang, H.; Hitzeroth, G.; Franke, P.; Vater, J.; Borriss, R. Structural and functional
characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens
strain FZB42. J. Bacteriol. 2004,186, 1084–1096. [CrossRef] [PubMed]
39.
Dimkic, I.; Stankovic, S.; Nišavic, M.; Petkovic, M.; Ristivojevic, P.; Fira, D.; Beric, T. The profile and antimicrobial activity of
Bacillus lipopeptide extracts of five potential biocontrol strains. Front. Microbiol. 2017,8, 925. [CrossRef] [PubMed]
Biology 2022,11, 137 23 of 26
40.
Mhammedi, A.; Peypoux, F.; Besson, F.; Michel, G. Bacillomycin f, a new antibiotic of iturin group: Isolation and characterization.
J. Antibiot. 1982,35, 306–311. [CrossRef] [PubMed]
41.
Phae, C.G.; Shoda, M.; Kubota, H. Suppressive effect of Bacillus subtilis and its products on phytopathogenic microorganisms. J.
Ferment. Bioeng. 1990,69, 1–7. [CrossRef]
42.
Moyne, A.L.; Shelby, R.; Cleveland, T.E.; Tuzun, S. Bacillomycin D: An iturin with antifungal activity against Aspergillus flavus.J.
Appl. Microbiol. 2001,90, 622–629. [CrossRef]
43.
Yu, G.Y.; Sinclair, J.B.; Hartman, G.L.; Bertagnolli, B.L. Production of iturin A by Bacillus amyloliquefaciens suppressing Rhizoctonia
solani.Soil Biol. Biochem. 2002,34, 955–963. [CrossRef]
44.
Aranda, F.J.; Teruel, J.A.; Ortiz, A. Further aspects on the haemolytic activity of the antibiotic lipopeptide iturin A. Biochim.
Biophys. Acta 2005,17, 51–56. [CrossRef] [PubMed]
45.
Besson, F.; Michel, G. Action of mycosubtilin, an antifungal antibiotic of Bacillus subtilis, on the cell membrane of Saccharomyces
cerevisiae.Microbios 1989,59, 113–121.
46.
Garcia-Gutierrez, L.; Zeriouh, H.; Romero, D.; Cubero, J.; Vicente, A.; Perez-Garcia, A. The antagonistic strain Bacillus subtilis
UMAF6639 also confers protection to melon plants against cucurbit powdery mildew by activation of jasmonate-and salicylic
acid-dependent defence responses. Microb. Biotechnol. 2013,6, 264–274. [CrossRef] [PubMed]
47.
Henry, G.; Deleu, M.; Jourdan, E.; Thonart, P.; Ongena, M. The bacterial lipopeptide surfactin targets the lipid fraction of the plant
plasma membrane to trigger immune-related defence responses. Cell Microbiol. 2011,13, 1824–1837. [CrossRef] [PubMed]
48.
Khong, N.G.; Randoux, B.; Tayeh, C.; Coutte, F.; Bourdon, N.; Tisserant, B.; Laruelle, F.; Jacques, P.; Reignault, P. Induction of
resistance in wheat against powdery mildew by bacterial cyclic lipopeptides. Commun. Agric. Appl. Biol. Sci.
2012
,77, 39–51.
[PubMed]
49.
Waewthongrak, W.; Leelasuphakul, W.; McCollum, G. Cyclic LIPopeptides from Bacillus subtilis ABS-S14 elicit defense-related
gene expression in citrus fruit. PLoS ONE 2014,9, e109386. [CrossRef]
50.
Chowdhury, S.P.; Hartmann, A.; Gao, X.; Borriss, R. Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42-a
review. Front. Microbiol. 2015,6, 780. [CrossRef] [PubMed]
51.
Li, Y.; Héloir, M.C.; Zhang, X.; Geissler, M.; Trouvelot, S.; Jacquens, L.; Henkel, M.; Su, X.; Fang, W.; Wang, Q.; et al. Surfactin and
fengycin contribute to the protection of a Bacillus subtilis strain against grape downy mildew by both direct effect and defence
stimulation. Mol. Plant Pathol. 2019,20, 1037–1050. [CrossRef]
52.
Sarwar, A.; Hassan, M.N.; Imran, M.; Iqbal, M.; Majeed, S.; Brader, G.; Sessitsch, A.; Hafeez, F.Y. Biocontrol activity of surfactin A
purified from Bacillus NH-100 and NH-217 against rice bakanae disease. Microbiol. Res. 2018,209, 1–13. [CrossRef]
53.
Wu, S.; Liu, G.; Zhou, S.; Sha, Z.; Sun, C. Characterization of antifungal lipopeptide biosurfactants produced by marine bacterium
Bacillus sp. CS30. Mar. Drugs 2019,17, 199. [CrossRef]
54.
Krishnan, N.; Velramar, B.; Velu, R.K. Investigation of antifungal activity of surfactin against mycotoxigenic phytopathogenic
fungus Fusarium moniliforme and its impact in seed germination and mycotoxicosis. Pestic. Biochem. Physiol.
2019
,155, 101–107.
[CrossRef] [PubMed]
55.
Hazarika, D.J.; Goswami, G.; Gautom, T.; Parveen, A.; Das, P.; Barooah, M.; Boro, R.C. Lipopeptide mediated biocontrol activity
of endophytic Bacillus subtilis against fungal phytopathogens. BMC Microbiol. 2019,19, 71. [CrossRef]
56.
Fan, H.; Zhang, Z.; Li, Y.; Zhang, X.; Duan, Y.; Wang, Q. Biocontrol of bacterial fruit blotch by Bacillus subtilis 9407 via surfactin-
mediated antibacterial activity and colonization. Front. Microbiol. 2017,8, 1973. [CrossRef] [PubMed]
57.
Xiong, H.Q.; Li, Y.T.; Cai, Y.F.; Cao, Y.; Wang, Y. Isolation of Bacillus amyloliquefaciens JK6 and identification of its lipopeptides
surfactin for suppressing tomato bacterial wilt. RSC Adv. 2015,5, 82042–82049. [CrossRef]
58.
Baise, H.P.; Fall, R.; Vivanco, J.M. Biocontrol of Bacillus subtilis against infection of Arabidopsis roots by Pseudomonas syringae is
facilitated by biofilm formation and surfactin production. Plant Physiol. 2004,134, 307–319. [CrossRef] [PubMed]
59.
Fernandes, P.; Arruda, I.R.; Santos, A.; Araújo, A.A.; Maior, A.M.; Ximenes, E. Antimicrobial activity of surfactants produced by
Bacillus subtilis r14 against multidrug-resistant bacteria. Braz. J. Microbiol. 2007,38, 704–709. [CrossRef]
60.
Patel, P.S.; Huang, S.; Fisher, S.; Pirnik, D.; Aklonis, C.; Dean, L.; Meyers, E.; Fernandes, P.; Mayerl, F. Bacillaene, a novel inhibitor
of procaryotic protein synthesis produced by Bacillus subtilis: Production, taxonomy, isolation, physico-chemical characterization
and biological activity. J. Antibiot. 1995,48, 997–1003. [CrossRef] [PubMed]
61.
Um, S.; Fraimout, A.; Sapountzis, P.; Oh, D.C.; Poulsen, M. The fungus-growing termite Macrotermes natalensis harbors
bacillaene-producing Bacillus sp. that inhibit potentially antagonistic fungi. Sci. Rep. 2013,3, 3250. [CrossRef] [PubMed]
62.
Muller, S.; Strack, S.N.; Hoefler, B.C.; Straight, P.; Kearns, D.B.; Kirby, J.R. Bacillaene and sporulation protect Bacillus subtilis from
predation by Myxococcus xanthus.Appl. Environ. Microbiol. 2014,80, 5603–5610. [CrossRef] [PubMed]
63.
Romero-Tabarez, M.; Jansen, R.; Sylla, M.; Lunsdorf, H.; Haussler, S.; Santosa, D.A.; Timmis, K.N.; Molinari, G. 7-O-malonyl
macrolactin A, a new macrolactin antibiotic from Bacillus subtilis active against methicillin-resistant Staphylococcus aureus,
vancomycin-resistant enterococci, and a small-colony variant of Burkholderia cepacia.Antimicrob. Agents Chemother.
2006
,50,
1701–1709. [CrossRef] [PubMed]
64.
Yuan, J.; Li, B.; Zhang, N.; Waseem, R.; Shen, Q.; Huang, Q. Production of bacillomycin- and macrolactin-type antibiotics by
Bacillus amyloliquefaciens NJN-6 for suppressing soilborne plant pathogens. J. Agric. Food Chem.
2012
,60, 2976–2981. [CrossRef]
[PubMed]
Biology 2022,11, 137 24 of 26
65.
Chen, X.H.; Scholz, R.; Borriss, M.; Junge, H.; Mogel, G.; Kunz, S.; Borriss, R. Difficidin and bacilysin produced by plant-associated
Bacillus amyloliquefaciens are efficient in controlling fire blight disease. J. Biotechnol. 2009,140, 38–44. [CrossRef] [PubMed]
66.
Wu, L.; Wu, H.; Chen, L.; Yu, X.; Borriss, R.; Gao, X. Difficidin and bacilysin from Bacillus amyloliquefaciens FZB42 have antibacterial
activity against Xanthomonas oryzae rice. Sci. Rep. 2015,5, 12975. [CrossRef] [PubMed]
67.
Wu, L.; Wu, H.; Chen, L.; Lin, L.; Borriss, R.; Gao, X. Bacilysin overproduction in Bacillus amyloliquefaciens FZB42 markerless
derivative strains FZBREP and FZBSPA enhances antibacterial activity. Appl. Microbiol. Biotechnol.
2015
,99, 4255–4263. [CrossRef]
[PubMed]
68.
Steinborn, G.; Hajirezaei, M.R.; Hofemeister, J. bac genes for recombinant bacilysin and anticapsin production in Bacillus host
strains. Arch. Microbiol. 2005,183, 71–79. [CrossRef]
69.
Mahlstedt, S.A.; Walsh, C.T. Investigation of anticapsin biosynthesis reveals a four-enzyme pathway to tetrahydrotyrosine in
Bacillus subtilis.Biochemistry 2010,49, 912–923. [CrossRef]
70.
Babasaki, K.; Takao, T.; Shimonishi, Y.; Kurahashi, K. Subtilosin A, a new antibiotic peptide produced by Bacillus subtilis 168:
Isolation, structural analysis, and biogenesis. J. Biochem. 1985,98, 585–603. [CrossRef] [PubMed]
71.
Stein, T.; Düsterhus, S.; Stroh, A.; Entian, K.D. Subtilosin production by two Bacillus subtilis subspecies and variance of the sbo-alb
cluster. Appl. Environ. Microbiol. 2004,70, 2349–2353. [CrossRef] [PubMed]
72.
Sutyak, K.E.; Wirawan, R.E.; Aroutcheva, A.A.; Chikindas, M.L. Isolation of the Bacillus subtilis antimicrobial peptide subtilosin
from the dairy product-derived Bacillus amyloliquefaciens. J. Appl. Microbiol. 2008,104, 1067–1074. [CrossRef] [PubMed]
73.
Shelburne, C.E.; An, F.Y.; Dholpe, V.; Ramamoorthy, A.; Lopatin, D.E.; Lantz, M.S. The spectrum of antimicrobial activity of the 18
bacteriocin subtilosin A. J. Antimicrob. Chemother. 2007,59, 297–300. [CrossRef] [PubMed]
74.
Wiedemann, I.; Breukink, E.; Kraaij, C.V.; Kuipers, O.S.; Bierbaum, G.; de Kruijff, B.; Sahl, H.G. Specific binding of nisin to the
peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity. J.
Biol. Chem. 2001,276, 1772–1779. [CrossRef] [PubMed]
75.
Thennarasu, S.; Lee, D.K.; Poon, A.; Kawulka, K.E.; Vederas, J.C.; Ramamoorthy, A. Membrane permeabilization, orientation, and
antimicrobial mechanism of subtilosin A. Chem. Phys. Lipids 2005,137, 38–51. [CrossRef] [PubMed]
76.
Zheng, G.; Hehn, R.; Zuber, P. Mutational analysis of sbo-alb locus of Bacillus subtilis: Identification of genes required for subtilosin
production and immunity. J. Bacteriol. 2000,182, 3266–3273. [CrossRef] [PubMed]
77.
González-Pastor, J.E.; Hobbs, E.C.; Losick, R. Cannibalism by sporulating bacteria. Science
2003
,301, 510–513. [CrossRef]
[PubMed]
78.
González-Pastor, J.E. Cannibalism: A social behavior in sporulating Bacillus subtilis.FEMS Microbiol. Rev.
2011
,35, 415–424.
[CrossRef] [PubMed]
79.
Lin, D.; Qu, L.J.; Gu, H.; Chen, Z. A 3.1-kb genomic fragment of Bacillus subtilis encodes the protein inhibiting growth of
Xanthomonas oryzae pv. oryzae.J. Appl. Microbiol. 2001,91, 1044–1050. [CrossRef]
80.
Guo, S.; Li, X.; He, P.; Ho, H.; Wu, W.; He, Y. Whole-genome sequencing of Bacillus subtilis XF-1 reveals mechanisms for biological
control and multiple beneficial properties in plants. J. Ind. Microbiol. Biotechnol. 2015,42, 925–937. [CrossRef]
81.
Peypoux, F.; Bonmatin, J.M.; Wallach, J. Recent trends in the biochemistry of surfactin. Appl. Microbiol. Biotechnol.
1999
,51,
553–563. [CrossRef]
82.
Bonmatin, J.M.; Laprévote, O.; Peypoux, F. Diversity among microbial cyclic lipopeptides: Iturins and surfactins. Activity-
structure relationships to design new bioactive agents. Comb. Chem. High Throughput Screen. 2003,6, 541–556. [CrossRef]
83.
Zeriouh, H.; de Vicente, A.; Pérez-García, A.; Romero, D. Surfactin triggers biofilm formation of Bacillus subtilis in melon
phylloplane and contributes to the biocontrol activity. Environ. Microbiol. 2013,16, 2196–2211. [CrossRef]
84.
Wipat, A.; Harwood, C.R. The Bacillus subtilis genome sequence: The molecular blueprint of a soil bacterium. FEMS Microbiol.
Ecol. 1999,28, 1–9. [CrossRef]
85.
Bezzate, S.; Aymerich, S.; Chambert, R.; Czarnes, S.; Berge, O.; Heulin, T. Disruption of the Paenibacillus polymyxa levansucrase
gene impairs its ability to aggregate soil in the wheat rhizosphere. Environ. Microbiol. 2000,2, 333–342. [CrossRef] [PubMed]
86.
Kearns, D.B.; Chu, F.; Rudner, R.; Losick, R. Genes governing swarming in Bacillus subtilis and evidence for a phase variation
mechanism controlling surface motility. Mol. Microbiol. 2004,52, 357–369. [CrossRef] [PubMed]
87.
Calvio, C.; Celandroni, F.; Ghelardi, E.; Amati, G.; Salvetti, S.; Ceciliani, F.; Galizzi, A.; Senesi, S. Swarming differentiation
and swimming motility in Bacillus subtilis are controlled by swrA, a newly identified dicistronic operon. J. Bacteriol.
2005
,187,
5356–5366. [CrossRef]
88.
Tsuge, K.; Ohata, Y.; Shoda, M. Gene yerP, involved in surfactin self resistance in Bacillus subtilis.Antimicrob. Agents Chemother.
2001,45, 3566–3573. [CrossRef] [PubMed]
89.
Yu, Y.; Yan, F.; Chen, Y.; Jin, C.; Guo, J.H.; Chai, Y. Poly-
γ
-glutamic acids contribute to biofilm formation and plant root colonization
in selected environmental isolates of Bacillus subtilis.Front. Microbiol. 2016,7, 1811. [CrossRef]
90.
Lazarevic, V.; Soldo, B.; Médico, N.; Pooley, H.; Bron, S.; Karamata, D. Bacillus subtilis alpha-Phosphoglucomutase is required for
normal cell morphology and biofilm formation. Appl. Environ. Microbiol. 2005,71, 39–45. [CrossRef] [PubMed]
91.
Branda, S.S.; González-Pastor, J.E.; Dervyn, E.; Ehrlich, S.D.; Losick, R.; Kolter, R. Genes involved in formation of structural
multicellular communities by Bacillus subtilis.J. Bacteriol. 2004,186, 3970–3979. [CrossRef]
92.
Kearns, D.B.; Chu, F.; Branda, S.S.; Kolter, R.; Losick, R. A master regulator for biofilm formation by Bacillus subtilis.Mol. Microbiol.
2005,55, 739–749. [CrossRef]
Biology 2022,11, 137 25 of 26
93.
Branda, S.S.; González-Pastor, J.E.; Ben-Yehuda, S.; Losick, R.; Kolter, R. Fruiting body formation by Bacillus subtilis.Proc. Natl.
Acad. Sci. USA 2001,98, 11621–11626. [CrossRef]
94.
Chu, F.; Kearns, D.B.; McLoon, A.; Chai, Y.; Kolter, R.; Losick, R. A novel regulatory protein governing biofilm formation in
Bacillus subtilis.Mol. Microbiol. 2008,68, 1117–1127. [CrossRef]
95.
Chu, F.; Kearns, D.B.; Branda, S.S.; Kolter, R.; Losick, R. Targets of the master regulator of biofilm formation in Bacillus subtilis.
Mol. Microbiol. 2006,59, 1216–1228. [CrossRef]
96.
Harman, G.E. Multifunctional fungal plant symbionts: New tools to enhance plant growth and productivity. New Phytol.
2011
,
189, 647–649. [CrossRef]
97.
Shafi, J.; Tian, H.; Ji, M. Bacillus species as versatile weapons for plant pathogens: A review. Biotechnol. Biotechnol. Equip.
2017
,31,
446–459. [CrossRef]
98.
Holtmann, G.; Bakker, E.P.; Uozumi, N.; Bremer, E. KtrAB and KtrCD: Two K+ uptake systems in Bacillus subtilis and their role in
adaptation to hypertonicity. J. Bacteriol. 2003,185, 1289–1298. [CrossRef]
99.
Miethke, M.; Klotz, O.; Linne, U.; May, J.J.; Beckering, C.L.; Marahiel, M.A. Ferri-bacillibactin uptake and hydrolysis in Bacillus
subtilis.Mol. Microbiol. 2006,61, 1413–1427. [CrossRef] [PubMed]
100.
Chen, K.; Tian, Z.; Luo, Y.; Cheng, Y.; Long, C. Antagonistic Activity and the Mechanism of Bacillus amyloliquefaciens DH-4 Against
Citrus Green Mold. Phytopathology 2018,108, 1253–1262. [CrossRef]
101.
Rajkumar, M.; Ae, N.; Prasad, M.N.; Freitas, H. Potential of siderophore-producing bacteria for improving heavy metal phytoex-
traction. Trends Biotechnol. 2010,28, 142–149. [CrossRef] [PubMed]
102.
Radzki, W.; Gutierrez Manero, F.J.; Algar, E.; Lucas Garcıa, J.A.; Garcıa-Villaraco, A.; Ramos Solano, B. Bacterial siderophores
efficiently provide iron to iron-starved tomato plants in hydroponics culture. Antonie Leeuwenhoek
2013
,104, 321–330. [CrossRef]
[PubMed]
103.
Lurthy, T.; Cantat, C.; Jeudy, C.; Declerck, P.; Gallardo, K.; Barraud, C.; Leroy, F.; Ourry, A.; Lemanceau, P.; Salon, C.; et al. Impact
of bacterial siderophores on iron status and ionome in pea. Front. Plant Sci. 2020,11, 730. [CrossRef]
104.
Arguelles-Arias, A.; Ongena, M.; Halimi, B.; Lara, Y.; Brans, A.; Joris, B.; Fickers, P. Bacillus amyloliquefaciens GA1 as a source of
potent antibiotics and other secondary metabolites for biocontrol of plant pathogens. Microb. Cell Fact. 2009,8, 63. [CrossRef]
105.
Yu, X.; Ai, C.; Xin, L.; Zhou, G. The siderophore-producing bacterium, Bacillus subtilis CAS15, has a biocontrol effect on Fusarium
wilt and promotes the growth of pepper. Eur. J. Soil Biol. 2011,47, 138–145. [CrossRef]
106.
Woo, S.M.; Kim, S.D. Structural identification of siderophore (AH18) from Bacillus subtilis AH18, a biocontrol agent of phytoph-
thora blight disease in red-pepper. Kor. J. Microbiol. Biotechnol. 2008,36, 326–335. [CrossRef]
107.
Tahir, H.A.; Gu, Q.; Wu, H.; Raza, W.; Hanif, A.; Wu, L.; Colman, M.V.; Gao, X. Plant Growth Promotion by Volatile Organic
Compounds Produced by Bacillus subtilis SYST2. Front. Microbiol. 2017,8, 171. [CrossRef] [PubMed]
108.
Lee, B.; Farag, M.A.; Park, H.B.; Kloepper, W.J.; Lee, S.H.; Ryu, C.M. Induced resistance by a long-chain bacterial volatile:
Elicitation of plant systemic defense by a C13 volatile produced by Paenibacillus polymyxa.PLoS ONE
2012
,7, e48744. [CrossRef]
109.
Park, Y.S.; Dutta, S.; Ann, M.; Raaijmakers, J.M.; Park, K. Promotion of plant growth by Pseudomonas fluorescens strain SS101 via
novel volatile organic compounds. Biochem. Biophys. Res. Commun. 2015,461, 361–365. [CrossRef] [PubMed]
110.
Tahir, H.A.; Gu, Q.; Wu, H.; Niu, Y.; Huo, R.; Gao, X. Bacillus volatiles adversely affect the physiology and ultra-structure of
Ralstonia solanacearum and induce systemic resistance in tobacco against bacterial wilt. Sci. Rep. 2017,7, 40481. [CrossRef]
111.
Zhang, X.; Zhang, R.; Bao, T.; Yang, T.; Xu, M.; Li, H.; Xu, Z.; Rao, Z. Moderate expression of the transcriptional regulator ALsR
enhances acetoin production by Bacillus subtilis.J. Ind. Microbiol. Biotechnol. 2013,40, 1067–1076. [CrossRef]
112.
Goupil-feuillerat, N.; Cocaign-Bousquet, M.; Godon, J.J.; Ehrlich, S.D.; Renault, P. Dual Role of
α
—Acetolactate Decarboxylase in
Lactococcus lactis subsp. Lactis.J. Bacteriol. 1997,179, 6285–6293. [CrossRef]
113.
Zhou, C.; Ma, Z.; Zhu, L.; Xiao, X.; Xie, Y.; Zhu, J.; Wang, J. Rhizobacterial strain Bacillus megaterium BOFC15 induces cellular
polyamine changes that improve plant growth and drought resistance. Int. J. Mol. Sci. 2016,17, 976. [CrossRef] [PubMed]
114.
Rooney, A.P.; Price, N.P.J.; Ehrhardt, C.; Sewzey, J.L.; Bannan, J.D. Phylogeny and molecular taxonomy of the Bacillus subtilis
species complex and description of Bacillus subtilis subsp. inaquosorum subsp. nov. Int. J. Syst. Evol. Microbiol.
2009
,59, 2429–2436.
[CrossRef]
115.
Yi, H.; Chun, J.; Cha, C.J. Genomic insights into the taxonomic status of the three subspecies of Bacillus subtilis.Syst. Appl.
Microbiol. 2014,37, 95–99. [CrossRef] [PubMed]
116.
Nakamura, L.K.; Roberts, M.S.; Cohan, F.M. Relationship of Bacillus subtilis clades associated with strains 168 and W23: A
proposal for Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis subsp. spizizenii subsp. nov. Int. J. Syst. Bacteriol.
1999
,
49, 1211–1215. [CrossRef]
117.
Brito, P.H.; Chevreux, B.; Serra, C.R.; Schyns, G.; Henriques, A.O.; Pereira-Leal, J.B. Genetic competence drives genome diversity
in Bacillus subtilis.Genome Biol. Evol. 2018,10, 108–124. [CrossRef] [PubMed]
118.
Dunlap, C.A.; Bowman, M.J.; Zeigler, D.I. Promotion of Bacillus subtilis subsp. inaquosorum,Bacillus subtilis subsp. spizizenii and
Bacillus subtilis subsp. stercoris to species status. Antonie Leeuwenhoek 2020,113, 1–12. [CrossRef] [PubMed]
119.
Weng, J.; Wang, Y.; Li, J.; Shen, Q.; Zhang, R. Enhanced root colonization and biocontrol activity of Bacillus amyloliquefaciens
SQR9 by abrB gene disruption. Appl. Microbiol. Biotechnol. 2012,97, 8823–8830. [CrossRef] [PubMed]
Biology 2022,11, 137 26 of 26
120.
Chowdhury, S.P.; Dietel, K.; Rändler, M.; Schmid, M.; Junge, H.; Borriss, R.; Hartmann, A.; Grosch, R. Effects of Bacillus
amyloliquefaciens FZB42 on lettuce growth and health under pathogen pressure and its impact on the rhizosphere bacterial
community. PLoS ONE 2013,8, e68818. [CrossRef]
121.
Zhu, M.L.; Wu, X.Q.; Wang, Y.H.; Dai, Y. Role of biofilm formation by Bacillus pumilus HR10 in biocontrol against pine seedling
damping-off disease caused by Rhizoctonia solani.Forests 2020,11, 652. [CrossRef]
122.
Steller, S.; Vater, J. Purification of the fengycin synthetase multienzyme system from Bacillus subtilis b213. J. Chromatogr. B Biomed.
Sci. Appl. 2000,737, 267–275. [CrossRef]
123.
Deleu, M.; Paquot, M.; Nylander, T. Fengycin interaction with lipid monolayers at the air-aqueous interface-implications for the
effect of fengycin on biological membranes. J. Colloid Interface Sci. 2005,283, 358–365. [CrossRef]
124.
Wang, J.; Liu, J.; Chen, H.; Yao, J. Characterization of Fusarium graminearum inhibitory lipopeptide from Bacillus subtilis IB. Appl.
Microbiol. Biotechnol. 2007,76, 889–894. [CrossRef] [PubMed]
125.
Hanif, A.; Zhang, F.; Li, P.; Li, C.; Xu, Y.; Zubair, M.; Zhang, M.; Jia, D.; Zhao, X.; Liang, J.; et al. Fengycin produced by Bacillus
amyloliquefaciens FZB42 inhibits Fusarium graminearum growth and mycotoxins biosynthesis. Toxins
2019
,11, 295. [CrossRef]
[PubMed]
126.
Ongena, M.; Jourdan, E.; Adam, A.; Paquot, M.; Brans, A.; Joris, B.; Arpigny, J.L.; Thonart, P. Surfactin and fengycin lipopeptides
of Bacillus subtilis as elicitors of induced systemic resistance in plants. Environ. Microbiol.
2007
,9, 1084–1090. [CrossRef] [PubMed]
127.
Cao, Y.; Xu, Z.; Ling, N.; Yuan, Y.; Yang, X.; Chen, L.; Shen, B.; Shen, Q. Isolation and identification of lipopeptides produced by B.
subtilis SQR 9 for suppressing Fusarium wilt of cucumber. Sci. Hortic. 2012,135, 32–39. [CrossRef]
128.
Yuan, J.; Raza, W.; Huang, Q.; Shen, Q. The ultrasound-assisted extraction and identification of antifungal substances from B.
amyloliquefaciensstrain NJN-6 suppressing Fusarium oxysporum.J. Basic Microbiol. 2012,52, 721–730. [CrossRef] [PubMed]
129.
Zhang, L.; Sun, C. Fengycins, cyclic lipopeptides from marine Bacillus subtilis strains, kill the plant-pathogenic fungus Magnaporthe
grisea by inducing reactive oxygen species production and chromatin condensation. Appl. Environ. Microbiol.
2018
,84, e00445-18.
[CrossRef] [PubMed]
130.
Cao, Y.; Pi, H.; Chandrangsu, P.; Li, Y.; Wang, Y.; Zhou, H.; Xiong, H.; Helman, J.D.; Cai, Y. Antagonism of two plant-growth
promoting Bacillus velezensis isolates against Ralstonia solanacearum and Fusarium oxysporum.Sci Rep. 2018,8, 4360. [CrossRef]
131.
Singh, B.; Satyanarayana, T. Microbial phytases in phosphorus acquisition and plant growth promotion. Physiol. Mol. Biol. Plants
2011,17, 93–103. [CrossRef]
132.
Idriss, E.E.; Makarewicz, O.; Farouk, A.; Rosner, K.; Greiner, R.; Bochow, H.; Richter, T.; Borriss, R. Extracellular phytase activity
of Bacillus amyloliquefaciens FZB45 contributes to its plant-growth-promoting effect. Microbiology
2002
,148, 2097–2109. [CrossRef]
133.
Kumar, V.; Singh, P.; Jorquera, M.A.; Sangwan, P.; Kumar, P.; Verma, A.K.; Agrawal, S. Isolation of phytase-producing bacteria
from Himalayan soils and their effect on growth and phosphorus uptake of Indian mustard (Brassica juncea). World J. Microbiol.
Biotechnol. 2013,29, 1361–1369. [CrossRef]
134.
Liu, L.; Li, A.; Chen, J.; Su, Y.; Li, Y.; Ma, S. Isolation of a phytase-producing bacterial strain from agricultural soil and its
characterization and application as an effective eco-friendly phosphate solubilizing bioinoculant. Commun. Soil Sci. Plant
2018
,49,
984–994. [CrossRef]
135.
Orozco-Mosqueda, M.C.; Duan, J.; DiBernardo, M.; Zetter, E.; Campos-García, J.; Glick, B.R.; Santoyo, G. The production of ACC
deaminase and trehalose by the plant growth promoting bacterium Pseudomonas sp. UW4 synergistically protect tomato plants
against salt stress. Front. Microbiol. 2019,10, 1392. [CrossRef] [PubMed]
136.
Duca, D.R.; Rose, D.R.; Glick, B.R. Indole acetic acid overproduction transformants of the rhizobacterium Pseudomonas sp. UW4.
Antonie Leeuwenhoek 2018,111, 1645–1660. [CrossRef] [PubMed]
137.
Moon, J.-H.; Won, S.-J.; Maung, C.E.H.; Choi, J.-H.; Choi, S.-I.; Ajuna, H.B.; Ahn, Y.S. Bacillus velezensis CE 100 Inhibits Root
Rot Diseases (Phytophthora spp.) and Promotes Growth of Japanese Cypress (Chamaecyparis obtusa Endlicher) Seedlings.
Microorganisms 2021,9, 821. [CrossRef]
