Genomic Insight into a Potential Biological Control Agent for Fusarium-Related Diseases in Potatoes: Bacillus cabrialesii Subsp. cabrialesii Strain PE1

Abstract

Bacillus strain PE1, which was isolated from potatoes harvested in the Yaqui Valley, Mexico, was evaluated as a potential biological control agent against Fusarium languescens. The draft genome sequence was obtained through Illumina NovaSeq sequencing, revealing a genomic size of 4,071,293 bp, with a G + C content of 44.13%, an N50 value of 357,305 bp, and 27 contigs. The taxonomic affiliation was confirmed by analyzing the 16S rRNA gene and overall genome relatedness indices (OGRIs) and constructing a phylogenomic tree based on the whole genome, which showed a close relationship to Bacillus cabrialesii subsp. cabrialesii. Genomic annotation using RAST and Prokka identified 4261 coding DNA sequences (CDSs) distributed across 331 subsystems, highlighting genes associated with biocontrol, stress response, and iron acquisition. AntiSMASH 7.1 was used for genome mining, revealing seven biosynthetic gene clusters that potentially produce biocontrol-related metabolites. In vitro assays confirmed the antagonistic activity of strain PE1 against Fusarium languescens CE2, demonstrating its potential to inhibit mycelial growth. The study provides a genomic basis for investigating B. cabrialesii subsp. cabrialesii PE1 as a potential biological control agent in potato production.

Full text

Citation: Valenzuela-Aragon, B.;
Montoya-Martínez, A.C.; Parra-Cota,
F.I.; de los Santos-Villalobos, S.
Genomic Insight into a Potential
Biological Control Agent for
Fusarium-Related Diseases in Potatoes:
Bacillus cabrialesii Subsp. cabrialesii
Strain PE1. Horticulturae 2024,10, 357.
https://doi.org/10.3390/
horticulturae10040357
Academic Editors: Rafael José
Carvalho Mendes, Leandro Pereira
Dias, Renato Lopes Gil and
Fernando Tavares
Received: 2 March 2024
Revised: 31 March 2024
Accepted: 3 April 2024
Published: 4 April 2024
Copyright: © 2024 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/).
horticulturae
Article
Genomic Insight into a Potential Biological Control Agent for
Fusarium-Related Diseases in Potatoes: Bacillus cabrialesii
Subsp. cabrialesii Strain PE1
Brenda Valenzuela-Aragon 1, Amelia C. Montoya-Martínez 1, Fannie Isela Parra-Cota 2
and Sergio de los Santos-Villalobos 1,*
1Instituto Tecnológico de Sonora, 5 de Febrero 818 sur, Ciudad Obregon 85000, Sonora, Mexico;
brendavalenzuelaaragon@gmail.com (B.V.-A.)
2Campo Experimental Norman E. Borlaug, Instituto Nacional de Investigaciones Forestales, Agrícolas y
Pecuarias, Ciudad Obregon 85000, Sonora, Mexico
*Correspondence: sergio.delossantos@itson.edu.mx
Abstract: Bacillus strain PE1, which was isolated from potatoes harvested in the Yaqui Valley, Mex-
ico, was evaluated as a potential biological control agent against Fusarium languescens. The draft
genome sequence was obtained through Illumina NovaSeq sequencing, revealing a genomic size
of 4,071,293 bp, with a G + C content of 44.13%, an N50 value of 357,305 bp, and 27 contigs. The
taxonomic affiliation was confirmed by analyzing the 16S rRNA gene and overall genome relatedness
indices (OGRIs) and constructing a phylogenomic tree based on the whole genome, which showed
a close relationship to Bacillus cabrialesii subsp. cabrialesii. Genomic annotation using RAST and
Prokka identified 4261 coding DNA sequences (CDSs) distributed across 331 subsystems, highlighting
genes associated with biocontrol, stress response, and iron acquisition. AntiSMASH 7.1 was used
for genome mining, revealing seven biosynthetic gene clusters that potentially produce biocontrol-
related metabolites.
In vitro
assays confirmed the antagonistic activity of strain PE1 against Fusarium
languescens CE2, demonstrating its potential to inhibit mycelial growth. The study provides a genomic
basis for investigating B. cabrialesii subsp. cabrialesii PE1 as a potential biological control agent in
potato production.
Keywords: Bacillus cabrialesii; biological control; genomic analysis; Fusarium;Solanum tuberosum;
biosynthetic gene cluster; secondary metabolites
1. Introduction
The potato (Solanum tuberosum L.) is a widely cultivated crop and is one of the top five
most produced crops worldwide [
1
]. However, unfavorable conditions such as diseases can
make potato production unsustainable, negatively impacting productivity and yield [
2
].
Fusarium is one of the most severe plant pathogens that attack potatoes, following late
blight. Fusarium-related diseases are serious soil-borne diseases that cause economic losses
worldwide. They can affect potatoes at any growth stage by inducing Fusarium wilt on
plants and Fusarium dry rot on tubers [
3
]. Several Fusarium species have been associated
with potatoes, mainly Fusarium sambucinum,F. solani,F. graminearum,F. oxysporum,F. verti-
cillioides, and F. oxysporum f. sp. tuberosi [
4
,
5
]. Fusarium species cause different symptoms
in potatoes. Fusarium oxysporum f. sp. tuberosi causes vascular wilt, and F. solani and
F. sambucinum lead to Fusarium dry rot in tubers [
3
–
6
]. Fusarium diseases are caused by
individual Fusarium or co-occurring species [
7
]. The relative importance of the Fusarium
species varies depending on factors such as local climate, agricultural practices, and host
susceptibility [
8
]. For instance, according to Montoya-Martinez and Cota-Barreras [
9
,
10
],
Fusarium languescens is a significant regional phytopathogen in Sonora, Mexico, that could
impact potato production.
Horticulturae 2024,10, 357. https://doi.org/10.3390/horticulturae10040357 https://www.mdpi.com/journal/horticulturae

Horticulturae 2024,10, 357 2 of 11
Currently, new alternative control methods to synthetic fungicides are being consid-
ered for sustainable agricultural systems, due to the development of fungicide resistance by
Fusarium strains [
11
–
13
], as well as the negative impact of these synthetic compounds on the
environment and health [
14
]. Thus, a promising approach that has gained attention is the
use of biological control agents (BCAs) [
15
,
16
]. Reports suggest that bacterial antagonists
can be used to combat phytopathogenic Fusarium species, for example, the inoculation of
Pseudomonas fluorescens to potato tuber seeds [
5
]. Similarly, postharvest studies using P.
fluorescens and Bacillus subtilis have shown antagonistic properties against dry rot caused
by F. sambucinum and F. solani, respectively [17].
Bacillus is a predominant bacterial genus, and numerous Bacillus species have been
reported as biocontrol agents [
18
,
19
]. Bacillus uses various direct and indirect mechanisms
to promote plant growth and control pathogen proliferation. These mechanisms include
solubilization and mineralization of nutrients such as phosphorus and potassium, nitrogen
fixation, production of 1-aminocyclopropne-1-carboxlyic acid (ACC), phytohormones,
antimicrobial compounds, hydrolytic enzymes, and siderophores, as well as abiotic-stress
tolerance [
18
,
20
]. However, identifying appropriate strains of potential biocontrol agents
and characterizing associated metabolites remains a complex and arduous task [
21
–
23
]. To
address this issue, bioinformatics tools such as genome mining for biosynthetic genes can
be used to rapidly predict the secondary metabolites produced by a BCA strain [
22
–
24
].
Thus, the identification of genetic blueprints is essential for recognizing bioactive secondary
metabolites and enzymes that mediate most biocontrol mechanisms [22].
In this context, there is a need to reduce the heavy reliance on the chemical control of
Fusarium diseases and explore the use of BCAs as a sustainable alternative by effectively
searching for beneficial strains [
25
]. This study aims to explore the ability of Bacillus cabriale-
sii subsp. cabrialesii strain PE1—isolated from potatoes—as a promising biological control
agent against Fusarium languescens CE2, a regional phytopathogen. Both microorganisms
were isolated from a commercial field in the Yaqui Valley, Mexico, the birthplace of the
Green Revolution in the 1950s. This strategy was carried out by sequencing, annotation,
and mining of the strain PE1 genome to identify biosynthetic gene clusters associated
with its biocontrol ability; in addition, this genomic insight was supported by testing
the antagonistic activity of extracellular metabolites produced by strain PE1 against F.
languescens CE2.
2. Materials and Methods
2.1. Bacteria Isolation and Culture Conditions
The bacterial strain PE1 was isolated from potatoes harvested from a commercial field
in the Yaqui Valley, Mexico (27
◦
17
′
43.7
′′
N 109
◦
51
′
44.1
′′
W). The site was selected because
the plants exhibited symptoms of disease. Potatoes without symptoms were selected
for sampling. The isolation process involved superficial disinfection of potatoes using
commercial sodium hypochlorite at a concentration of 1.5% for 15 min. Then, disinfected
potatoes were thrice washed with sterilized water before being cut and macerated. The
serial dilution method (1:10) was subsequently employed up to 10
−6
. Thus, 1 mL of each
dilution was evenly spread on nutrient agar (NA) Petri dishes and incubated at 28
◦
C
for 2 days. After incubation, strain PE1 was isolated, purified, and characterized based
on its morphological traits, such as cell and colony shape, color, elevation, and opacity.
Following these assessments, the strain was cryopreserved at
−
80
◦
C, using a nutrient
broth (NB) culture medium containing glycerol (30%), at the Colección de Microorganismos
Edáficos y Endófitos Nativos (COLMENA, itson.edu.mx/micrositios/COLMENA, accessed
on 18 February 2024).
2.2. Genomic Analysis
High-quality genomic DNA was extracted from a fresh culture of strain PE1 grown
in nutrient broth (NB), under growth conditions set at 30
◦
C for 24 h and at 121 rpm,
obtaining 1
×
10
6
Colony Forming Units (CFU/mL). Thus, 40
µ
L of the cell suspension

Horticulturae 2024,10, 357 3 of 11
was lysed with 120
µ
L of TE buffer containing lysozyme (final concentration 0.1 mg/mL)
and RNase A (final concentration 0.1 mg/mL), incubated for 25 min at 37
◦
C. Proteinase K
(final concentration 0.1 mg/mL) and SDS (final concentration 0.5% v/v) were added and
incubated for 5 min at 65
◦
C. Genomic DNA was purified using an equal volume of SPRI
beads and resuspended in EB buffer (10 mM Tris-HCl, pH 8.0). The extracted total DNA
(DNA
≥
1
µ
g, concentration
≥
20 ng/
µ
L) was then quantified with the Quant-iT dsDNA
HS kit (ThermoFisher Scientific) assay in a plate reader and diluted as appropriate.
The Illumina NovaSeq platform (2
×
250 bp) was used for DNA sequencing, and
library preparation was carried out using the Nextera XT Library Prep Kit, following the
manufacturer’s protocol, but with the following modifications: input DNA was increased
2-fold, and PCR elongation time was increased to 45 s. DNA quantification and library
preparation were carried out on a Hamilton Microlab STAR automated liquid handling
system (Hamilton Bonaduz AG, Reno, Nevada, USA). Libraries were sequenced on an
Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA), using a 250 bp paired-end protocol.
Genomic information analysis was performed following the workflow reported by Ortega-
Urquieta, 2022 [
26
]. Trimmomatic version 0.30 was used to remove adapter sequences and
eliminate low-quality bases. The SPAdes version 3.15.4 generated a de novo assembly, and
the contigs were ordered regarding the genome of Bacillus cabrialesii subsp. cabrialesii TE3
T
(GenBank accession number GCA_004124315.2), using Mauve Contig Mover version 2.4.0.
Plasmid detection was performed with PlasmidFinder 2.1. The genome sequence of strain
PE1 was analyzed for contamination using CheckM version 1.0.18 [27].
To affiliate the strain PE1 at a species level, its genome was compared to its more closely
related strains (Table 1; 16S rRNA similarity
≥
99.6%) by using the overall genome related-
ness indices (OGRIs): average nucleotide identity (ANI) by the OrthoANI algorithm [
28
]
and the Genome to Genome Distance Calculator (GGDC) version 3.0 by BLAST [
29
]. A
whole-genome-based phylogenetic tree was constructed using Type (strain) Genome Server
(TYGA) (https://tygs.dsmz.de/, accessed on 10 January 2024) [29].
Table 1. 16S rRNA gene and OGRIs-based taxonomic affiliation of strain PE1.
Taxon Name Strain GenBank Accession
Number
Similarity
(%) OrthoANI GGDC
(Formula 2)
Bacillus cabrialesii
subsp. cabrialesii TE3 TMK462260 100 100 100
Bacillus inaquosorum KCTC 13429 TAMXN01000021 100 93.95 54.5
Bacillus tequilensis KCTC 13622 TAYTO01000043 99.86 93.58 52.7
Bacillus stercoris JCM 30051 TMN536904 99.86 92.19 46.4
Bacillus spizizenii NRRL B-23049 TCP002905 99.86 93.76 53.4
Bacillus subtilis NCIB 3610 TABQL01000001 99.8 92.42 47.8
Bacillus halotolerans ATCC 25096 TLPVF01000003 99.73 87.46 33.4
Bacillus mojavensis RO-H-1 TJH600280 99.66 87.57 33.2
TType strain.
2.3. Genome Annotation and Genome Mining
The genome annotation for strain PE1 was performed using the Rapid Annotation
Using Subsystem Technology (RAST) server version 2.0 and the RASTtk pipeline based
on the PathoSystems Resource Integration Center (PATRIC). A second annotation was
conducted using Proksee, which incorporates the Rapid Prokaryotic Genome Annotation
(Prokka), and this process generated the circular chromosome map of strain PE1, including
coding sequences (CDSs), tRNAs, rRNAs, guanine–cytosine (GC), and skew content. To
identify biosynthetic gene clusters associated with biocontrol, the genome of strain PE1 was
submitted to the Antibiotics & Secondary Metabolite Analysis Shell (AntiSMASH) 7.1 web
server (https://antismash.secondarymetabolites.org, accessed on 18 February 2024) [
30
],
under the ‘relaxed’ parameter.

Horticulturae 2024,10, 357 4 of 11
2.4. Evaluation of the Antagonistic Activity of Strain PE1 against Fusarium languescens CE2
through Extracellular Metabolites
Fusarium languescens CE2 was obtained from a national microbial culture collection,
named COLMENA (Coleccion de Microorganismos Edaficos y Endofitos Nativos, [
31
]).
This is a phytopathogenic strain previously isolated from the Yaqui Valley, Mexico [
9
]. In
this study, the strain CE2 was used as a model for Fusarium. This assay was carried out
following the methods described by Montoya-Martinez et al. (2023) [
9
] and Baard et al.
(2023) [
32
], with modifications. Thus, a liquid bacterial culture was prepared by inoculating
a 1
×
10
4
CFU of strain PE1 into 20 mL of NB. The culture was incubated at 30
◦
C, with
constant shaking at 120 rpm, for 72 h. Afterward, the bacterial culture was centrifuged
at 5000 rpm for 10 min, and the resulting supernatant was collected and filtered through
a hydrophilic syringe filter (0.22
µ
m). The effectiveness of the cell-free supernatant (CF)
against F. languescens CE2 was tested using two methods: a well-diffusion method and
casting agar plates with 50% CF. To perform the well-diffusion method, a 0.6 cm diameter
agar plug containing growing mycelia from F. languescens CE2 was placed at the center of
a fresh Potato Dextrose Agar (PDA) plate. Then, three paper discs, each with a diameter
of 0.6 cm, were placed equidistantly on the plate, and 25
µ
L of cell-free supernatant was
added over each disc. The plates were sealed and incubated at 30
◦
C for three days. The
antagonistic effect of the cell-free supernatant on F. languescens CE2 mycelial growth was
measured in terms of area (mm
2
) using ImageJ 1.54g [
33
]. For the second method, agar
plates with 50% cell-free supernatant were used; thus, Potato Dextrose Agar (PDA) was
prepared at double concentration and sterilized, and then the cell-free supernatant was
added to a 50% concentration v/v. Then, a 0.6 cm diameter agar plug containing growing
mycelia from F. languescens CE2 was placed at the center of each plate. The inoculated
plates were then incubated at 30
◦
C for six days, and the area (mm
2
) with mycelial growth
was measured. For the control treatment, the cell-free supernatant was replaced with sterile
distilled water.
The experiment was conducted with three replicates. Statistical analyses were per-
formed using R Studio Version 4.3.0 [
34
]. Data were analyzed using a one-way ANOVA
(
p≤0.05
). The presented values represent the mean between replicates, and the bars
represent the standard deviation (SD).
3. Results
3.1. Genomic Analysis
The bacterial DNA was subjected to sequencing, yielding a total of 1,017,168 paired-
end reads (2
×
250 bp). Subsequent assembly of the obtained reads resulted in the draft
genome of strain PE1, comprising 27 contigs (
≥
500 bp). The assembled genome encom-
passed a total length of 4,071,293 bp, with a G + C content of 44.13%, an N50 value of 357,
305 bp, and 5 L50. Importantly, plasmids were not detected within the genome of strain
PE1, indicating its genomic stability and absence of extrachromosomal genetic elements.
Based on the 16S rRNA gene, strain PE1 was taxonomically affiliated with the genus
Bacillus, showing 100% similarity to Bacillus cabrialesii subsp. cabrialesii TE3
T
, 100% to B.
inaquosorum KCTC 13429
T
, and 99.86% to B. tequilensis KCTC 13622
T
(Table 1). Based on the
OGRIs analysis, this strain was strongly affiliated with Bacillus cabrialesii subsp. cabrialesii
due to these values being higher than those delimiting the species affiliation (
ANI ≥95–96%
and GGDC
≥
70%) (Table 1). Finally, this taxonomic affiliation was confirmed by the
construction of a whole-genome-based phylogenomic tree (Figure 1), showcasing the close
evolutionary relationship of strain PE1. Thus, this strain belongs to Bacillus cabrialesii
subsp. cabrialesii.

Horticulturae 2024,10, 357 5 of 11
Horticulturae 2024, 10, x FOR PEER REVIEW 5 of 12
rmed by the construction of a whole-genome-based phylogenomic tree (Figure 1),
showcasing the close evolutionary relationship of strain PE1. Thus, this strain belongs to
Bacillus cabrialesii subsp. cabrialesii.
Figure 1. Phylogenetic relationship between strain PE1 and closely related species based on ge-
nome sequences constructed by TYGS. Tree inferred with FastME 2.1.6.1 [35] from GBDP distances
calculated from genome sequences. The branch lengths are scaled in terms of the GBDP distance
formula d5. The numbers above the branches are GBDP pseudo-bootstrap support values > 60%
from 100 replications, with an average branch support of 73.0%. The tree was rooted at the mid-
point [36].
3.2. Genome Annotation
According to the RAST prediction, the genome of B. cabrialesii subsp. cabrialesii PE1
contains 94 RNA sequences and 4261 protein-coding DNA sequences (CDSs) distributed
across 331 subsystems, as shown in Figure 2. Notable subsystems in the genome of strain
PE1 include coding sequences related to biocontrol, such as virulence, disease, and de-
fense (36 CDSs). These are further subdivided into resistance to antibiotics and toxic
compounds, invasion and intracellular resistance, and bacteriocins and ribosomally
synthesized antibacterial peptides. Additionally, the iron acquisition and metabolism (30
CDSs) subsystem includes genes related to siderophores, which function as iron chela-
tors, decreasing the accessibility of iron to phytopathogenic microorganisms. The car-
bohydrates subsystem (272 CDSs) contains coding sequences related to the production of
acetoin and butanediol. These volatile components are capable of inducing systemic re-
sistance in plants [37]. The cell wall and capsule (86 CDSs) subsystem contains coding
sequences for exopolysaccharide biosynthesis (EpsC and EpsD), which are genes related
to colonization [22]. Additionally, subsystems related to bacterial resilience for the de-
velopment of agricultural bioproducts were identied, including the stress response (43
CDSs), which covers osmotic and oxidative stress. Furthermore, complementing these
results, the circular chromosome map based on Prokka predicted a total of 5395 CDSs,
130 tRNAs, and 1 tmRNA (Figure 3).
Figure 1. Phylogenetic relationship between strain PE1 and closely related species based on genome
sequences constructed by TYGS. Tree inferred with FastME 2.1.6.1 [
35
] from GBDP distances cal-
culated from genome sequences. The branch lengths are scaled in terms of the GBDP distance
formula d5. The numbers above the branches are GBDP pseudo-bootstrap support values > 60% from
100 replications, with an average branch support of 73.0%. The tree was rooted at the midpoint [
36
].
3.2. Genome Annotation
According to the RAST prediction, the genome of B. cabrialesii subsp. cabrialesii PE1
contains 94 RNA sequences and 4261 protein-coding DNA sequences (CDSs) distributed
across 331 subsystems, as shown in Figure 2. Notable subsystems in the genome of strain
PE1 include coding sequences related to biocontrol, such as virulence, disease, and defense
(36 CDSs). These are further subdivided into resistance to antibiotics and toxic compounds,
invasion and intracellular resistance, and bacteriocins and ribosomally synthesized antibac-
terial peptides. Additionally, the iron acquisition and metabolism (30 CDSs) subsystem
includes genes related to siderophores, which function as iron chelators, decreasing the
accessibility of iron to phytopathogenic microorganisms. The carbohydrates subsystem
(272 CDSs) contains coding sequences related to the production of acetoin and butanediol.
These volatile components are capable of inducing systemic resistance in plants [
37
]. The
cell wall and capsule (86 CDSs) subsystem contains coding sequences for exopolysaccharide
biosynthesis (EpsC and EpsD), which are genes related to colonization [
22
]. Additionally,
subsystems related to bacterial resilience for the development of agricultural bioproducts
were identified, including the stress response (43 CDSs), which covers osmotic and oxida-
tive stress. Furthermore, complementing these results, the circular chromosome map based
on Prokka predicted a total of 5395 CDSs, 130 tRNAs, and 1 tmRNA (Figure 3).

Horticulturae 2024,10, 357 6 of 11
Horticulturae 2024, 10, x FOR PEER REVIEW 6 of 12
Figure 2. Pie chart of the subsystem category distribution of CDSs from Bacillus cabrialesii subsp.
cabrialesii PE1 constructed by RAST server version 2.0 and R studio version 4.3.0. CDSs, 4261; and
subsystems, 331.
Figure 3. Circular chromosome map of Bacillus cabrialesii subsp. cabrialesii PE1, including the dis-
tribution of CDS, tRNAs, rRNAs, and GC content skew generated through genome annotation
from PROKKA. Additionally, the identied biosynthetic gene clusters associated with biocontrol
by AntiSMASH [30] are also shown.
3.3. Genome Mining
The AntiSMASH 7.1 web server was used for genome mining on Bacillus cabrialesii
subsp. cabrialesii PE1, revealing seven distinct genomic regions. Notable biosynthetic
gene clusters were detected in regions 5, 6, 14, 20, and 21 (Table 2 and Figure 3).
Figure 2. Pie chart of the subsystem category distribution of CDSs from Bacillus cabrialesii subsp.
cabrialesii PE1 constructed by RAST server version 2.0 and R studio version 4.3.0. CDSs, 4261; and
subsystems, 331.
Horticulturae 2024, 10, x FOR PEER REVIEW 6 of 12
Figure 2. Pie chart of the subsystem category distribution of CDSs from Bacillus cabrialesii subsp.
cabrialesii PE1 constructed by RAST server version 2.0 and R studio version 4.3.0. CDSs, 4261; and
subsystems, 331.
Figure 3. Circular chromosome map of Bacillus cabrialesii subsp. cabrialesii PE1, including the dis-
tribution of CDS, tRNAs, rRNAs, and GC content skew generated through genome annotation
from PROKKA. Additionally, the identied biosynthetic gene clusters associated with biocontrol
by AntiSMASH [30] are also shown.
3.3. Genome Mining
The AntiSMASH 7.1 web server was used for genome mining on Bacillus cabrialesii
subsp. cabrialesii PE1, revealing seven distinct genomic regions. Notable biosynthetic
gene clusters were detected in regions 5, 6, 14, 20, and 21 (Table 2 and Figure 3).
Figure 3. Circular chromosome map of Bacillus cabrialesii subsp. cabrialesii PE1, including the
distribution of CDS, tRNAs, rRNAs, and GC content skew generated through genome annotation
from PROKKA. Additionally, the identified biosynthetic gene clusters associated with biocontrol by
AntiSMASH [30] are also shown.
3.3. Genome Mining
The AntiSMASH 7.1 web server was used for genome mining on Bacillus cabrialesii
subsp. cabrialesii PE1, revealing seven distinct genomic regions. Notable biosynthetic gene
clusters were detected in regions 5, 6, 14, 20, and 21 (Table 2and Figure 3).

Horticulturae 2024,10, 357 7 of 11
Table 2. Biosynthetic gene clusters found in B. cabrialesii subsp. cabrialesii PE1 obtained from genome
mining by the antiSMASH web server.
Region From To BGCs Type Most Similar Known Cluster Similarity (%)
5.1 37,565 57,940 Phosphonate Rhizocticin A 100
6.1 121,022 186,413 NRPS Surfactin 86
14.1 230,573 345,237 TransAT-PKSm PKS-like, T3PKS, NRPS Bacillaene 100
14.2 424,703 487,830 NRPS, betalactone Fengycin/plipastatin 100
20.1 82,439 134,222 NRP-metallophore, NRPS Bacillibactin; paenibactin 100
20.2 662,012 682,579 Sactipectide Subtilosin A 100
21.1 3451 44,869 Other Bacilyn 100
3.4. Antagonistic Activity of Bacillus cabrialesii subsp. cabrialesii PE1 against Fusarium
Languescens CE2, through Extracellular Metabolites
Extracellular metabolites produced by B. cabrialesii subsp. cabrialesii PE1 significantly
inhibited the growth of F. languescens CE2, as demonstrated by the reduction of 41.6% in the
mycelial growth area, from 969.60
±
55.72 mm
2
(control treatment) to
566.20 ±29.54 mm2
(Figure 4a). On the other hand, 50% of the cell-free supernatant (CF) obtained from B. cabri-
alesii subsp. cabrialesii PE1 also showed a significant inhibitory effect on the mycelial growth
of F. languescens CE2, from 1835.18
±
125.09 mm
2
(control treatment) to
83.31 ±6.32 mm2
95.4% (Figure 4b).
Horticulturae 2024, 10, x FOR PEER REVIEW 7 of 12
Table 2. Biosynthetic gene clusters found in B. cabrialesii subsp. cabrialesii PE1 obtained from ge-
nome mining by the antiSMASH web server.
Region
From
To
BGCs Type
Most Similar Known Cluster
Similarity (%)
5.1
37,565
57,940
Phosphonate
Rhizocticin A
100
6.1
121,022
186,413
NRPS
Surfactin
86
14.1
230,573
345,237
TransAT-PKSm PKS-like, T3PKS, NRPS
Bacillaene
100
14.2
424,703
487,830
NRPS, betalactone
Fengycin/plipastatin
100
20.1
82,439
134,222
NRP-metallophore, NRPS
Bacillibactin; paenibactin
100
20.2
662,012
682,579
Sactipectide
Subtilosin A
100
21.1
3451
44,869
Other
Bacilyn
100
3.4. Antagonistic Activity of Bacillus cabrialesii subsp. cabrialesii PE1 against Fusarium
Languescens CE2, through Extracellular Metabolites
Extracellular metabolites produced by B. cabrialesii subsp. cabrialesii PE1 signicantly
inhibited the growth of F. languescens CE2, as demonstrated by the reduction of 41.6% in
the mycelial growth area, from 969.60 ± 55.72 mm2 (control treatment) to 566.20 ± 29.54
mm2 (Figure 4a). On the other hand, 50% of the cell-free supernatant (CF) obtained from
B. cabrialesii subsp. cabrialesii PE1 also showed a signicant inhibitory eect on the myce-
lial growth of F. languescens CE2, from 1835.18 ± 125.09 mm2 (control treatment) to 83.31 ±
6.32 mm2 95.4% (Figure 4b).
Figure 4. Antagonistic activity of extracellular metabolites from B. cabrialesii subsp. cabrialesii PE1
against F. languescens CE2 in PDA: (a) well-diusion method, 3 days of incubation; and (b) cell-free
supernatant 50% v/v, 6 days of incubation. The antagonistic eect of the cell-free supernatant on F.
languescens CE2 mycelial growth was measured in terms of area (mm2). * Statistically signicant
dierence (p ≤ 0.05). F: F. languescens CE2. S: cell-free supernatant from B. cabrialesii subsp. cabrialesii
PE1.
4. Discussion
Sustainable plant disease management faces the challenge of meeting the global
demand for safe and diversied food. Thus, bioprospecting is a key tool to reach this
goal, which involves isolating microorganisms from the habitat where they will be used
Figure 4. Antagonistic activity of extracellular metabolites from B. cabrialesii subsp. cabrialesii PE1
against F. languescens CE2 in PDA: (a) well-diffusion method, 3 days of incubation; and (b) cell-free
supernatant 50% v/v, 6 days of incubation. The antagonistic effect of the cell-free supernatant on
F. languescens CE2 mycelial growth was measured in terms of area (mm
2
). * Statistically signifi-
cant difference (p
≤
0.05). F: F. languescens CE2. S: cell-free supernatant from B. cabrialesii subsp.
cabrialesii PE1.
4. Discussion
Sustainable plant disease management faces the challenge of meeting the global
demand for safe and diversified food. Thus, bioprospecting is a key tool to reach this goal,
which involves isolating microorganisms from the habitat where they will be used and

Horticulturae 2024,10, 357 8 of 11
directly screening their potential against phytopathogens, under specific edaphoclimatic
conditions [
38
,
39
]. Strain PE1 was isolated and genomically characterized under this
objective. This work establishes the basis for studying and evaluating Bacillus cabrialesii
subsp. cabrialesii PE1 as a biological control agent against Fusarium languescens in potato
production in the Yaqui Valley, Mexico.
Biological control offers several opportunities for improved disease control, partic-
ularly where conventional approaches are limited or compromised; this is particularly
relevant for Fusarium-related diseases, where chemical resistance and the potential harm to
human and environmental health from fungicide use are major concerns [
5
,
40
]. In this sense,
there are four main routes of action underlying biological control of plant diseases: (i) ex-
ploitation competition for resources, (ii) interference competition for space via antibiosis or
toxic secondary metabolites, (iii) hyperparasitism, and (iv) induced resistance [
39
]. Bacillus
cabrialesii subsp. cabrialesii PE1 demonstrated biocontrol potential against F. languescens
CE2 by producing secondary metabolites, including potentially subtilosin A, bacillibactin,
bacillaene, fengycin, surfactin, bacilyn, and rhizocticin A, as revealed by genome mining
(Figures 2and 3; Table 2). The genus Bacillus is well-characterized and exhibits multiple
beneficial properties in plant nutrition and antimicrobial activity against phytopathogens.
Among diverse species of this genus, B. velezensis,B. subtilis,B. amyloliquefaciens, and B.
cereus have been reported to be active against Fusarium [
41
,
42
]. Additionally, Bacillus cabri-
alesii subsp. Tritici TSO2
T
and Bacillus cabrialesii subsp. cabrialesii TE3
T
have been reported
as biocontrol agents [
9
,
43
]. The biocontrol capacity of these biological control strains is
largely attributed to their ability to produce extracellular enzymes (cellulase, glucanases,
proteases, chitinases, beta-xylosidase, chitin deacetylases, catalase, peroxide dismutase,
and peroxidase); and antimicrobial secondary metabolites such as organic compounds
(1,2-benzenedicarboxylic acid, 6-undecylamine, 2-methyloctacosane, 9-octadecenoic acid
and 1-tetradecanamine, and N,N-dimethyl), lipopeptides (mycosubtilin, fengycin B, iturin
A, surfactin A, iturin, bacillomycin, bacilysin, and mersacidin) and siderophores [41,44].
In vitro
assays of B. cabrialesii subsp. cabrialesii PE1 demonstrated a reduction in the
growth of F. languescens CE2 by 41.6% and 95.4%, respectively, depending on the concentra-
tion strategy using CF (Figure 4). This validates the production and diversity of secondary
metabolites produced by B. cabrialesii subsp. cabrialesii PE1 and highlights the importance
of exploring the action modes to quantify their roles in the biocontrol efficacy [
45
]. Similar
metabolites found in the genome information have been associated with Fusarium bio-
control. For instance, Bacillus subtilis SG6 has been reported to act against F. graminearum
by producing fengycins and surfactins [
42
]. Additionally, under field conditions, Bacillus
velezensis LM2303 inhibited F. graminearum, where the observed inhibition was attributed
to the presence of three antifungal metabolites (fengycin B, iturin A, and surfactin A) and
eight antibacterial metabolites, including bacillaene [
44
], which were also identified in the
strain PE1 genome. Another report showed strong antifungal activity by disrupting the cell
walls, membranes, and cytoskeleton of Fusarium oxysporum f. sp. cucumerinum hyphae due
to plipastatin [
46
], which was found in region 14.2 of the strain PE1 genome. Thus, the pro-
duction of lipopeptides such as fengycin/plipastatin and surfactin, as well as polyketides
such as bacillaene by strain PE1, may be involved in the demonstrated biocontrol activity
in the
in vitro
assay. However, further deeper studies, such as metabolomics, are necessary
to complement these findings.
Additionally, the iron acquisition and metabolism subsystem in the genome of B. cabri-
alesii subsp. cabrialesii PE1 includes 15 CDSs related to siderophores, which are iron chelators
that decrease the accessibility of iron to phytopathogenic microorganisms. Siderophores are
classified into three functional groups based on their structure: hydroxamate, catecholate,
and carboxylate [
47
]. Bacillus sp. produces catecholate as its main siderophore, including
bacillibactin [
48
], which has also been identified through genome mining in strain PE1.
Siderophores play a crucial role in the biological control of Fusarium wilt of pepper [
49
],
and they could be applicable in the case of biocontrol of Fusarium affecting potato.

Horticulturae 2024,10, 357 9 of 11
The genome annotation also suggests that strain PE1 may possess additional bio-
control mechanisms. For example, the strain may produce acetoin and butanediol, which
are volatile compounds that can induce systemic resistance in plants [
37
]. Furthermore, the
strain may be associated with exopolysaccharide biosynthesis, which enhances colonization
and biocontrol [
22
]. In this context, strain PE1 may have multiple biocontrol mechanisms
that could be expressed based on the environment. Identifying the responsible mechanisms
is a significant challenge due to the involvement of biotic and abiotic factors that can affect
the performance of biocontrol agents and their mechanisms of action [
50
]. In many cases,
complex interactions between plants, biocontrol agents, and pathogens involve various
mechanisms [
51
,
52
]. Characterizing the genome of selected strains is a crucial step in
developing biological control agents (BCAs), as it provides valuable information about
their potential as biopesticides [
22
,
52
]. This process allows further analysis of strains based
on their key attributes. Identifying and characterizing the genotypic traits of strains is
essential in determining their potential as biocontrol agents.
5. Conclusions
This research showcases the biocontrol capabilities of Bacillus cabrialesii subsp. cabriale-
sii PE1 against the causal agent that affects potato cultivation, Fusarium languescens CE2.
Genome mining identified seven biosynthetic gene clusters associated with biocontrol
within this strain, providing insights into potential genomic determinants of its biocontrol
ability. The in vitro assessments suggest that lipopeptides, such as fengycin and surfactin,
as well as polyketides like bacillaene, may contribute to the observed biocontrol activity.
However, further investigations are required, particularly in the field of metabolomics, to
improve and refine these findings. Additionally, exploring the diverse functional activities
and genes of strain PE1 is crucial to effectively use it as a biocontrol agent in the field.
Author Contributions: Conceptualization, S.d.l.S.-V. and B.V.-A.; methodology, formal analysis, all
authors, and data curation, B.V.-A. and A.C.M.-M.; writing—original draft preparation, all authors;
writing—review and editing, all authors; supervision, project administration, and funding acquisition,
S.d.l.S.-V. All authors have read and agreed to the published version of the manuscript.
Funding: We acknowledge funding from GetGenome (a newly formed charitable organization that
provides equitable access to genomics technology for early career researchers all over the world)
and the Instituto Tecnológico de Sonora (PROFAPI_2024_0001). In addition, A.C.M.-M. was funded
by Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT) for a postdoctoral
fellowship (application number: 2306476).
Data Availability Statement: The complete genome sequence was deposited in DDBJ/ENA/GenBank
and is openly available in NCBI under accession number JBAMMW000000000, under BioProject
number PRJNA1080047, and BioSample number SAMN40099177.
Acknowledgments: The authors thank all members of the LBRM-COLMENA for their support in the
development of this research project, mainly Abraham Ruíz Castrejón.
Conflicts of Interest: The authors declare no conflict of interest.
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