microorganisms

Article
Integrated Genomic and Metabolomic Analysis Illuminates Key
Secreted Metabolites Produced by the Novel Endophyte
Bacillus halotolerans Cal.l.30 Involved in Diverse Biological
Control Activities
Polina C. Tsalgatidou 1,2 , Eirini-Evangelia Thomloudi 1 , Eirini Baira 3 , Konstantinos Papadimitriou 4 ,
Aggeliki Skagia 1 , Anastasia Venieraki 5, * and Panagiotis Katinakis 1, *

1 Laboratory of General and Agricultural Microbiology, Crop Science Department,

Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece; [email protected] (P.C.T.);
[email protected] (E.-E.T.); [email protected] (A.S.)
2 Department of Agriculture, University of the Peloponnese, 24100 Kalamata, Greece
3 Laboratory of Toxicological Control of Pesticides, Scientific Directorate of Pesticides’ Control and
Phytopharmacy, Benaki Phytopathological Institute (BPI), Kifissia, 14561 Athens, Greece; [email protected]
4 Department of Food Science and Technology, University of the Peloponnese, 24100 Kalamata, Greece;
[email protected]



5

 Laboratory of Plant Pathology, Crop Science Department, Agricultural University of Athens, Iera Odos 75,
11855 Athens, Greece
Citation: Tsalgatidou, P.C.;
* Correspondence: [email protected] (A.V.); [email protected] (P.K.)
Thomloudi, E.-E.; Baira, E.;
Papadimitriou, K.; Skagia, A.;
Abstract: The endophytic strain Cal.l.30, isolated from the medicinal plant Calendula officinalis, was
Venieraki, A.; Katinakis, P. Integrated
Genomic and Metabolomic Analysis
selected among seven Bacillus strains with plant growth promoting activity and strong biological
Illuminates Key Secreted Metabolites potential against the postharvest fungal pathogen Botrytis cinerea. Treatment by inoculating Cal.l.30
Produced by the Novel Endophyte bacterial cell culture or cell free supernatant on harvested grapes and cherry tomato fruits, signifi-
Bacillus halotolerans Cal.l.30 Involved cantly reduced gray mold disease severity index and disease incidence. Based on 16S rRNA sequence
in Diverse Biological Control analysis and whole genome phylogeny, Cal.l.30 was identified as Bacillus halotolerans. Genome mining
Activities. Microorganisms 2022, 10, revealed that B. halotolerans Cal.l.30 is endowed with a diverse arsenal of secondary metabolite biosyn-
399. https://doi.org/10.3390/ thetic gene clusters (SM-BGCs) responsible for metabolite production with antimicrobial properties.
microorganisms10020399
A sub-set of the identified SM-BGCs (mojavensin A, ‘bacillunoic acid’) appears to be the result of
Academic Editors: Hera Karayanni, recent horizontal gene transfer events. Its genome was also mined for CAZymes associated with
Katherine Maria Pappas, antifungal activity. Further UHPLC-HRMS analysis indicated that Cal.l.30 synthesizes and secretes
Chrysoula Tassou secondary metabolites with antimicrobial activity, including the lipopeptides, fengycin, surfactin and
and Danae Venieri mojavensin A, bacillaene isoforms, L-dihydroanticapsin and bacillibactin. Other compounds with
Received: 11 January 2022
known antimicrobial activity were also detected, such as azelaic acid, 15- hydroxypentadecanoid acid
Accepted: 4 February 2022 and 2-hydroxyphenylacetic acid. The genomic and metabolomic features of the B. halotolerans Cal.l.30
Published: 9 February 2022 provided new perspectives on the exploitation of novel Bacillus sp. as a biocontrol agent.

Publisher’s Note: MDPI stays neutral

Keywords: postharvest biocontrol; biosynthetic gene clusters; horizontal gene transfer; plant defense
with regard to jurisdictional claims in
elicitors; grapes; tomato; Botrytis cinerea
published maps and institutional affil-
iations.

1. Introduction
Copyright: © 2022 by the authors. The extensive use of chemical pesticides, herbicides and fertilizers has a significant
Licensee MDPI, Basel, Switzerland. impact on environmental pollution. Due to this, there has been much attention given to the
This article is an open access article study and implementation of sustainable, efficient and environmentally friendly products
distributed under the terms and in agriculture [1]. The application of beneficial microorganisms, and especially bacteria, has
conditions of the Creative Commons
gained a lot of interest due to their multiple plant growth promoting and plant protection
Attribution (CC BY) license (https://
mechanisms, making it necessary to find strong and effective biological control agents
creativecommons.org/licenses/by/
(BCAs) [2]. Plant growth promoting rhizobacteria (PGPR) and plant growth promoting
4.0/).

Microorganisms 2022, 10, 399. https://doi.org/10.3390/microorganisms10020399 https://www.mdpi.com/journal/microorganisms

Microorganisms 2022, 10, 399 2 of 27

endophytic bacteria (PGPEB) interact directly with their host plant and compete with other
microbial antagonists, either in the rhizosphere or endophytically, and are considered as
useful tools to develop efficient biocontrol strategies [1,3].
Plant associated bacteria of the genus Bacillus have been extensively studied for their
successful antagonistic activity against a wide range of plant pathogenic microorganisms,
including bacteria, fungi and viruses [4,5]. Bacillus species are known for their potential to
biosynthesize and secrete multiple secondary metabolites, being the fundamental producers
of lipopeptide compounds [6,7]. Depending on the biosynthetic pathway, the bioactive
compounds are separated either into small molecular peptides synthesized ribosomally
(ribosomal peptides, RPs), into peptide compounds synthesized by the non-ribosomal
pathway (non-ribosomal peptides, NRPs), such as lipopeptides (LPs) and polyketides
(PKs), or into hybrids of PKs and NRPs, such as bacillaene [8]. Genes involved in the
biosynthesis of antibiotics and secondary metabolites are organized in clusters and encode
multifunctional enzyme complexes, with the gene clusters polyketide synthase (PKS) and
non-ribosomal peptide synthetase (NRPS) being the most well studied [9]. The bioactive
secondary metabolites of Bacillus species biosynthesized by the PKS/NRPS pathways
include surfactin, iturin, fengycin, mycosubtilin, bacilysin and bacillaene compounds [8].
The most studied and exploited Bacillus species are Bacillus subtilis, Bacillus velezen-
sis and Bacillus amyloliquefaciens, due to their suppressive effect against several plant
diseases [5,10]. However, an interesting but less studied Bacillus species that has recently
gained interest for its plant growth effect and antimicrobial activity is Bacillus halotoler-
ans [11–13]. As other Bacillus species, many B. halotolerans strains can biosynthesize and
secrete several bioactive secondary metabolites and lytic enzymes, suppressing multiple
phytopathogenic fungi such as Botrytis cinerea, Rhizoctonia bataticola, Fusarium oxysporum,
Alternaria alternate, Sclerotinia sclerotiorum and Phytophthora infestans under in vitro or ex
vivo conditions [11,12,14–18].
The phytopathogen B. cinerea is a potent necrotrophic fungus able to infect a significant
number of fruits and berries, such as strawberry, tomato, grape, kiwi, cucumber, apples,
peaches, etc., causing the postharvest gray mold disease [19]. The application of beneficial
bacterial strains on plant tissues is an important method of selecting potential BCAs able
to suppress phytopathogens causing postharvest diseases. The ex vivo biological control
method places both the BCA candidate and the phytopathogenic microorganisms in more
natural environmental conditions, being an alternative approach to study the mechanisms
for combating postharvest diseases in fruits and vegetables [10,20]. The detached fruit
method has been implemented either as an initial selection method, or after the dual culture
method for the detection of competitive BCAs with strong biocontrol ability in vitro [21–24].
The present study focuses on B. halotolerans strain Cal.l.30, a strong BCA candidate
among four B. halotolerans strains (Cal.f.4, Cal.l.11, Cal.f.2.1, Cal.r.11), one B. velezensis
(Cal.r.29) and one B. subtilis strain (Cal.r.19), as previously isolated from different plant
parts of the medicinal plant C. officinalis [25]. We therefore studied its antagonistic activity
for the suppression of the postharvest pathogen Botrytis cinerea, under in vitro and ex
vivo conditions on detached tomato fruit and grape berries. Furthermore, genome and
metabolomic analyses revealed the presence of different biosynthetic gene clusters (BGCs)
of several secondary metabolites and the potential of strain Cal.l.30 to biosynthesize and
secrete them.

2. Materials and Methods

2.1. Dual Culture Assay
For the detection of antagonistic activity of the seven selected endophytic bacterial
strains against the phytopathogenic fungus B. cinerea, an in vitro dual culture method was
performed as described by Nifakos et al., 2021 [26]. A mycelial disc of 5-mm diameter from
the edge of a 7-day-old colony of the phytopathogenic fungus was cut out using a sterile
cork borer and placed onto an NA plate. After 18 h of bacterial incubation in Nutrient Broth
(NB), 10 µL of bacterial culture was inoculated at a 3 cm distance from the fungus. NA
Microorganisms 2022, 10, 399 3 of 27

plates inoculated with the antagonistic bacterial strains and the phytopathogen as well as
the phytopathogen alone (control plate), were maintained at 25 ◦ C for 10 d. The antagonistic
effect was detected by measuring the radial growth of B. cinerea in control plates and after
interaction with the selected bacterial strains. The inhibition rate of mycelium growth was
calculated using the formula

(A1 − A2)
% Inhibition rate of fungus = × 100 (1)
A1
where A1 represents the colony diameter of control and A2 represents the colony diameter
of treatment [27,28].

2.2. Drop Collapse Assay

Drop collapse assay for the seven selected bacterial strains was performed with small
modifications, as described by Townsley et al. (2018) [29], in order to determine their ability
to biosynthesize and secrete biosurfactants in liquid culture. Briefly, bacterial cultures after
grown for 2 days at 28 ◦ C in Nutrient Broth were centrifuged at 10,000 rpm for 10 min. A
25 µL aliquot of the supernatant was placed on the parafilm, and after 5 min the droplet
was examined visually. A small quantity (1 µL) of Evans Blue dye was added to each
droplet to better visualize and capture the presence of biosurfactants affecting the droplet
shape. Distilled water was used as negative control.

2.3. Detached Fruit Assay

The selected endophytic bacterial strain Cal.l.30 was further evaluated for its antago-
nistic activity against B. cinerea on tomato fruit (Solanum lycopersicum L. cv. Lobello) and
grape berries (Vitis vinifera L. cv. Sultanina), using the ex vivo modified detached fruit
assay as described by Shi and Sun, 2017 [30]. Tomato fruits and grape berries were surface
sterilized with 3% v/v NaClO for 15 min, rinsed four times with sterilized dH2 O, air dried
for 1 h at room temperature and wounded with a sterile cork borer (3 mm diameter) in one
side. An aliquot of 10 µL of bacterial culture (1 × 108 CFU/mL), 10 µL of bacterial cell free
supernatant of the same culture and 10 µL of sterilized distilled water as control treatment
were inoculated on the surface of each wound and incubated at room temperature for 1 h
before applying the fungus. Conidial suspension was prepared by flooding Potato Dextrose
Agar (PDA) plates of a 10-day-old solid culture with sterilized dH2 O to gently remove the
conidia and adjust the concentration approximately to 1 × 105 spores/ml. Finally, 10 µL of
fungal spore suspension was injected into each wound. Inoculated tomato fruit and grape
berries were placed into plastic boxes to maintain high relative humidity (approximately
80%) and incubated in a dark growth chamber at 25 ◦ C for 5 d. The experiment was
conducted three times (20 fruit/ replicate). The percentage of infected tomato fruits and
grape berries by B. cinerea were calculated to assess Disease Incidence (DI) according to the
following equation

Number of infected fruits

% Disease Incidence, (DI) = × 100 (2)
Total number of fruits
Growth area of the phytopathogenic fungus on each fruit surface was calculated
using ImageJ software analysis tool in order to determine Disease Severity (DS) using the
following equation

Infected fruit area

% Disease Severity, (DS) = × 100 (3)
Total fruit area
From the calculated disease severity, a percentage scale of the disease severity (Rating
scale) is obtained, based on the modified Horsfall–Barratt scale method [31–33]. Disease
Severity Index (DSI) was scored on a 0 to 9 rating scale, with 0 = healthy fruits, 1 = 1–10%,
Microorganisms 2022, 10, 399 4 of 27

3 = 11–25%, 5 = 26–50%, 7 = 51–75% and 9 = >75% infected fruit area and was calculated
according to the formula

∑ (n × i)
% Disease Severity Index, (DSI) = × 100 (4)
N×Z
where n: number of fruit in a specific value of disease rating scale, i: the corresponding
value of the scale, N: total number of fruit, Z: highest value of disease rating scale.

2.4. Colonization on Wounded Grape Berries and Tomato Fruits

To evaluate the colonization of bacterial strain Cal.l.30 when inoculated alone on the
wounds of grape berries and tomato fruits, tissue samples were cut out of the surfaces
with a sterile cork borer (0.4 cm diameter and 0.4 cm deep) and transferred into a falcon
tube containing 10 mL of sterile phosphate-buffered saline (PBS: KH2 PO4, 0.27 g/L; KCl
0.2 g/L; NaCl 8 g/L; Na2 HPO4 1.42 g/L; pH 7.0). After shaking for 1 h at 200 rpm, the
suspensions were diluted by 10- fold serial dilutions, and 100 µL of the last dilution was
plated in NA supplemented with 50 µg/mL cycloheximide and was then incubated at
30 ◦ C for 48 h in order to calculate bacterial colony-forming units. Four samples were
collected of each fruit at 0, 24, 48, 72, 96 and 120 h post inoculation and the experiment was
repeated independently three times.

2.5. Extraction of Agar-Diffusible Secondary Metabolites of Cal.l.30

The protocol used for the extraction of the agar-diffusible secondary metabolites
(ADSM) of Cal.l.30 either secreted when grown singly (ADSM1), or during interaction
with B. cinerea (ADSM2) was conducted as previously described by Nifakos et al., 2021 [28].
Firstly, an aliquot of 200 µL of pre-grown bacterial culture of strain Cal.l.30 was inoculated
in NA plates (1.5% (w/v) agar) in an artificial well (0.5 cm × 6 cm) against a mycelial disc
(5 mm diameter) of the 10-day-old phytopathogenic fungus B. cinerea. Plates inoculated
only with the bacterial strain were used as control treatment. All NA plates were maintained
in 25 ◦ C for 6 d until the creation of a clear inhibition zone between the fungus and strain
Cal.l.30. Afterwards, NA medium blocks from the intermediate zone of inhibition created
during interaction, as well as the zone in front of the bacterial control treatment, were
removed and cut into smaller pieces. Ethyl acetate and 0.1% formic acid were added to
NA pieces and thoroughly mixed. Samples were transferred to a water-bath sonicator
(Elmasonic S30H, Elma Schmidbauer GmbH, Germany) and were incubated for 30 min at
room temperature. After separation of the organic phase, samples were transferred to a
speed vacuum evaporator (Rotavapor R-114, BÜCHI Labortechnik AG, Flawil, Switzerland)
to dry. Finally, samples were dissolved in HPLC grade methanol, filtered and stored at
−80 ◦ C, until further use.

2.6. Antagonistic Activity of Agar-Diffusible Secondary Metabolites of Cal.l.30 against B. cinerea

In Vitro
Antagonistic activity of agar-diffusible secondary metabolites of Cal.l.30 (ADSM1 and
ADSM2) against B. cinerea mycelia growth was evaluated by the disc diffusion method. A
mycelia disc (5-mm diameter) from the colony edge of a 10-day-old B. cinerea culture was
applied in the center of a NA plate and three round filter papers were placed approximately
at 1 cm distance around the phytopathogenic fungus. An aliquot of 20 µL of each ADSM1
and ADSM2 was inoculated in the two filters while in the third filter 20 µL of methanol was
applied as negative control. Plates were incubated at 25 ◦ C for 5 d and antimicrobial activity
was determined by the creation of an inhibition zone between the filters and B. cinerea.

2.7. TLC-bioautography Assay of Cal.l.30 Secreted Compounds

Thin-layer chromatography (TLC) in combination with bioautography assay of the
agar-diffusible secreted compounds of Cal.l.30 was conducted as previously described
by Nifakos et al., 2021 and Calvo et al., 2019 [26,34]. ADSM1 and ADSM2 samples were
Microorganisms 2022, 10, 399 5 of 27

separated by TLC after spotted in an aliquot of 20 µL onto silica gel 60 F254 TLC aluminum
sheets (20 × 20 cm; layer thickness, 0.20 mm; Merck, Darmstadt, Germany) with mobile
phase consisting of chloroform–methanol–water at 65:25:4, v/v/v. After drying the TLC
plate at room temperature, the bioautography assay was performed with B. cinerea being
the indicator strain. PDA (0.8% agar w/v) semi-solid medium inoculated with approxi-
mately 105 spores/ mL of B. cinerea was poured onto the TLC plate and then incubated at
25 ◦ C overnight. The inhibition zones indicating the presence of antifungal compounds be-
came visible after spraying with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) at a concentration of 5 mg/ml. The Rf values were calculated using the formula

Distance travelled by the solute

R f value = (5)
Distance travelling by the solvet

2.8. Ultra-High Performance Liquid Chromatography Orbitrap High-Resolution Mass

Spectrometry Analysis
The chemical profiling of ADSM1 extract was performed as previously described by
Nifakos et al., 2021 [28]. Specifically, the Q Exactive Orbitrap platform (Thermo Fisher
Scientific, San Jose, CA, USA) was coupled to a Dionex Ultimate 3000 UHPLC system
(Thermo Scientific™ Dionex™, Sunnyvale, CA, USA) and the separation was performed
using a Hypersil Gold UPLC C18 (2.1 × 150 mm, 1.9µm) reversed phased column (Thermo
Fisher Scientific, San Jose, CA, USA). For mass detection, two electrospray ionization
modes were carried out, the positive (ESI+) and negative (ESI−). Eluent A consisting of
ultrapure water with 0.1% formic acid and eluent B consisting of acetonitrile were used in a
gradient mode of 30 min, under the following program: 0–21 min: 95% A–5% B; 21–24 min:
5% A–95% B; 24–30 min: 95% A–5% B, at 0.22 mL/min flow rate. Data acquisition was
performed on a mass range of 115–1500 Da on profile mode. The HRMS parameters for
both ion modes were set as follows: spray voltage, 2.7 kV; capillary temperature, 350 ◦ C;
aux. gas heater temperature, 50 ◦ C; sheath gas flow, 40 arb. units; S-lense Rf level, 50 V;
aux gas flow, 5 arb. units. The resolution for full scan analysis was set on 70,000, whereas
for the data dependent acquisition mode the resolution was 35,000 allowing for MS/MS
fragmentation of the three most intense ions. Stepped normalized collision energy was set
at 35, 60 and 100. The column temperature was set at 40 ◦ C and the sample tray temperature
at 4 ◦ C.
The resulting data were processed through the Compound Discovered version 2.1
(Thermo Fisher Scientific, San Jose, CA, USA). The online mzCloud library, the public
chemical database PubChem (NCBI) and the Norine platform were used for metabolite
annotation, taking into consideration the isotopic pattern and applying m/z tolerance of
5 ppm.

2.9. Genome Sequencing

Genome sequencing of B. halotolerans strains Cal.l.30, Cal.f.4 and Cal.l.11 was con-
ducted as previously described by Nifakos et al., 2021 [26]. Briefly, genomic DNA of the bac-
terial strains was isolated from an overnight culture using PureLink®Genomic DNA Mini
Kit (Thermo Fisher Scientific, Carlsbad, CA, USA) and sequenced by SNPsaurus (Eugene,
OR, USA) using an Illumina HiSeq 2000 platform, generating 2 × 150-bp paired-end reads.
A library was created using the Nextera XT DNA Library Prep Kit. Trimming of adaptors
was performed with BBDuk and assembly with SPAdes-3.12.0 using default parameters [35].
This workflow generated a total of 1.546.685 (Cal.l.30), 1.251.500 (Cal.f.4), 1.442.900 (Cal.l.11)
trimmed paired reads with coverage >60-fold. The final de novo genome of strain Cal.l.30
(NCBI accession: JAEACK000000000) assembled in 9 scaffolds with size of 4.206.058 bp, of
strain Cal.f.4 (NCBI accession: JAEACI000000000) assembled in 12 scaffolds with size of
4.283.396 bp, Cal.l.11 (NCBI accession: JAEACJ000000000) assembled in 11 scaffolds with
size of 4,212,585 bp.
The whole genome data of strains Cal.l.30, Cal.f.4 and Cal.l.11 have been deposited at
DDBJ/EMBL/GeneBank under the accession numbers JAEACK000000000, JAEACI000000000
Microorganisms 2022, 10, 399 6 of 27

and JAEACJ000000000, respectively. The identification of all three strains was established
using the Type (Strain) Genome server (TYGS) platform (https://tygs.dsmz.de, accessed on
15 December 2021) [36] and by calculating the values of digital DNA-DNA hybridization
(dDDH) using the genome to genome distance calculator (GGDC 2.1) [37] and the values
of orthologous average nucleotide identity (OrthoANI) [38].

2.10. Functional Genome Analysis

The secondary metabolite BGCs were identified using the online server antiSMASH
6.0 [39]. The ClusterBlast and KnownClusterBlast modules integrated into antiSMASH 6.0
were also used for comparative gene cluster analysis based on the NCBI GenBank and the
‘Minimum Information about a Biosynthetic Gene Cluster’ (MIBiG) data standard, respec-
tively [39–41]. The server dbCAN2 and the combination of HMMER (E-Value < 1 × 10−15
coverage > 0.35), DIAMOND (E-Value < 1 × 10−102 ) and Hotpep 117 (Frequency > 2.6,
Hits > 6 tools, were used to predict the carbohydrate-active enzymes (CAZymes) in the
core genome [42]. CAZyme domains predicted by at least two of the three algorithms
(DIAMOND, HMMER and Hotpep) employed by dbCAN2 were considered as a true
annotation for carbohydrate-active enzymes [42].

2.11. Statistical Analysis

For statistical analysis, IBM SPSS Statistics for Windows, version 25 (IBM7Corp.,
Microorganisms 2021, 9, x FOR PEER REVIEW of 27
Armonk, NY, USA) software was used while plots were carried out with Sigma Plot,
version 12.0 (Systat Software, San Jose, CA, USA). The heatmaps were created using Mi-
crosoft Excel 2010. To evaluate bacterial population, data were transformed to the loga-
strains
rithmic(Figure 1B). to
scale prior The radial
Tukey mycelial
analysis growth< of
(p-value B. cinerea
0.05). towards the
Data expressed antagonistic were
as percentages iso-
lates showed
arcsine significant
transformed priordifferences between(p-value
to Tukey analysis them, with the highest inhibition rate ob-
< 0.05).
served for strain Cal.r.29 (48.95%). Strains Cal.l.30 and Cal.r.11 followed with the second
3. Results
highest inhibition rates at 39.18% and 39.02%, respectively, while strain Cal.f.4 presented
the
3.1.third highest
Biological valueActivity
Control (24.49%) of (Figure
Bacillus 1C). Finally,
Strains strains Cal.l.11,
from Calendula Cal.f.2.1 and Cal.r.19,
officinalis
presented the lowest inhibition rates in comparison to the others with
All seven selected Bacillus isolates were tested for biosurfactants secretion. values at 22.05%,
The cell
21.75% and 20.45%,ofrespectively
free supernatant all studied (Figure 1C). Similar
strains showed resultsdrop
excellent werecollapse
also observed
abilitywhen
with in-
the
oculated against out
drop flattening phytopathogenic
to different extendfungiamong
Rhizoctonia solani and
the different Fusarium
strains, oxysporum
indicating f.sp.
adequate
lycopersici under
biosurfactant in vitro dual
production culture
(Figure 1). assays.

Figure 1. (A) Detection of secreted biosurfactants in the cell free supernatant of the seven studied
Figure 1. (A) Detection of secreted biosurfactants in the cell free supernatant of the seven studied
bacterial strains using drop collapse assay. (B) Direct antifungal activity of endophytic Bacillus
bacterial strains using drop collapse assay. (B) Direct antifungal activity of endophytic Bacillus strains
strains against B. cinerea by an in vitro dual culture assay. (C) Percentage of inhibition of mycelial
growth B. cinerea
against(IMG) afterbyeach
an in vitro dual
bacterial culture assay.
formulation (C)B.Percentage
against of inhibition
cinerea in vitro. of mycelial
Data values growth
represent the
(IMG) after each bacterial formulation against B. cinerea in vitro. Data values represent the
mean of 4 biological replicates ± SD. Letters indicate the statistical difference between treatments mean of
4 biological replicates ± SD.
after Tukey analysis (p < 0.05). Letters indicate the statistical difference between treatments after Tukey
analysis (p < 0.05).
3.2. Biological Control Potential of Endophytic Bacillus Strain Cal.l.30 Against Botrytis cinerea
Strain Cal.l.30 was selected among others of the dominant endophytic B. halotolerans
species for further study on its antagonistic activity against B. cinerea. Bacterial strain
Cal.l.30 presented a strong inhibitory effect against the postharvest pathogenic fungi, as
observed after ex vivo inoculation on grape berries and tomato fruits (Figure 2A and 2B).
Microorganisms 2022, 10, 399 7 of 27

The selected endophytic bacterial strains of the genus Bacillus (Cal.r.29, Cal.l.30,
Cal.l.11, Cal.f.4, Cal.r.11, Cal.f.2.1 and Cal.r.19), presented a strong antagonistic activity
against B. cinerea in a dual culture assay, until after 10 d of co-inoculation with the fungus
(Figure 1B). The antagonistic strains suppressed B. cinerea mycelial growth generating a
clear inhibition zone of differential width, by an in vitro dual culture assay, indicating ade-
quate production of agar-diffusible antifungal metabolites by all studied bacterial strains
(Figure 1B). The radial mycelial growth of B. cinerea towards the antagonistic isolates
showed significant differences between them, with the highest inhibition rate observed
for strain Cal.r.29 (48.95%). Strains Cal.l.30 and Cal.r.11 followed with the second highest
inhibition rates at 39.18% and 39.02%, respectively, while strain Cal.f.4 presented the third
highest value (24.49%) (Figure 1C). Finally, strains Cal.l.11, Cal.f.2.1 and Cal.r.19, presented
the lowest inhibition rates in comparison to the others with values at 22.05%, 21.75%
and 20.45%, respectively (Figure 1C). Similar results were also observed when inoculated
against phytopathogenic fungi Rhizoctonia solani and Fusarium oxysporum f.sp. lycopersici
under in vitro dual culture assays.

3.2. Biological Control Potential of Endophytic Bacillus Strain Cal.l.30 against Botrytis cinerea
Strain Cal.l.30 was selected among others of the dominant endophytic B. halotolerans
species for further study on its antagonistic activity against B. cinerea. Bacterial strain
Cal.l.30 presented a strong inhibitory effect against the postharvest pathogenic fungi, as
observed after ex vivo inoculation on grape berries and tomato fruits (Figure 2A,B). Strain
Cal.l.30 was able to successfully colonize both of the fruits tested, presenting a high density
until the fifth day of observation (Figure 2E,H).
The tomato fruits when inoculated for five days only with the presence of B. cinerea as
negative control, presented fruits with extremely severe mycelial growth on top of them,
while grape berries developed brown lesion and visible mycelia growth around the point of
formulation (Figure 2(Aiii,Biii)). Strain Cal.l.30 suppressed fungal growth after inoculation
of liquid bacterial cell culture (BCC) as well as liquid cell free culture (CFC) on both grape
berries and tomato fruits (Figure 2(Ai,ii,Bi,ii)).
However, treatment with BCC (liquid bacterial cell culture) containing both living
bacterial cells and multiple secreted secondary metabolites/ biosurfactants was more effi-
cient than CFC in suppressing gray mold in both fruits studied. Specifically, BCC treatment
on tomato fruit significantly reduced disease incidence by 14.63% in comparison to CFC
inoculation (18.52%) and the negative control (73.52%), whereas disease severity index
was equally reduced after both BCC and CFC formulations (Figure 2A,B). As described
in Figure 2G, infected grape berries were significantly reduced in both treatments, with
disease incidence being 77.78% and 88.89% after BCC and CFC inoculation, respectively, in
comparison to the control treatment (97.78%). In addition, disease severity index was also
significantly reduced in both BCC and CFC treatments by 28.89% and 33.33%, respectively,
in comparison to the control treatment (85.93%) (Figure 2F).
Microorganisms 2021, 9, x FOR PEER REVIEW 8 of 27

Microorganisms 2022, 10, 399 8 of 27

was also significantly reduced in both BCC and CFC treatments by 28.89% and 33.33%,
respectively, in comparison to the control treatment (85.93%) (Figure 2F).

Figure
Figure2.2.Antagonistic
Antagonisticactivity
activityofofendophytic
endophyticbacterial
bacterialstrain
strainCal.l.30
Cal.l.30against
againstgraygraymold
molddisease
diseaseon on
tomato fruits and grape berries. (A and B) Suppression of gray mold disease after ex vivo formula-
tomato fruits and grape berries. (A,B) Suppression of gray mold disease after ex vivo formulation on
tion on grape berries and tomato fruits, respectively, after liquid bacterial cell culture (BCC) (i),
grape berries and tomato fruits, respectively, after liquid bacterial cell culture (BCC) (i), liquid cell
liquid cell free culture (CFC) (ii) and individual formulation of B. cinerea as control treatment (iii).
free
(C culture
and (CFC) (ii)
D) Disease and individual
severity index (%)formulation B. cinerea as
and DiseaseofIncidence (%)control treatment
on tomato (iii).
fruits, (C,D) Disease
respectively. (E
severity
and index severity
F) Disease (%) and Disease
index (%) Incidence (%) on
and Disease tomato fruits,
incidence (%) onrespectively.
grape berries, (E,F) Disease severity
respectively. Bars
index (%)the
represent and Disease
mean ± SDincidence (%) on grape
of 3 independent berries,
biological respectively.
replicates. Bars
Letters represent
indicate thethe mean ±
statistical SD of
differ-
3 independent
ences biological replicates.
between treatments Letters
at 4 days after indicate the
inoculation statistical
(DAI) differences
after Tukey between
analysis treatments
(p < 0.05). (G andat
H) Timeafter
4 days growth curve of (DAI)
inoculation Cal.l.30 (Log10
after TukeyCFU/ wound)
analysis (p <after inoculation
0.05). (G,H) Time in growth
tomato curve
fruit and grape
of Cal.l.30
berry wounds (one wound/ tomato or grape), respectively, without the presence
(Log10 CFU/ wound) after inoculation in tomato fruit and grape berry wounds (one wound/ tomatoof phytopathogenic
fungus B. cinerea.
or grape), Bars represent
respectively, without the thepresence
mean ± ofSDphytopathogenic
of 4 independent biological
fungus replicates
B. cinerea. after Tukey
Bars represent the
analysis. Letters indicate the statistical difference per hour (p < 0.05).
mean ± SD of 4 independent biological replicates after Tukey analysis. Letters indicate the statistical
difference per hour (p < 0.05).
3.3. Genomic Features and Taxonomic Classification of Cal.l.30, Cal.f.4 and Cal.l.11
3.3. To
Genomic Features
ascertain and Taxonomic
the taxonomic Classification
position of Cal.l.30,
of bacterial strainsCal.f.4 andCal.f.4
Cal.l.30, Cal.l.11and Cal.l.11,
phylogenetic analysis
To ascertain using whole
the taxonomic genome
position sequences
of bacterial was Cal.l.30,
strains conducted using
Cal.f.4 andtheCal.l.11,
Type
(strain) Genome
phylogenetic Server using
analysis (TYGS) bioinformatics
whole platform (https://tygs.dsmz.de,
genome sequences was conducted using accessedthe Type
on 15 December
(strain) 2021). (TYGS)
Genome Server The data indicated that
bioinformatics strains(https://tygs.dsmz.de,
platform Cal.l.30, Cal.f.4 and Cal.l.11
accessedareon
15 December
closely 2021).
affiliated to B.The data indicated
halotolerans speciesthat strains
(Figure Cal.l.30,
3). To confirmCal.f.4 and Cal.l.11 are
this classification, allclosely
three
genomes to B. halotolerans
affiliated were species (Figure
further compared 3). To confirmANI
by alignment-based this classification,
and digital DDH all three genomes
to other ge-
were further
nomes compared strains.
of B. halotolerans by alignment-based
The dDDH valuesANI and digitalstrain
between DDH Cal.l.30,
to other Cal.f.4
genomes andof
B. halotolerans strains. The dDDH values between strain Cal.l.30, Cal.f.4 and Cal.l.11 and
other B. halotolerans strains ranged between 80.70% and 94.70%, indicating that all three
strains are closely affiliated to B. halotolerans species as shown in Figure 4. The range
 Cal.l.11 and other B. halotolerans strains ranged between 80.70% and 94.70%, indicating
that all three strains are closely affiliated to B. halotolerans species as shown in Figure 4.
The range
Microorganisms between
2022, 10, 399 97.91 and 99.33% of ANI values showed agreement between the two 9 of 27
methods for species discrimination and established classification of strains Cal.l.30, Cal.f.4
and Cal.l.11 as B. halotolerans. Although, the B. halotolerans Cal.l.30 isolate demonstrated a
very high level of genome between conservation
97.91 and 99.33%with of B.
ANIhalotolerans Cal.l.11
values showed and Cal.f.4,
agreement betweenwith
the twoANImethods
and dDDH values exceeding 99% and 92%, respectively, a close inspection/comparison of and
for species discrimination and established classification of strains Cal.l.30, Cal.f.4
Cal.l.11 as B. halotolerans. Although, the B. halotolerans Cal.l.30 isolate demonstrated a very
the annotated genes revealed that the genes encoding the proteins of type I restriction-
high level of genome conservation with B. halotolerans Cal.l.11 and Cal.f.4, with ANI and
modification DNA-methyltransferases
dDDH values exceeding hsdMSR
99% andand 92%,Mrr proteins
respectively, (cryptic
a close type IV re-
inspection/comparison of
striction endonucleases with specificity
the annotated for methylated
genes revealed DNA),
that the genes werethe
encoding exclusively
proteins of found in
type I restriction-
Cal.l.30. modification DNA-methyltransferases hsdMSR and Mrr proteins (cryptic type IV restriction
endonucleases with specificity for methylated DNA), were exclusively found in Cal.l.30.

Figure 3. Whole genome phylogenomic

Figure tree ofphylogenomic
3. Whole genome Bacillus strainstreeclosely related
of Bacillus to closely
strains B. halotolerans
related tostrains
B. halotolerans
Cal.l.30, Cal.f.4 and Cal.l.11, constructed
strains Cal.l.30, Cal.f.4using the type
and Cal.l.11, (strain)using
constructed genome server—TYGS.
the type (strain) genomeThe num- The
server—TYGS.
bers at the branches arenumbers
pseudo-bootstrap
at the branchessupport values inferred
are pseudo-bootstrap fromvalues
support 100 replicates.
inferred fromThe100
two col- The
replicates.
ored columns to the right
twoof each columns
colored name referto thetoright
the of
genome-based
each name referspecies and subspecies
to the genome-based clusters,
species and subspecies
respectively, as determined byrespectively,
clusters, dDDH cut-off of 70 and
as determined 79%, cut-off
by dDDH respectively.
of 70 andB. halotolerans
79%, respectively.strains
B. halotolerans
strains Cal.l.30, Cal.l.11 and
Cal.l.30, Cal.l.11 and Cal.f.4 are highlighted in red. Cal.f.4 are highlighted in red.
Microorganisms 2021, 9, x FOR PEER REVIEW
Microorganisms 2022, 10, 399
10 of 27
10 of 27

Figure 4. Heatmap
Figure ofofAverage
4. Heatmap Average Nucleotide Identity(ANI)
Nucleotide Identity (ANI) and
and digital
digital DNA–DNA
DNA–DNA Hybridization
Hybridization
(dDDH) scores among B. halotolerans strains.
(dDDH) scores among B. halotolerans strains.

3.4.3.4. Genomic
Genomic Insightsinto
Insights intothe
theAntifungal
Antifungal Activity
Activityand
andBiocontrol Potential
Biocontrol of Cal.l.30
Potential of Cal.l.30
ToTo gain an insight of the secondary metabolite biosynthetic gene clusters (SM-BGCs)
gain an insight of the secondary metabolite biosynthetic gene clusters (SM-BGCs)
associated with the biosynthesis of metabolites with antimicrobial properties of B. halotol-
associated with the biosynthesis of metabolites with antimicrobial properties of B. halotol-
erans strain Cal.l.30, a bioinformatic approach based on the antiSMASH and ClusterBlast
erans
algorithmCal.l.30,
strain platformawasbioinformatic
used. approach based on the antiSMASH and ClusterBlast
algorithm platformgenome
AntiSMASH was used.mining for SM-BGCs revealed that B. halotolerans Cal.l.30 harbors
10 BGCs, indicating that amining
AntiSMASH genome for SM-BGCs
large proportion revealed
(12.56%) of its that B. halotolerans
genome is dedicatedCal.l.30
for the har-
bors 10 BGCs, indicating
biosynthesis of secondary that a large proportion
metabolites (Table 1 and(12.56%) of its Among
Figure 5A–G). genomethe is predicted
dedicated for
BGCs, four NRPS clusters, associated with the synthesis of surfactin, fengycin,
the biosynthesis of secondary metabolites (Table 1 and Figure 5A–G). Among the pre- bacillaene
and BGCs,
dicted bacillibactin, one RiPP
four NRPS cluster,associated
clusters, one subtilosin
withAthe
andsynthesis
one bacilysin gene cluster,
of surfactin, were ba-
fengycin,
detected (Table 1). BGC of mojavensin A was not present as an individual cluster but
cillaene and bacillibactin, one RiPP cluster, one subtilosin A and one bacilysin gene clus-
was detected adjacent to the fengycin gene cluster. The remaining BGCs, including this
ter, were detected (Table 1). BGC of mojavensin A was not present as an individual cluster
of kalimantacin A (bearing only 20% resemblance to the BGC encoding kalimantacin A),
butappear
was detected adjacent
to be involved tobiosynthesis
in the the fengycin gene cluster.
of metabolites The remaining
of unknown BGCs, including
function.
this of kalimantacin A (bearing only 20% resemblance to the BGC encoding kalimantacin
A), appear to be involved in the biosynthesis of metabolites of unknown function.

Table 1. Biosynthetic gene clusters (BGCs) involved in biosynthesis of secondary metabolites of bacterial strain B. halotol-
erans Cal.l.30, detected by antiSMASH server.

Cluster BGC Type Compound MIBiG ID (Similarity %) Predicted Size (bp)

1 NRPS Surfactin BGC0000433_c1 (86%) 65.394
2 T1PKS-NRPS Kalimantacin A BGC0001532_c1 (20%) 117.825
Microorganisms 2021,10,
Microorganisms2022, 9, 399
x FOR PEER REVIEW 11 of
12 of 27
27

Figure5.5. Secondary
Figure Secondary metabolite
metabolite biosynthetic
biosyntheticgene
geneclusters
clusters(BGCs)
(BGCs)ofof
Bacillus halotolerans
Bacillus Cal.l.30
halotolerans us-
Cal.l.30
ing antiSMASH. Eight BGCs for the possible production of secondary metabolites: (A) Surfactin, (B)
using antiSMASH. Eight BGCs for the possible production of secondary metabolites: (A) Surfactin,
Mojavensin A/ Fengycin, (C) FAS-PKS, (D) Bacillaene, (E) Bacilysin, (F) Subtilosin A and (G) Bacil-
(B) Mojavensin A/ Fengycin, (C) FAS-PKS, (D) Bacillaene, (E) Bacilysin, (F) Subtilosin A and (G) Bacil-
libactin were predicted. Genes with different color represent different functions as described in the
libactin
coloredwere predicted. Genes with different color represent different functions as described in the
blocks.
colored blocks.
Microorganisms 2022, 10, 399 12 of 27

Table 1. Biosynthetic gene clusters (BGCs) involved in biosynthesis of secondary metabolites of

bacterial strain B. halotolerans Cal.l.30, detected by antiSMASH server.

Cluster BGC Type Compound MIBiG ID (Similarity %) Predicted Size (bp)

1 NRPS Surfactin BGC0000433_c1 (86%) 65.394
2 T1PKS-NRPS Kalimantacin A BGC0001532_c1 (20%) 117.825
3 Terpene Unknown - 20.426
4 TransAT-PKS-NRPS Bacillaene BGC0001089_c1 (100%) 114.235
5 TransAT-PKS-NRPS Fengycin/ Mojavensin A BGC0001095_c1 (100%) 129.752
6 Terpene Unknown - 21.898
7 T3PKS Unknown - 41.097
8 NRPS Bacillibactin BGC0000309_c1 (100%) 49.738
9 Other Bacilysin BGC0001184_c1 (100%) 41.418
10 Sactipeptide Subtilosin A BGC0001184_c1 (100%) 21.612

Genome mining using antiSMASH with the aid of ClusterBlast algorithm showed that
the kalimantacin A BGC has a high similarity to a novel FAS-PKS (Fatty Acid Synthases-
Polyketide Synthases) biosynthetic gene cluster identified in B. velezensis SQR9 and, there-
fore, will be referred to as FAS-PKS BGC (Figure 5C). Furthermore, a large part of the core
genes of the Cal.l.30 FAS-PKS BGC was identified in a BGC of B. halotolerans NRLLB-41618
(Figure 5C). Using the Cal.l.30 BGC nucleotide sequences as a Blastn query in the NCBI
database, it was realized that the core genes of Cal.l.30 in comparison to strains’ SQR9,
AP183 and NRLLB-41618 novel BGCs were collinearly homologous with a high similarity
of 97.05%, 96.25% and 99.61%, respectively. In contrast, the up and downstream flanking
DNA regions of Cal.l.30 FAS-PKS core BGC in comparison to those of the SQR9 strain, were
partly identified and showed a relatively less nucleotide identity, ranging from 85.32% to
85.53% (upstream) and 80.05% to 80.32% (downstream), respectively (Figure 5C). How-
ever, in strain NRLLB-41618 both down and upstream flanking DNA regions were highly
homologous with the corresponding genes of Cal.l.30 with a range of 98.00% and 99.59,
respectively (Figure 5C).
Furthermore, analysis of Cal.l.30 fengycin’s gene cluster revealed that adjacent to the
fengycin/plipastatin BGC, there is a BGC encoding for the biosynthesis of the iturinic
type lipopeptide, known as mojavensin A (Figure 5B). Genome mining (antiSMASH anal-
ysis) revealed that the recently published genomes of B. halotolerans strains F41-3 [43],
ZB201702 [44] and KKD1 [44] harbor only the plipastatin BGC, while B. halotolerans FJAT-
2398 harbors part of the core genes (ppsC, ppsD and ppsE) involved in the biosynthesis
of fengycin/plipastatin but also the whole BGC for the biosynthesis of mojavensin A
(Figure 5B). The up and downstream flanking DNA regions of Cal.l.30 and FJAT-2398
mojavensin A’s core BGC are found upstream of the F43-1 plipastatin core BGC joined in
a sequential mode (Figure 5B). In addition, Bacillus sp. TSO2 and Bacillus cereus MBGJa3
carry both plipastatin/fengycin and mojavensin A BGCs. The mojavensin A biosynthetic
gene cluster in strain Cal.l.30 is homologous to TSO2 and MBGJa3 corresponding BGCs,
with similarity rates of 99.34% and 97.8%, respectively, (Figure 5B). Phylogenetic analysis
indicated that both Bacillus sp. TSO2 and B. cereus MBGJa3 are classified as Bacillus cabrialesii,
a bacterial species phylogenetically diverse from B. halotolerans (Figure 3).
Further genome mining revealed that strain Cal.l.30 harbors the genes alsS, alsD, ilvB,
ilvH and bdhA involved in the biosynthesis of the well-known metabolites, 3-hydroxy-2-
butanone and 2, 3-butanediol that act as elicitors and promote antimicrobial activity by
inducing systemic resistance against plant pathogens [45].

3.5. Predicted Secondary Metabolite BGC Richness in B. halotolerans Genomes

To determine which secondary metabolites are encoded in the B. halotolerans strains
genome, we used antiSMASH 6.0 and ClusterBlast analysis to identify the BGCs in
21 sequenced genomes (Figure 6). Because in some strains BGC is spread in two or more
contigs and estimating the precise cluster boundaries is a critical step when computationally
 3.5. Predicted Secondary Metabolite BGC Richness in B. halotolerans Genomes
To determine which secondary metabolites are encoded in the B. halotolerans strains
Microorganisms 2022, 10, 399 genome, we used antiSMASH 6.0 and ClusterBlast analysis to identify the BGCs in 1321of 27
sequenced genomes (Figure 6). Because in some strains BGC is spread in two or more
contigs and estimating the precise cluster boundaries is a critical step when computation-
allycomparing
comparingBGCs, BGCs, all
all these
these clusters
clustersdetected
detectedby byantiSMASH
antiSMASH were
weremanually
manually assembled
assembled
using the most similar cluster identified in antiSMASH ClusterBlast analysis
using the most similar cluster identified in antiSMASH ClusterBlast analysis [46,47]. [46,47]. An-An-
tiSMASH outputs indicated that all the B. halotolerans strains harbor fengycin,
tiSMASH outputs indicated that all the B. halotolerans strains harbor fengycin, surfactin, surfactin,
bacillaene,
bacillaene,bacilysin,
bacilysin,bacillibactin
bacillibactinand
andsubtilosin
subtilosin A A BGC
BGCwhile
whilea fraction
a fraction (8 out
(8 out of 21ofstrains)
21
strains) harbors
harbors a mojavensin
a mojavensin A BGCA BGC (Figure
(Figure 6). Mojavensin
6). Mojavensin A production
A production hashasbeenbeen con-
confirmed
firmed
in B.inhalotolerans
B. halotolerans strains
strains Cal.l.30,
Cal.l.30, FJAT-2398,
FJAT-2398, NRLL NRLL B-41618T,
B-41618T, NRLL NRLL B-41617T
B-41617T andand ATCC
ATCC25096T [48]. All the B. halotolerans strains harboring or lacking the mojavensin A A
25096T [48]. All the B. halotolerans strains harboring or lacking the mojavensin BGC
BGC cluster
cluster onon neighboring
neighboring butbut distinct
distinct branches
branches of of
thethe phylogenetic
phylogenetic tree
tree (Figure
(Figure 6). 6).
TheTheinter-
inter-genomic relatedness
genomic relatedness among
among thethe B. halotolerans
B. halotolerans strains
strains (ANI(ANI and dDDH
and dDDH values)values)
showed
showed significant
significant variation.
variation. All B.All B. halotolerans
halotolerans strainsstrains harboring
harboring the mojavensin
the mojavensin A BGCAexhibit
BGC a
exhibit a relatively
relatively very highvery highofvalue
value dDDH of(92–93%)
dDDH (92–93%)
and ANI and ANI (98–99%)
(98–99%) among them, among
whilethem,
exhibit
while exhibit a lower
a relatively relatively
dDDH lower dDDHvalue
(80-82%) (80-82%)
withvalue with
all the all the strains
bacterial bacterial strains
(e.g. (e.g. B.
B. halotolerans
F43-1, B. F43-1,
halotolerans halotolerans
B. halotolerans KKD1)the
KKD1) lacking lacking the mojavensin
mojavensin A BGC4).
A BGC (Figure (Figure 4).

Figure 6. The
Figure 6. secondary metabolic
The secondary gene clusters
metabolic evolution.
gene clusters (I) Whole(I)
evolution. genome
Wholephylogeny of B. halo- of
genome phylogeny
tolerans strains highlighting the endophytic bacterial strains Cal.l.30, Cal.f.4 and Cal.l.11. The
B. halotolerans strains highlighting the endophytic bacterial strains Cal.l.30, Cal.f.4 and Cal.l.11.num-The
bersnumbers
above branches are pseudo-bootstrap support values from 100 replications. Inferred clusters
above branches are pseudo-bootstrap support values from 100 replications. Inferred clusters
are for B. halotolerans strains harboring (Cluster B) or lacking (Cluster A) the iturin/ mojavensin A
are for B. halotolerans strains harboring (Cluster B) or lacking (Cluster A) the iturin/ mojavensin A
BGC. (II) Number and distribution heatmap of the secondary metabolite biosynthetic gene clusters
BGC. (II) Number and distribution heatmap of the secondary metabolite biosynthetic gene clusters
(iturin/ mojavensin A, fengycin/ plipastatin, surfactin, bacillaene, bacilysin, bacillibactin, subtilosin
(iturin/
A and FAS-PKS)mojavensin A, fengycin/
detected (green) orplipastatin, surfactin,
not (red) among bacillaene, bacilysin,
B. halotolerans strains. B.bacillibactin, subtilosin
halotolerans strains
A andCal.l.11
Cal.l.30, FAS-PKS)
and detected
Cal.f.4 are(green) red. among B. halotolerans strains. B. halotolerans strains
or notin(red)
highlighted
Cal.l.30, Cal.l.11 and Cal.f.4 are highlighted in red.

3.6. Analysis of CAZyme Genes in B. halotolerans Stain Cal.l.30 Genome

Genome mining revealed the presence of 119 CAZymes encoding enzymes that target
key components of the plant and fungal cell wall mainly cellulose, chitin and hemicellulose.
There were 54 glycoside hydrolases (GHs), 38 glycosyltransferases (GTs), 7 polysaccharide
lyases (PLs), 14 carbohydrate esterases (CEs), and 1 auxiliary activity (AAs), as well as
13 carbohydrate-binding modules (CBMs) (Table 2). A large fraction (25.2%) of the CAZyme
Microorganisms 2022, 10, 399 14 of 27

proteins possess amino-terminal signal peptides to mediate protein export through the
cytoplasmic membrane, indicating that these are secreted enzymes. The genome of Cal.l.30
is characterized by an abundance of genes encoding for possible antifungal CAZymes
(secreted and non-secreted) in the GH families, such as chitinase (GH18), chitosanase
(GH46), glucanase (GH16, GH51, GH64) and β-glucosidase (GH1, GH3), which have the
potential to inhibit the growth of plant fungal pathogens (Table 2).

Table 2. Distribution of CAZymes gene families in the genome of B. halotolerans Cal.l.30.

CAZyme Categories Family Activity Gene Copy Numbers

GH0 - 1
GH1 β-glucosidase 4
GH3 β-glucosidase 1
GH4 maltose-6-phosphate glucosidase 4
GH5 cellulase 1
GH11 endo-β-1,4-xylanase 1
GH13 α-amylase 9
GH16 xyloglucan:xyloglucosyltransferase 1
GH18 chitinase 4
GH23 lysozyme type G 4
GH26 β-mannanase 1
Glycoside Hydrolases GH30 endo-β-1,4-xylanase 1
(GH) GH32 invertase 3
GH42 β-galactosidase 2
GH43 β-xylosidase 4
GH51 endoglucanase 2
GH53 endo-β-1,4-galactanase 1
GH46 chitosanase 2
GH65 α,α-trehalase 1
GH68 levansucrase 1
GH73 lysozyme 2
GH105 unsaturated rhamnogalacturonyl hydrolase 2
GH126 α-amylase 1
GH171 peptidoglycan β-N-acetylmuramidase 1
GT0 - 4
GT1 UDP-glucuronosyltransferase 3
GT2 cellulose synthase 13
GT4 sucrose synthase 7
Glycosyl Transferases GT5 glycogen glucosyltransferase 1
(GT) GT8 α-1,3-galactosyltransferase 1
GT26 β-N-acetyl mannosaminuronyltransferase 1
GT28 1,2-diacylglycerol 3-β-galactosyltransferase 3
GT35 glycogen 1
GT51 murein polymerase 4
CE4 acetyl xylan esterase 7
CE7 acetyl xylan esterase 1
Carbohydrate Esterases CE9 N-acetylglucosamine 6-phosphate deacetylase 2
(CE) CE12 pectin acetylesterase 3
N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-
CE14 1
glucopyranoside deacetylase
PL1 pectate lyase 2
PL3 pectate lyase 1
Polysaccharide Lyases
PL9 pectate lyase 1
(PL)
PL11 rhamnogalacturonan endolyase 2
PL26 rhamnogalacturonan exolyase 1
Auxiliary Activities
AA10 lytic polysaccharide monooxygenases 1
(AA)

Carbohydrate-Binding CBM16 binding to cellulose and glucomannan 1

Modules (CBM) attached to various enzymes from families GH18,
CBM50 11
GH19, GH23, GH24, GH25 and GH73
CBM63 bind cellulose 1
 Auxiliary Activities
AA10 lytic polysaccharide monooxygenases 1
(AA)
Carbohydrate-Binding Mod- CBM16 binding to cellulose and glucomannan 1
ules attached to various enzymes from families GH18, GH19,
CBM50 11
(CBM)
Microorganisms 2022, 10, 399 GH23, GH24, GH25 and GH73 15 of 27
CBM63 bind cellulose 1

3.7.
3.7.Constitutive
ConstitutiveProduction
Productionand
andSecretion
SecretionofofAgar-Diffusible
Agar-DiffusibleAntifungal
AntifungalMetabolites
Metabolitesof
of Cal.l.30
Cal.l.30
when
WhenGrown
Grownon onaaSolid
SolidSurface
Surface
Cal.l.30
Cal.l.30ethyl
ethylacetate
acetateextracts
extractsof
ofagar-diffusible
agar-diffusiblesecreted
secretedmetabolites
metabolitesproduced
producedwhenwhen
Cal.l.30
Cal.l.30isisgrown
grownsingly
singly(ADSM1)
(ADSM1)or orduring
duringconfrontation
confrontationwithwithB.
B.cinerea
cinerea(ADSM2),
(ADSM2),werewere
evaluated
evaluatedfor fortheir
theirantifungal
antifungalactivity using
activity agar-plate
using diffusion
agar-plate basebase
diffusion assay and TLC-bio-
assay and TLC-
autography
bioautography assay. BothBoth
assay. ADSM1 and ADSM2
ADSM1 and ADSM2 couldcould
effectively constrain
effectively the growth
constrain and
the growth
expansion of fungal mycelium (Figure 7A). Furthermore, TLC-bioautography,
and expansion of fungal mycelium (Figure 7A). Furthermore, TLC-bioautography, using using B.
cinerea as an
B. cinerea indicator,
as an revealed
indicator, revealedthat both
that bothADSM1
ADSM1and andADSM2
ADSM2formformaastrong
stronginhibition
inhibition
spot
spot(suppressed
(suppressedconidial
conidialgermination)
germination)with
withidentical
identicalRf
Rfvalues
values(0.36
(0.36and
and0.40),
0.40),showing
showing
that
thatCal.l.30
Cal.l.30 biosynthesizes
biosynthesizes and and secretes
secretesbioactive
bioactivemetabolites
metaboliteswhen
whengrown
grown individually
individually on
on a solid
a solid surface
surface (Figure
(Figure 7B).
7B).

Figure
Figure7.7.Inhibitory
Inhibitoryeffect of agar-diffusible
effect secondary
of agar-diffusible metabolites
secondary (ADSM)
metabolites of Cal.l.30
(ADSM) against
of Cal.l.30 B.
against
cinerea. (A) Confrontation well-diffusion assay at 3 d (i) and 6 d (ii) of interaction. ADSM1 and
B. cinerea. (A) Confrontation well-diffusion assay at 3 d (i) and 6 d (ii) of interaction. ADSM1
ADSM2 represent agar-diffusible secondary metabolites of Cal.l.30 when grown singly and to-
and ADSM2 represent agar-diffusible secondary metabolites of Cal.l.30 when grown singly and
gether with B. cinerea, respectively, while M is the solvent of methanol used as negative control.
together
(B) with B. cinerea,using
TLC-bioautography respectively,
ADSM1 while M is the
and ADSM2 of solvent
Cal.l.30.of methanol used as negative control.
(B) TLC-bioautography using ADSM1 and ADSM2 of Cal.l.30.
Ultra-high-performance liquid chromatography coupled to Q Exactive Orbitrap
Ultra-high-performance liquid chromatography coupled to Q Exactive Orbitrap High-
High-Resolution Mass Spectrometry (UHPLC-HRMS) was employed for the putative an-
Resolution Mass Spectrometry (UHPLC-HRMS) was employed for the putative annotation
notation of secondary (antifungal) metabolites biosynthesized and secreted by strain
of secondary (antifungal) metabolites biosynthesized and secreted by strain Cal.l.30 when
Cal.l.30 when grown singly on a solid surface. The UHPLC-HRMS chemical analysis re-
grown singly on a solid surface. The UHPLC-HRMS chemical analysis revealed the pres-
vealed the presence of different types of secondary metabolites with antimicrobial/ anti-
ence of different types of secondary metabolites with antimicrobial/ antifungal activity,
fungal activity, including the lipopeptides, mojavensin A, fengycin and surfactin, the pol-
including the lipopeptides, mojavensin A, fengycin and surfactin, the polyene diamide
yene diamide
polyketide polyketide
bacillaene, bacillaene,
the the intermediate
intermediate metabolite ofmetabolite
bacilysin of bacilysin biosynthetic
biosynthetic pathway, L-
pathway, L-dihydroanticapsin and the siderophore bacillibactin
dihydroanticapsin and the siderophore bacillibactin (Table 3). (Table 3).
The compounds were putatively annotated based on isotope distribution and the
accurate mass (±5 ppm). The precise masses obtained from ions m/z 1082.5617 [M–H]- and
m/z 1461.7893 [M–H]- revealed the presence of two compounds with molecular formulas
C50 H77 N13 O14 and C72 H110 N12 O20 annotated as mojavensin A and fengycin A, respectively
(Figure 8A,B). In addition, HRMS analysis revealed the presence of ions with m/z 1006.6447
[M–H]- and m/z 1034.6760 [M–H]- corresponding to surfactin A analogues with molecular
formula of C51 H89 N7 O13 (Surfactin A C13 ) and C53 H93 N7 O13 (Surfactin A C15 ), respectively
(Figure 8C,D). The compounds with m/z 881.2492 [M–H]- and m/z 200.0923 [M–H]- were as-
signed to the siderophore bacillibactin and L-dihydroanticapsin, respectively (Figure 8E,K).
The compounds with m/z 579.3441 [M–H]-, m/z 581.3597 [M–H]-, m/z 741.3978 [M–H]- and
m/z 843.4293 [M–H]- were assigned to the four analogues of bacillaene, known as bacillaene
A, dihydrobacillaene, bacillaene B and bacillaene C, with molecular formulas C34 H48 N2 O6 ,
C34 H50 N2 O6 , C40 H58 N2 O11 and C44 H64 N2 O14 , respectively (Figure 8G–J).
Microorganisms 2022, 10, 399 Microorganisms 2021, 9, x FOR PEER REVIEW
16 17
of of
27 27

Figure 8. Cont.
Microorganisms 2022, 10, 399 17 of 27
Microorganisms 2021, 9, x FOR PEER REVIEW 18 of 27

Figure 8. Agar-diffusible secondary metabolites secreted by B. halotolerans Cal.l.30 and annotated

through UHPLC-HRMS analysis. The compounds were putatively annotated based on isotope
distribution and the accurate mass (±5 ppm). The precise masses revealed the presence of compounds
(A) mojavensin A, (B) fengycin A, (C) Surfactin A C13, (D) Surfactin A C15 , (E) L-dihydroanticapsin
(K) bacillibactin, (G–J) four analogues of bacillaene, known as bacillaene A, dihydrobacillaene,
bacillaene B and bacillaene C, respectively, (F,L,M) azelaic acid, 15-hydroxypentadecanoid acid and
2-hydroxyphenylacetic acid, respectively.
Microorganisms 2022, 10, 399 18 of 27

Table 3. Secondary metabolites biosynthesized by B. halotolerans Cal.l.30 and putatively annotated

through UHPLC-HRMS analysis.

Antibiotic Molecular Experimental RT Reference

∆ppm Adduct
Compounds Formula m/z (min) or Source
Mojavensin A C50 H77 N13 O14 1082.5617 14.66 -1.13 [M–H]- [49]
Surfactin A C13 C51 H89 N7 O13 1006.6447 22.699 1.23 [M–H]- [50]
Surfactin A C15 C53 H93 N7 O13 1035.683 23.945 0.57 [M–H]- [51]
Fengycin A C16 C72 H110 N12 O20 1461.7893 16.51 1.19 [M–H]- [52]
Bacillaene A C34 H48 N2 O6 579.3441 16.365 2.13 [M–H]- [53]
dihydrobacillaene A C34 H50 N2 O6 581.3597 16.24 2.04 [M–H]- [53]
Bacillaene B C40 H58 N2 O11 741.3978 14.357 2.85 [M–H]- [54]
Bacillaene C C44 H64 N2 O14 843.4293 14.984 2.27 [M–H]- [54]
L-dihydroanticapsin C9 H15 NO4 200.0923 11.395 2.83 [M–H]- [55]
Azelaic acid C9 H16 O4 187.0969 11.022 2.21 [M–H]- [56]
Bacillibactin C39 H42 N6 O18 881.2497 11.88 2.85 [M–H]- [57]
15-hydroxypentadecanoid acid C15 H30 O3 257.2124 17.953 4.97 [M–H]- [58]
2-hydroxyphenylacetic acid C8 H8 O3 151.0391 9.381 0.86 [M–H]- [59]

In addition, other compounds, such as azelaic acid, and the fatty acids 15-hydroxypen-
tadecanoid acid and 2-hydroxyphenylacetic acid, with known antioxidant, antibiotic
and/or antifungal activity were also detected in the ADSM1 fraction (Table 3). Ion signals
with m/z 187.0969 [M–H]-, m/z 257.2124 [M–H]- and m/z 151.0391 [M–H]- were assigned to
azelaic acid, 15-hydroxypentadecanoid acid and 2-hydroxyphenylacetic acid with molecu-
lar formulas C9 H6 O4 , C15 H30 O3 and C8 H8 O3 , respectively (Figure 8F,L,M).

4. Discussion
The ecological challenges posed by the extensive use of chemical fungicides to control
plant pathogens have reinforced the need for replacing them with biopesticides against
plant pathogens [60]. The application of microorganisms and especially bacteria in agri-
culture has significantly increased in recent years, making it necessary to find strong and
effective BCAs. Endophytic bacteria derived from medicinal plants are an important source
of potential BCAs, due to the diverse competing mechanisms they have developed in order
to adapt and survive endophytically [44,61]. C. officinalis is one of the most important
medicinal plants, used in traditional medicine since 12 BC, due to its multiple antimicrobial
properties [62]. As far as we know, only one study has been conducted on the endophytic
bacterial community of the medicinal plant C. officinalis, from which a very small number
of bacteria (five strains) were isolated and only from the leaves and shoots of the plant [63].
As presented in a previous study of ours, a total number of 119 cultivable endophytic
bacterial strains were isolated from different plant parts such as roots, leaves and flowers of
the medicinal plant Calendula officinalis, from which twenty-four morphologically distinct
endophytic bacterial strains of the genus Bacillus were identified based on 16S rRNA
sequencing analysis [25]. After co-cultivation with Arabidopsis thaliana seedlings, the
majority of Bacillus strains positively affected plants’ morphological characteristics such as
total fresh weight, primary root length, lateral root number and root hair number [25]. In
the current study, the most promising Bacillus isolates, B. halotolerans strains Cal.l.30, Cal.f.4,
Cal.l.11, Cal.f.2.1, Cal.r.11, B. velezensis strain Cal.r.29 and B. subtilis strain Cal.r.19, were
further selected and studied for their possible antifungal activity. Among them, Bacillus
velezensis Cal.r.29, B. halotolerans Cal.l.30 and B. halotolerans Cal.r.11 prevented B. cinerea
more efficiently by creating the strongest inhibition zone in between, under in vitro dual
culture assay. Bacteria of the genus Bacillus stand out for their antagonistic activity due
to their multiple antimicrobial secondary metabolites and modes of action, with secreted
lipopeptides being the most crucial [2,8]. Previous studies have shown that the inhibition
zone created during the microbial interaction consists of lytic enzymes and accumulated
secreted antimicrobial compounds with biosurfactants, mainly lipopeptides, being mostly
responsible for phytopathogen suppression [52]. The secretion of biosurfactants takes place
Microorganisms 2022, 10, 399 19 of 27

not only on solid and semi-solid substrates, but also on liquid growth medium, maintaining
their strong antimicrobial activity [52]. Biosurfactants are molecules that play an important
role in antagonistic attributes of microbial BCAs because they facilitate colonization, they
may have direct antimicrobial properties or elicit an immune response in plant tissues that
makes the host less susceptible to subsequent infection [10]. In the present study, all seven
Bacillus strains that created a strong inhibition zone against B. cinerea, were also positive for
secreting biosurfactants into their liquid culture with their supernatant showing excellent
drop collapse ability.
Although endophytic bacteria of the Bacillus species have emerged as an alternative
promising strategy to control plant pathogens [8], the discovery and scrutiny of novel
endophytic Bacillus spp. can provide new options in the field of food and crop protection,
specifically for the development of efficient microbial biocontrol agents to suppress post
and preharvest fungal plant diseases [10,20]. In the current study, B. halotolerans was the
predominant species among the Bacillus species isolated from C. officinalis, with strain
Cal.l.30 being the most promising BCA candidate. B. halotolerans is a newly characterized
species within the Bacillus genus [64]. Thus far, a limited number of plant-associated
B. halotolerans strains have been isolated, and even fewer have been evaluated for their
potential to act as BCAs against plant pathogens [11–13,18].
In the present study, an integrative approach coupling genome mining and metabolic
profiling has been applied to decipher the potential of the novel endophyte B. halotol-
erans Cal.l.30 as a BCA against the postharvest pathogen B. cinerea. Genome mining
revealed that a considerable part (13%) of the Cal.l.30 genome is predicted to be devoted
to the biosynthesis of secondary metabolites compared to the approximately 9% identi-
fied in the other B. halotolerans strains [11,12]. B. halotolerans Cal.l.30, in addition to the
fengycin/plipastatin, surfactin, bacillaene, bacilysin, bacillibactin and subtilosin A BGCs
found in all B. halotolerans strains, contains also both the mojavensin A and FAS-PKS gene
clusters. Phylogenomic analysis revealed that all the B. halotolerans strains harboring or lack-
ing the mojavensin A BGC cluster in well-separated distinct branches of the phylogenetic
tree. Furthermore, B. halotolerans strains harboring the mojavensin A BGC share above 91%
dDDH similarities with each other but only 80–82% with other B. halotolerans strains, a value
very close to the boundary of 79–80% dDDH for subspecies delineation [65]. Thus, it could
be argued that the acquisition of gene clusters may lead to ecological diversification and
speciation [66,67]. Whether this speciation process may be related to endophytic behavior
is not yet known [68].
The mojavensin A and FAS-PKS BGCs’ diversity across B. halotolerans strains suggests
that both BGCs may have been acquired via recombination and horizontal gene transfer
processes [69–71]. Our in silico analysis revealed that B. halotolerans strains contain ‘hot
spots’ at specific location of their genome, allowing the preferential recombination at non-
homologous regions that are flanked by homologous regions with the net result being the
incorporation of mojavensin A BGC during a horizontal gene transfer (HGT) event [72].
The mechanism only requires housekeeping of recombination functions and exogenous
DNA with homology to the flanking core genes [72,73]. The evolutionary origin of these
novel BGCs is not known; however, the presence of almost identical mojavensin A BGC in
both B. halotolerans Cal.l.30 and the B. cabrialesii genome, and the identical FAS-PKS BGC in
B. halotolerans Cal.l.30 and B. velezensis SQR9, clearly indicates that these clusters may have
originated via an HGT event from a donor closely related to the B. subtilis species.
Horizontal BGC transfer usually results in the production of an existing compound
in a new, typically distantly related organism [74]. Thus, the acquisition of BGCs by the
Cal.l.30, as well as the potential endowment with new traits, also provides the opportunity
to improve upon an existing trait, generating synergistic effects between the corresponding
secondary metabolites. As a result, strain Cal.l.30 may compete with other members of the
population, and the associated genome can theoretically sweep through the population [75].
The ecological role of mojavensin A BGC and the FAS-PKS BGC products in Cal.l.30
is not yet known. A few studies have aimed to explore the ecological role of the biosyn-
Microorganisms 2022, 10, 399 20 of 27

thetic products of mojavensin A and FAS-PKS BGCs [51,76]. Purified mojavensin A from
B. mojavensis B0621A displayed moderate antagonism and dose-dependent activity against
F. oxysporum [76], while mojavensin A produced by the actinomycete Gordonia strain WA
4-31 showed adequate antimicrobial activity against both Aspergillus nidulans and Escherichia
coli [77]. Interestingly, mojavensin A and a cyclic (leucine–leucine) dipeptide had a syner-
gistic effect and showed enhanced antimicrobial activity [77]. On the other hand, the novel
cluster (a FAS-PKS biosynthetic gene cluster) found in SQR9 appeared to be involved in the
biosynthesis of the novel long-chain fatty acid(s) (C15 H30 O3 and/or C17 H34 O3 ) such as bacil-
lunoic acid(s), presenting an adequate antifungal and antibacterial activity [51]. Furthermore,
inactivation of the FAS-PKS BGC appeared to reduce root colonization ability of B. velezensis
SQR9 over closely related rhizobacteria [51]. Following the aforementioned observation,
similar or identical compounds may be produced by Cal.l.30 since 15-hydroxypentadecanoid
acid with the chemical formula C15 H30 O3 was identified.
In comparison to other B. halotolerans strains, Cal.l.30 is a novel and powerful strain
due to its ability to biosynthesize and secrete a significant number of secondary metabolites
and strong antimicrobial compounds. Among the compounds identified after UHPLC-
ESI HRMS analysis are C16 fengycin A, surfactin A (C13 surfactin A and C15 surfactin A),
mojavensin A, four analogues of the antibiotic polyketide bacillaene (bacillaene A1, bacil-
laene B1, bacillaene C2 and dihydrobacillaene A), the iron chelating compound bacillibactin,
the active substance azelaic acid, and the fatty acids 15-hydroxypentadecanoid acid and
2-hydroxyphenylacetic acid. In addition, the secondary metabolite L-dihydroanticapsin
was identified in B. halotolerans Cal.l.30, which is the precursor of the potent antibiotic
bacilycin [55]. Bacilycin is an important dipeptide with strong antibacterial and antifungal
activity [51].
One of the most important observations in our study was the ability of Cal.l.30 to
secrete the iturinic-type lipopeptide mojavensin A. Metabolic analysis of Cal.l.30, con-
firmed the results of genomic analysis corresponding to the lipopeptide of mojavensin A
(C50 H77 N13 O14 ), with an m/z value of 1082.5617. This result is in agreement with Ma and
colleagues who first isolated and identified mojavensin A in B. mojavensis B0621A [49].
Of particular interest was the detection of the polyketide bacillaene, biosynthesized
by the endophyte Cal.l.30. Although it is a chemically unstable secondary metabolite,
sensitive to light, oxygen and even room temperature [54], we were able to detect four
analogues of the compound, identified as bacillaene A, dihydrobacillaene A, bacillaene B
and bacillaene C [54]. Due to the sensitivity of the polyketide, a limited number of lit-
erature reports have been able to highlight the antibacterial activity of the compound
and its analogues, as well as their inhibitory effect against fungi in vitro [78–81]. Further-
more, bacillaene can accelerate self-biofilm development of B. subtilis at sub-inhibitory
concentrations [54], inhibit biofilm formation and disperse pre-established biofilm by other
bacteria [82], protect B. subtilis from predation by other bacteria [83] and may modulate the
production of secondary metabolites by competing bacteria [84].
One of the most significant compounds secreted and identified in B. halotolerans Cal.l.30
is the active substance azelaic acid (AzA). Thus far, only a few reports have been found
on the biosynthesis of AzA by bacteria, and only two by Bacillus species and, specifically,
B. velezensis Bvel1 and B. halotolerans Hil4 [18,26,56]. AzA is a nine-carbon dicarboxylic
acid, which has been reported primarily in plants as a mobile signaling molecule that
activates plant defense mechanisms [85]. Specifically, the produced azelaic acid triggers
the accumulation of SA in plants by activating SAR in order to treat phytopathogenic
microorganisms, either in the contact tissue or systemically [86,87].
The current study establishes the antagonistic endophytic bacterial strain Cal.l.30
as a strong inhibitor of the necrotrophic phytopathogenic fungus B. cinerea, under both
in vitro (dual culture assay) and ex vivo conditions (detached fruit assay). The application
of bacteria on plant tissues is an important method of selecting competing BCAs, able
to protect plants from pathogenic microorganisms. Strain Cal.l.30 was able to suppress
B. cinerea and significantly reduce disease incidence and disease severity index of gray
Microorganisms 2022, 10, 399 21 of 27

mold when applied on tomato cherry fruits and grape berries, in comparison to control
treatments. Moreover, the application of Cal.l.30 cell free supernatant on both the fruit
tissues significantly inhibited the spread of the phytopathogen, confirming the existence of
bioactive secondary metabolites in its liquid culture.
A necessary step for an efficient BCA inoculation and biological control is the suc-
cessful colonization of the BCA candidate at the point of formulation and its effective
competition for nutrients and space against the host microbial community [24,88,89]. In the
present study, strain Cal.l.30 successfully colonized both of the plant tissues tested, keeping
a high cell density until the last day of observation. It is known that the cyclic lipopeptide
surfactin, produced by bacteria of the genus Bacillus, is directly involved in the movement,
biofilm formation and colonization of bacteria on surfaces and is less referred to as a strong
antimicrobial compound [90–92]. Among surfactin’s compounds, isoform C15 surfactin A,
which was also identified in strain Cal.l.30, is a particularly potent antibiotic lipopeptide
against a large number of phytopathogenic fungi, including B. cinerea [93–95]. In contrast
to the surfactin lipopeptide, which is mainly involved in biofilm formation and bacterial
colonization, the cyclic lipopeptides of the iturin and fengycin family are the major antimi-
crobial compounds of the Bacillus species [49,50,91,92,96]. Both genomic and metabolomic
analysis of Cal.l.30 indicated the production of surfactin (C13 surfactin A and C15 surfactin
A), fengycin (C16 fengycin A) and mojavensin A biosurfactants, enhancing their role in the
colonization of Cal.l.30 on tomato fruits and grape berries and antagonistic activity. It is
worth mentioning that bacillaene compounds, in addition to their antimicrobial activity,
accelerate the production of biofilm for a better bacterial cell self-protection [54]. Therefore,
the production of bacillaene may have played a crucial self-protective role for strain Cal.l.30,
increased biofilm formation for a successful ex vivo colonization and combated potential
competing microorganisms.
As a result of successful colonization, Cal.l.30 likely reduced B. cinerea access to essential
nutrients, such as iron. In silico analysis of strain Cal.l.30 highlighted the genes involved for
the biosynthesis of the siderophore bacillibactin, while metabolomics enhanced the above
result by identifying the compound (C39 H42 N6 O18 ). Iron is one of the key ingredients in the
formation of the bacterial biofilm as it stabilizes the matrix of polysaccharides involved in
its formation [97]. Therefore, the binding of available iron significantly increases biofilm
formation and, consequently, the colonization of bacteria at the site of inoculation. Absorp-
tion of available iron is carried out by the production of bacterial iron chelating compounds,
which significantly reduce the available iron to phytopathogenic fungi, an element necessary
for their spore germination, growth and, ultimately, their infectivity [98–100]. Thus, the
production of bacterial iron chelating compounds in comparison to the total number of
antimicrobial compounds, enhances their competitiveness [101].
A significant number of scientific reports have focused on the emergence of bacte-
rial lipopeptides (LPs), polyketides (PKs), lytic enzymes, phytohormones, volatiles, iron
compounds and various proteins (e.g. flagellin, flg22) as strong ISR elicitors [10,102–104].
Waewthongrak and colleagues presented ISR induction of mandarin fruits after the appli-
cation of both the endospores and bioactive supernatant of B. subtilis ABS-S14, resulting in
an efficient protection against the pathogenic fungus Penicillium digitatum [105]. Fragments
of chitosan, as a result of chitinolytic enzymes action, are known to elicit plant defense
reactions both locally and systemically, including the stimulation of the production of small
molecules such as jasmonic acid and abscisic acid [106]. Additionally, lipopeptides, such
as plipastatin, bacillomycin L and surfactin produced by antagonistic Bacillus sp., have
also been reported as elicitors for inducing disease resistance within plant hosts [107,108].
Phenylacetic acid produced and secreted by B. fortis IAGS162 may act as an ISR elicitor con-
tributing to tomato plants’ resistance against Fusarium wilt disease [109]. Furthermore, a re-
cent study demonstrated that medium-chain 3-hydroxy fatty acids ((R)-3-hydroxydecanoic
acid) trigger immunity in Arabidopsis [110].
Microorganisms 2022, 10, 399 22 of 27

5. Conclusions
This study introduced the novel B. halotolerans Cal.l.30 strain, which is endowed with
a rich arsenal of secondary metabolite biosynthetic gene clusters, equipped with a large
repertoire of CAZyme genes involved in antifungal activity, and armed with the capacity to
produce and secrete secondary metabolites and other novel compounds that are involved in
antibiosis and/or host plant systemic resistance induction and the successful colonization
of host plant tissues. In many cases, fungal pathogens such as B. cinerea and Colletotrichum
acutatum infest fruits (at the flower stage) in the field, and these latent infections may
become active at pre and/or postharvest stage [111]. Based on in vitro and ex vivo assays,
B. halotolerans Cal.l.30 effectively controlled the ingression and expansion of the postharvest
fungal pathogen B. cinerea in both tomato fruits and grape berries. In this context, it will
be of interest to explore the prospects of the endophytic strain B. halotolerans Cal.l.30 as a
biological control agent of pre and postharvest fungal diseases.

Author Contributions: Conceptualization, P.C.T., A.V. and P.K.; methodology, P.C.T., E.-E.T., A.V.
and E.B.; investigation and formal analysis, P.C.T., E.-E.T., E.B. and K.P.; validation: P.C.T.; writing—
original draft, P.C.T. and P.K.; elaborating the research questions, analyzing the data, formal analysis,
software, writing and reviewing the article: P.C.T., E.-E.T., E.B., A.S., K.P., A.V. and P.K.; supervision
and funding acquisition, P.K. and A.V. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was partially funded by the project «The foremost flagship in Greece ‘vine-
yards roads’» (Code number 2018ΣE01300000/Public Investments Program General Secretariat for
Research and Technology), Greek Ministry of Education and Religious Affairs. The research of Polina
Tsalgatidou was co-financed by Greece and the European Union (European Social Fund, ESF) through
the Operational Programme «Human Resources Development, Education and Lifelong Learning» in
the context of the project ‘Strengthening Human Resources Research Potential via Doctorate Research,
2nd cycle’ (MIS-5000432), implemented by the State Scholarships Foundation (IKΥ).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The Bacillus halotolerans strains Cal.l.30, Cal.f.4 and Cal.l.11 whole
genome projects are available in the NCBI database under the accession numbers JAEACK000000000,
JAEACI000000000 and JAEACJ000000000(GenBank), respectively, and SAMN16949411, SAMN16949417,
SAMN16949418 (BioSample) and PRJNA681331, PRJNA681333, PRJNA681332 (BioProject), respectively.
Conflicts of Interest: The authors declare no conflict of interest.

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