Multidisciplinary Collaborative Journal
|
Vol
.
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.
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ISSN:
3073
-
1356
1
Artic
le
Efficacy of
Trichoderma
sp. and
Bacillus subtilis
as
biocontrol agents against
Pseudocercospora fijiensis
in the
cultivation of banana (
Musa × paradisiaca
)
Angel Virgilio Cedeño Moreira
1
,
Kimberly López Cedeño
2
,
Mauricio
Renato
Morejón Centeno
3
,
Juan Antonio Torres Rodríguez
4
,
Ketty Vanessa Arellano Ibarra
5
,
Jorge Alberto Alejandre Rosas
6
and
Alejandra Alvarado Mávil
7
*
1
Facultad
de Ciencias Pecuarias y Biológicas, Universidad Técnica Estatal de
Quevedo, Av. Quito km 1.5 vía a Santo Domingo, Quevedo 120501, Ecuador
;
https://orcid.org/0000
-
0002
-
6564
-
5569
;
acedenom@uteq.edu.ec
2
Instituto Superior Tecnológico La Maná, Av. Amazonas entre Miguel Iturralde y
Héroes del Cenepa, La Maná 050202, Ecuador
;
https://orcid.org/0000
-
0002
-
6838
-
1474
;
klopez@istlm.edu.ec
3
Facultad de Ciencias Agrarias y Forestales, Universidad Técnica Estatal de
Quevedo, Av. Quito km 1.
5 vía a Santo Domingo, Quevedo 120501, Ecuador
;
https://orcid.org/0000
-
0002
-
2621
-
2306
;
mmorejonc@uteq.edu.ec
4
Facultad
de Ciencias Agrarias y Forestales, Universidad Técnica Estatal de
Quevedo, Av. Quito km 1.5 vía a Santo Domingo, Quevedo 120501, Ecuador
;
https://orcid.org/0000
-
0003
-
3326
-
4371
;
jatorres@uteq.edu.ec
5
Facultad de Ciencias Pecuarias y Biológicas, Universidad Técnica Estatal de
Quevedo, Av. Quito km 1.5 vía a Santo Domingo, Quevedo 120501, Ecuador
;
https://orcid.org/0000
-
0001
-
7168
-
7485
;
Ketty.arellano2017@uteq.edu.ec
6
Facultad de Ciencias Químicas de Orizaba, Universidad Veracruzana, Oriente 6
No. 1009, Colonia Rafael Alvarado, CP. 94340 Orizaba, Mexico;
https://orcid.org/0000
-
0002
-
1252
-
4966
;
jalejandre@uv.mx
7
Facultad
de Ciencias Químicas de Orizaba, Universidad Veracruzana, Oriente 6
No. 1009, Colonia Rafael Alvarado, CP. 94340 Orizaba, Mexico;
https://orcid.org/0009
-
0009
-
3041
-
8997
;
aalvarado@uv.mx
*
Correspondence:
aalvarado@uv.mx
https://doi.org/10.70881/mcj/v4/n2/145
Abstract:
This study evaluated the efficacy of
Trichoderma
sp. and
Bacillus
subtilis
as biocontrol agents against
Pseudocercospora fijiensis
, the fungus
responsible for Black Sigatoka in bananas (
Musa × paradisiaca
). Under
laboratory conditions, the inhibition of ascospore germination
and radial
growth was evaluated by applying
Trichoderma
sp.
and
B. subtilis
metabolites
at concentrations of 5% and 10 %. At a 10 % concentration,
B. subtilis
achieved 100% inhibition of ascospore germination and a 90 % reduction in
radial growth of
P
. fi
jiensis
, outperforming
Trichoderma
, which achieved 60
% inhibition in both tests at the same concentration. In greenhouse trials,
disease incidence and severity were measured in Cavendish banana
seedlings inoculated with
P
. fijiensis
and treated with weekl
y foliar
applications of the biocontrol agents. At a 10 % concentration,
B. subtilis
reduced disease severity to 10 %, while
Trichoderma
at 10 % achieved a 30
% reduction in severity, compared to the control, which maintained a constant
severity of around
81 %. These results highlight the potential of
B. subtilis
as
a robust biocontrol agent against
P
. fijiensis
, providing a sustainable and
effective alternative for managing Black Sigatoka in agricultural systems, thus
reducing dependency on chemical fungic
ides and promoting environmentally
responsible cultivation practices.
Cit
ation
:
Cedeño Moreira, A. V.,
López Cedeño, K., Morejón
Centeno, M. R., Torres Rodríguez,
J. A., Arellano Ibarra, K. V.,
Alejandre Rosas, J. A., & Alvarado
Mávil, A. (2026). Eficacia de
Trichoderma sp. y Bacillus subtilis
como agentes de control biológico
contra
Pseudocercospora fijiensis
en el cultivo del plátano (Musa ×
paradisiaca).
Multidisciplinary
Collaborative Journal
,
4
(2), 1
-
15.
https://doi.org/10.70881/mcj/v4
/n2/145
Received:
02
/03/202
6
Revised:
06
/04
/2026
Accepted:
09
/04/2026
Published:
14
/04/2026
Copyright:
2026
by the authors.
This article is an open access
article distributed under the terms
and conditions of the
Creative
Commons License, Attribution
-
NonCommercial 4.0 International
(CC BY
-
NC).
(
htt
ps://creativecommons.org/licen
ses/by
-
nc/4.0/
)
Multidisciplinary Collaborative Journal
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2
Palabras clave:
biocontrol,
ascospore germination, incidence, metabolites,
radial growth
Resumen
:
Este estudio evaluó la eficacia de
Trichoderma
sp. y
Bacillus subtilis
como
agentes de biocontrol contra
Pseudocercospora fijiensis
, el hongo responsable de la
Sigatoka Negra en banano (
Musa × paradisiaca
). Bajo condiciones de laboratorio, se
evaluó la inhibición de la germinación de ascosporas y el crecimiento radial mediante
la
aplicación de metabolitos de
Trichoderma
sp
.
y
B. subtilis
en concentraciones de
5% y 10%. A una concentración de 10%,
B. subtilis
logró una inhibición del 100% de
la germinación de ascosporas y una reducción del 90% en el crecimiento radial de
P
.
fijiensi
s
, superando a
Trichoderma
, que logró una inhibición del 60% en ambas
pruebas a la misma concentración. En ensayos de invernadero, se midió la incidencia
y severidad de la enfermedad en plántulas de banano Cavendish inoculadas con
P
.
fijiensis
y tratadas c
on aplicaciones foliares semanales de los agentes de biocontrol.
Con una concentración del 10 %,
B. subtilis
redujo la gravedad de la enfermedad al
10 %, mientras que
Trichoderma
, al 10 %, logró una reducción del 30 % en la
gravedad, en comparación con el
control, que mantuvo una gravedad constante de
alrededor del 81 %. Estos resultados resaltan el potencial de
B. subtilis
como un
potente agente de biocontrol contra
P
. fijiensis
, ofreciendo una alternativa sostenible
y eficaz para el manejo de la Sigatoka
Negra en sistemas agrícolas, reduciendo así
la dependencia de fungicidas químicos y promoviendo prácticas de cultivo
respetuosas con el medio ambiente.
Keywords:
Biocontrol, germinación de ascosporas, incidencia, metabolitos,
crecimiento radial
1.
Introduction
Banana (
Musa × paradisiaca
) cultivation represents one of the most important economic
and nutritional pillars in tropical and subtropical regions worldwide (Sau et al., 2023).
This crop is a fundamental source of income and employment for mill
ions of farmers and
rural communities, in addition to being an important source of carbohydrates and other
nutrients in human diets (Alonso et al., 2020). However, banana crops are severely
threatened by various diseases, among which Black Sigatoka, caused
by the fungus
Pseudocercospora fijiensis
, is particularly prominent (Esguera et al., 2024).
Despite its agricultural and socioeconomic importance, banana cultivation is highly
susceptible to foliar fungal diseases, which impair the plant’s
photosynthetic capacity
and significantly affect fruit yield and quality (Da Silva et al., 2023). Among these, leaf
spot diseases caused by the fungus
Pseudocercospora fijiensis
(formerly
Mycosphaerella fijiensis
) represent the most serious threat to the b
anana industry, as
this
phyto
pathogen has been the principal constraint on banana production over the past
fifty years (Arango Isaza et al., 2016; Strobl & Mohan, 2020). This phytopathogen causes
yield losses estimated at 33% to 70% in banana and plantain
crops, and its management
requires substantial investment in phytosanitary inputs (Arango Isaza et al., 2016
; Chang
et al., 2016
).
This disease causes premature leaf necrosis, drastically reducing the plant’s
photosynthetic capacity and thereby
affecting fruit yield and quality (Da Silva et al.,
2023). Farmers facing Black Sigatoka are often forced to increase the use of chemical
fungicides to control the disease, which, in the long term, leads to environmental,
economic, and public health conseq
uences (Esguera et al., 2024). Moreover, the
excessive use of these products has fostered the emergence of resistant strains of
P.
fijiensis
, thereby reducing the effectiveness of traditional chemical control methods
(Palmieri et al., 2022).
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In recent year
s, sustainable agriculture has gained prominence as a preferred approach
to disease management, emphasizing the importance of reducing dependence on
chemical products and promoting biological alternatives that enhance the natural
resistance of plants (
Torr
es
-
Rodriguez et al., 2021;
Collinge et al., 2022; Elnahal et al.,
2022). In this context, biocontrol agents such as
Trichoderma
spp. and
B. subtilis
have
emerged as promising solutions because of their antagonistic properties and their ability
to inhibit a
wide range of phytopathogens (Lahlali et al., 2022).
The genus
Trichoderma
includes fungi that have demonstrated efficacy in controlling
fungal
phyto
pathogens through mechanisms such as competition for space and
nutrients, mycoparasitism, and the producti
on of antifungal compounds (Ty;kiewicz et
al., 2022). Similarly,
B. subtilis
, a beneficial bacterium, has shown great potential for
disease control through the production of metabolites that inhibit phytopathogen growth
and stimulate systemic resistance in
plants (Dimkić et al., 2022).
The use of
Trichoderma
spp. and
B. subtilis
not only represents an effective strategy to
reduce the incidence of Black Sigatoka, but also promotes environmentally responsible
and cost
-
effective management (Dadrasnia et al., 2
020). Previous studies have
documented that both biocontrol agents can adapt well to diverse conditions, persist in
the environment, and provide continuous control of phytopathogens (Lahlali et al., 2022).
However, the effectiveness of these species as bio
control agents against
P. fijiensis
in
banana cultivation requires thorough analysis under both laboratory and field conditions
in order to evaluate their benefits and limitations in a real agricultural context (Cuellar et
al., 2021).
The objective of the
present study is to evaluate the efficacy of
Trichoderma
spp. and
B.
subtilis
as biocontrol agents for the management of
P. fijiensis
. Through this research,
we aim to generate useful knowledge about the potential of these microorganisms in the
biological
control of Black Sigatoka, offering a sustainable and safe alternative that
contributes to reducing the use of chemical fungicides in banana cultivation. This
approach could improve the sustainability of agricultural production, reduce disease
management c
osts, and contribute to the ecological and socioeconomic well
-
being of
banana
-
producing regions.
2.
Methodology
Strain Acquisition
The
Bacillus subtilis
and
Trichoderma
s
p
. strains used in this study were obtained from
the strain bank of the Biology
and Microbiology Laboratory at the Technical State
University of Quevedo (UTEQ). These strains had been previously isolated,
characterized, and preserved under controlled conditions to ensure their viability and
purity. The
Trichoderma
sp. strain was inoc
ulated on potato dextrose agar (PDA; Difco,
39 g L
¹
)
and incubated at 25
°
C in darkness for 7 days.
B
.
subtilis
was inoculated on
nutrient agar (NA) and incubated at 30
°
C for 24 h.
On the other hand, the
P
. fijiensis
strains used in this study were isola
ted from a
commercial banana plantation located in Valencia, Ecuador. The
P
. fijiensis
strains were
cultured on potato dextrose agar (PDA) and maintained at 25 °C in darkness for 15 days.
Each strain was purified using the hyphal tip method, which allowed
the acquisition of
pure cultures by selecting and transferring only the tips of actively growing hyphae. This
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purification process ensured the elimination of potential contaminants, thereby
guaranteeing the purity of the strains for use in the inhibition a
ssays.
Molecular identification of
Pseudocercospora fijiensis
For molecular identification, an rDNA fragment encompassing the ITS region (ITS1
–
5.8S
–
ITS2) and extending toward the large subunit (2
8
S/LSU) was amplified using the
specific primers
MF137 (5
-
GGCGCCCCCGGAGGTCTCCTT
-
3) and R635 (5
-
GGTCCGTGTTTCAAGACGG
-
3), as described by Johanson and Jeger (1993), which
generate an expected amplicon of approximately 1.0 kb (~1018 bp). The PCR reaction
was performed in a final volume of 25 µL containi
ng 1× buffer, MgCl
(
1.5
–
2.5 mM),
dNTPs (0.2 mM each), primers (0.2
–
0.5
µ
M each), Taq DNA polymerase (approximately
1 U), and template DNA (approximately 20
–
50 ng).
Amplification was carried out with an initial denaturation at 94 °C, followed by 35 cycles
of 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 45 s, with a final
extension at 72 °C for 10 min. The amplified products were separated by electrophoresis
on a 1.5% agarose gel in 1× TAE buffer at 90
–
110 V for 40
–
60 min and visu
alized by
staining with ethidium bromide or SYBR Safe under a UV transilluminator. Amplicon size
was estimated by comparison with a 100 bp DNA ladder. A negative control without
template DNA (C0) was included to rule out contamination.
Determination of the
incidence and severity of
Pseudocercospora
fijiensis
in
banana plantlets
The incidence and severity of
P. fijiensis
were assessed under controlled greenhouse
conditions. Cavendish banana plantlets obtained through in vitro propagation,
approximately 30 cm in height, were used in the experiment. The plantlets were
transplanted into 26
-
cm
-
diameter pots at a density of one plant per pot and allowed to
acclimatize for 14 days. The experimental design included an inoculated treatment and
a no
n
-
inoculated control, with 10 replicates per treatment.
For inoculation, plantlets assigned to the inoculated treatment were sprayed with a spore
suspension of
P. fijiensis
adjusted to 1 × 10d spores mL
¹
an
d uniformly applied to both
adaxial and abaxial l
eaf surfaces. Immediately after inoculation, the plants were
maintained in a humid chamber for 48 h to promote infection. Control plants were
sprayed with sterile distilled water only and kept under the same environmental
conditions
(Torres
-
Rodriguez et al
., 2025)
. Disease incidence was evaluated six weeks
after inoculation and expressed as the percentage of infected plantlets relative to the
total number of evaluated plants, according to the following equation:
DI (%) = (IP
/
TP) × 100
where IP represents
the number of infected plants and TP the total number of evaluated
plants.
Disease severity was determined in each of the 10 replicates by recording the total
number of functional leaves and estimating the percentage of leaf area affected by Black
Sigatok
a symptoms on each leaf. Severity was scored using a seven
-
class visual scale
(0
–
6) adapted from Orjeda (1998), where 0 = no symptoms; 1 = less than 1% infected
leaf area; 2 = 1
–
5%; 3 = 6
–
15%; 4 = 16
–
33%; 5 = 34
–
50%; and 6 = 51
–
100%. The
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disease severity i
ndex (DSI) was then calculated according to Craenen (1998) using the
following equation:
DSI (%) = [
%(
nb)
/
((N −
1) T
)] × 100
where n is the number of leaves at each severity grade, b is the numerical value assigned
to each grade, N is the total number of
categories in the scale (7), and T is the total
number of leaves evaluated per plant.
Inhibition of
Pseudocercospora
fijiensis
ascospore germination by metabolites
produced by
Trichoderma
sp. and
Bacillus subtilis
The inhibition of
P
. fijiensis
ascospore germination was assessed under laboratory
conditions using metabolites produced by
Trichoderma
s
p
. and
B. subtilis
. Four
treatments were evaluated: two concentrations of
Trichoderma
sp.
metabolites (5% and
10%) and two concentrations of
B. subtil
is
metabolites (5% and 10%). The metabolites
were obtained from liquid cultures and filtered through Whatman No. 1 filter paper.
For each treatment, 20
:
L of an
P
. fijiensis
ascospore suspension (1 × 10d spores mL
¹
)
w
as mixed with 20
:
L of the correspond
ing metabolite solution in a sterile Petri dish. The
negative control consisted of 20
:
L of the ascospore suspension mixed with 20
:
L of
sterile distilled water.
Each treatment, including the control, was replicated ten times, and the plates were
incubate
d at 25 °C in darkness for 48 h.
Following incubation, ascospore germination
was assessed by light microscopy at 40× magnification. For each replicate, five
microscopic fields were randomly selected, and at least 100 spores were counted per
treatment. Germ
ination inhibition was calculated according to the following equation:
AGI (%) = (UA / TA) × 100
where AGI represents the percentage of ascospore germination inhibition, UA is the
number of ungerminated ascospores, and TA is the total number of ascospores
observed.
Radial Growth Inhibition of
Pseudocercospora
fijiensis
The inhibitory effect of metabolites produced by
Trichoderma
sp. and
B
.
subtilis
on the
radial growth of
P
. fijiensis
colonies was evaluated under controlled laboratory
conditions. Two
concentrations of each metabolite (5% and 10%) were assessed, using
dilutions prepared from liquid culture extracts of both microorganisms.
In each Petri dish containing potato dextrose agar (PDA), a mycelial disc of
P
. fijiensis
was placed at the center,
and 50
:
L of each metabolite solution was applied at
equidistant points surrounding the disc. Plates were incubated at 25 °C in darkness, and
colony radial growth was measured after
7
days. Radial growth inhibition was expressed
as a percentage relative t
o the untreated control.
The percentage of radial growth
inhibition was calculated according to the following equation (Torres
-
Rodr
i
guez et al.,
2024):
RGI (%) = [(R1 − R2) / R1] × 100
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confirming both the specificity of the primers and the absence of contamination in the
assay.
Figure 1
Molecular identification of Pseudocercospora fijiensis by PCR amplification of the ITS
region
Note:
(A) Genomic DNA extract
ed from
Pseudocercospora fijiensis
isolates (M1
–
M4).
(B) PCR amplification of the ITS region using the species
-
specific primers MF137 and
R635, showing the expected ~1018 bp amplicon (red arrows) in isolates M1
–
M4. MP,
DNA ladder; C0, no
-
template
negative control.
Infectivity of
Pseudocercospora fijiensis
in Banana Seedlings
The incidence and severity of
P
.
fijiensis
varied significantly among the evaluated strains
(Figure 2). M4 was the most virulent strain, showing the highest incidence (70%) an
d
severity (80%), whereas M3 exhibited the lowest values for both variables, with 30%
incidence and 40% severity. M1 showed intermediate but relatively high levels of
affectation, reaching 60% incidence and 64% severity, while M2 presented 42%
incidence an
d 58% severity. These findings reveal substantial variability in pathogenicity
among the tested strains and identify M4 as the most aggressive isolate in banana
seedlings.
Figure 2
Incidence and severity of Pseudocercospora fijiensis in four strains (M1, M
2, M3, and
M4) evaluated in banana seedlings
b
cd
d
a
b
b
c
a
0
50
100
M1
M2
M3
M4
Afectation (%)
Strain
Incidence
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Note:
Dark bars represent incidence, and light bars represent severity. Different letters
above the bars indicate significant differences among strains within each variable
according to Tukey’s
test (P < 0.05).
Ascospore Germination Inhibition
Ascospore germination of
P. fijiensis
was significantly affected by the microbial
metabolites evaluated (Figure 3).
B
.
subtilis
showed the highest inhibitory activity at both
concentrations, reaching 80% i
nhibition at 5% and complete inhibition (100%) at 10%. In
contrast, metabolites from
Trichoderma
sp. produced lower inhibition values, with 47%
at 5% and 60% at 10%. In both microorganisms, inhibition increased with metabolite
concentration; however,
B. su
btilis
consistently outperformed
Trichoderma
sp., indicating
a stronger suppressive effect on ascospore germination.
Figure 3
Inhibition of Pseudocercospora fijiensis ascospore germination by microbial metabolites
Note:
(A)
Percentage of ascospore germination inhibition induced by metabolites
produced by
Trichoderma
sp. and
B. subtilis
at 5% and 10%. Different letters above the
bars indicate significant differences among treatments according to Tukey’s multiple
comparison tes
t (P < 0.05). (B) Representative microscopic images showing the effect
of each treatment on ascospore germination. B1,
B. subtilis
at 10%; B2,
B. subtilis
at 5%;
B3,
Trichoderma
sp. at 10%; and B4,
Trichoderma
sp. at 5%.
Radial Growth Inhibition
Radial gro
wth of
P
.
fijiensis
differed significantly among the evaluated treatments (Figure
4). The strongest inhibition was observed with metabolites from
B
.
subtilis
at 10%, which
reduced colony growth by 90% relative to the control. Metabolites from
Trichoderma
sp.
at the same concentration produced 61% inhibition. At 5%,
Trichoderma
sp. showed
moderate inhibitory activity (56%), whereas
B. subtilis
exhibited the lowest effect (26%).
Overall, inhibition increased with concentration for
B. subtilis
, wh
ile
Trichoderma
sp.
maintained intermediate inhibition at both concentrations. These results indicate that
B.
c
c
b
a
0
20
40
60
80
100
120
5%
10%
Inhibition (%)
Metabolite concentration
Trichoderma
B. subtilis
A
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ı
subtilis
, particularly at 10%, was the most effective treatment for suppressing mycelial
growth of
P. fijiensis
.
Representative images further sho
wed that metabolite application induced evident
morphological changes in
P. fijiensis
colonies, including irregular colony development
and altered pigmentation at the margins. Such responses are consistent with fungal
stress and reinforce the inhibitory ac
tivity of the evaluated microbial metabolites against
the
phyto
pathogen.
Figure 4
Effect of microbial metabolites on the radial growth of Pseudocercospora fijiensis
Note:
(A) Radial growth inhibition (%) of
P. fijiensis
colonies treated with metabolites
produced by
Trichoderma
sp. and
B
.
subtilis
at 5% and 10%. Different letters above the
bars indicate significant differences among treatments according to Tukey’s multiple
comparison test (P < 0.05). (B) Representative col
ony morphology of
P. fijiensis
under
each treatment. B1,
B. subtilis
at 10%; B2,
B. subtilis
at 5%; B3,
Trichoderma
sp. at 10%;
and B4,
Trichoderma
sp. at 5%.
Reduction of Disease Severity in Banana Seedlings
The severity of
P. fijiensis
in banana seedlings was significantly reduced by the
application of microbial treatments, although the magnitude of the effect depended on
both the biological agent and the concentration used (Figure 5). At the 5% concentration,
Trichoderma
sp. reduced di
sease severity to 62%, whereas
B. subtilis
reduced it to 59%.
Both treatments showed lower severity than the phytopathogen
-
inoculated control, which
reached 84%.
A stronger reduction in disease severity was observed at the 10% concentration.
B.
subtilis
wa
s the most effective treatment, reducing severity to 10%, while
Trichoderma
sp. reduced it to 41%. In contrast, the control maintained a high severity value of 81%.
Statistical analysis showed significant differences among treatments (P < 0.05), with
B.
su
btilis
at 10% showing the greatest suppressive effect on disease development. Overall,
these results indicate that both
B. subtilis
and
Trichoderma
sp. have biocontrol potential
b
b
c
a
0
10
20
30
40
50
60
70
80
90
100
5%
10%
Inhibition (%)
Metabolite concentration
Trichoderma
B. subtilis
B
B
B
B
A
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against
P. fijiensis
in banana seedlings, although
B. subtilis
, particularly a
t 10%, was the
most effective treatment.
Figure 5
Severity of Pseudocercospora fijiensis in banana seedlings treated with Trichoderma sp.
and Bacillus subtilis
Not
e
:
(A) Severity (%) of
P. fijiensis
in banana seedlings treated with
Trichoderma
sp.
and
Bacillus subtilis
at 5% and 10%, relative to the
phyto
pathogen
-
inoculated control.
Different letters above the bars indicate significant differences among treatments
according to Tukey’s multiple compar
ison test (P < 0.05). The control is shown in both
concentration groups for comparative purposes, although it represents a single
treatment. (B) Representative leaf symptoms of
P. fijiensis
under each treatment. B1,
B.
subtilis
at 10%; B2,
Trichoderma
sp.
at 10%; B3,
B. subtilis
at 5%; B4,
Trichoderma
sp.
at 5%; and B5
–
B6,
phyto
pathogen
-
inoculated control treated only with sterile water.
4.
Discussion
The variability in incidence and severity observed among
P
.
fijiensis
strains is likely
associated with dif
ferences in virulence
-
related traits among isolates. Previous studies
have shown that
P. fijiensis
exhibits substantial genetic diversity, which enables rapid
adaptation to different environmental conditions and host defense responses, thereby
influencing
its pathogenicity (Souleymane et al., 2022
; Esguera et al., 2024
). This genetic
variability may be reflected in differences in the production of cell wall
-
degrading
enzymes, toxins, and other infection
-
related compounds that facilitate host colonization
an
d tissue damage (Noar et al., 2022).
Previous reports have indicated that some
P. fijiensis
isolates may have a greater
capacity to produce effector molecules that suppress host defenses and increase
disease severity (Pinheiro
et al., 2022). In particular, highly virulent isolates, such as M4
in the present study, may possess specific genes involved in the regulation of
pathogenicity
-
related compounds and enzymes that promote tissue colonization and
lesion development (Olivares
et al., 2021). In contrast, less aggressive isolates, such as
d
b
cd
a
e
e
0
10
20
30
40
50
60
70
80
90
100
5%
10%
Severity (%)
Treatments
Trichoderma
B. subtilis
Control
A
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M3, may express these virulence
-
related factors at lower levels, which could explain their
reduced ability to infect host tissues and cause severe symptoms.
The results of the present study are
consistent with previous research reporting the
effectiveness of
B. subtilis
as a biocontrol agent against phytopathogens through the
production of secondary metabolites such as surfactins, iturins, and fengycins, which
directly inhibit spore germination
and pathogen growth (Zhu et al., 2020; Mahmood et
al., 2022). These antimicrobial compounds disrupt cell membrane integrity, which may
explain the strong inhibitory effect against
P. fijiensis
observed in this study. At the higher
concentration tested (10%
),
B. subtilis
achieved complete inhibition, likely due to the
increased availability of these bioactive metabolites, further supporting its potential as an
effective biocontrol agent (Dimkić et al., 2022).
Metabolites produced by
Trichoderma
sp. also show
ed inhibitory activity against the
pathogen, although their effect was less pronounced than that of
B. subtilis
. Previous
studies have shown that
Trichoderma
spp. produce hydrolytic enzymes, such as
glucanases and chitinases, which weaken phytopathogen cel
l walls and reduce their
capacity to infect host tissues (Konappa et al., 2020; Dutta et al., 2023). Although this
mode of action may not result in complete inhibition as efficiently as the antibiotic
compounds produced by
B. subtilis
, it can still substan
tially reduce pathogen
development, as observed in the present study, where inhibition reached 60% at the
10% concentration.
The greater efficacy of
B. subtilis
compared with
Trichoderma
sp. in reducing disease
severity in banana seedlings may be explained
by differences in their modes of action.
B. subtilis
is widely recognized for its ability to produce antimicrobial metabolites,
including surfactins, iturins, and fengycins, which directly affect pathogen cells by altering
membrane permeability and suppre
ssing mycelial growth (Elsharkawy et al., 2022; Ajuna
et al., 2024). In addition,
B. subtilis
may activate induced systemic resistance in plants,
thereby enhancing host defense against subsequent infections (Rabari et al., 2023; Jinal
et al., 2024). The co
mbined effect of direct antagonism and host defense stimulation
likely explains the marked reduction in disease severity observed at the 10%
concentration, where
B. subtilis
reduced severity to 10%.
In contrast,
Trichoderma
sp. acts mainly through the prod
uction of hydrolytic enzymes
and through competition for space and nutrients, which can limit
phyto
pathogen
establishment and development. In addition, some
Trichoderma
strains are known to
promote plant defense responses and contribute indirectly to disea
se suppression
(Contreras et al., 2020; Poveda et al., 2020). These mechanisms may explain the
reductions in severity observed at both 5% and 10%, although their effect was lower than
that achieved with
B. subtilis
, particularly at the higher concentration
.
5.
Conclusions
The results demonstrated significant variability in virulence among the evaluated
Pseudocercospora fijiensis
strains, with M4 showing the highest incidence and severity
in banana seedlings. Among the microbial treatments,
Bacillus
subtilis
exhibited the
strongest antagonistic activity, particularly at the 10% concentration, where it achieved
complete inhibition of ascospore germination, the highest radial growth inhibition, and
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12
the greatest reduction in disease severity. In contrast
,
Trichoderma
sp. also showed
inhibitory activity, although its effectiveness was consistently lower than that of
B. subtilis
.
In addition to suppressing pathogen development, the microbial metabolites induced
visible morphological alterations in
P. fijien
sis
colonies, suggesting a direct antifungal
effect. Under greenhouse conditions, the marked reduction in disease severity observed
in banana seedlings treated with
B. subtilis
, especially at 10%, confirms its high potential
as a biocontrol agent against B
lack Sigatoka. Overall, these findings support the use of
B. subtilis
as a promising biological alternative for the management of
P. fijiensis
in
banana production systems.
Author Contributions:
Conceptualization,
A.V.C.M.
and
J.A.T.R
.; methodology,
A.V.C.
M
.,
J.A.A.R
and
A.A.M
.; formal analysis,
M.R.M.C.
,
K.L.C.
and
K.V.A.I
.;
investigation,
A.V.C.M
.,
K.V.A.I
.
and
J.A.T.R
.; resources,
A.V.C.M.
and
K.V.A.I
.;
writing
—
original draft preparation,
A.A.M
.; writing
—
review and editing,
J.A.A.R. and
A.A.M.
; visualiza
tion,
M.R.M.C.
and
K.L.C.;
supervision,
J.A.T.R
.;
All authors have read
and agreed to the published version of the manuscript.
Funding:
This research did not receive external funding.
Acknowledgments:
We would like to thank the
Universidad
Técnica
Estatal de
Quevedo
(UTEQ) and the UTEQ Research Department for their ongoing support.
Special thanks to the entire team at the UTEQ microbiology laboratory.
Data availability statement:
The data are available upon reasonable request from the
correspondin
g author at
:
aalvarado@uv.mx
Conflicts of interest:
The authors declare no conflict of interest.
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