© The Authors, 2022, Published by the Universidad del Zulia*Corresponding author: jholguin04@cibnor.mx
Keywords:
Chemical combat
Fusarium
Tobacco
Sensitivity
Fungitoxicity
Wilt
Growth inhibition of Fusarium oxysporum f. sp. nicotianae by two in vitro poisoning methods
with fungicides of different toxicological groups
Inhibición del crecimiento de Fusarium oxysporum f. sp. nicotianae por dos métodos de
envenenamiento in vitro con fungicidas de diferentes grupos toxicológicos
Inibição de Fusarium oxysporum f. sp. nicotianae por dois métodos de envenenamento in vitro com
fungicidas de diferentes grupos toxicológicos
University of Zulia, Faculty of Agronomy
Bolivarian Republic of Venezuela
1
Universidad de Granma, Facultad de Ciencias Agropecuarias,
Departamento Agronomía, Bayamo. Carretera de Manzanillo
km 17 ½, Bayamo, Provincia de Granma, Cuba. CP 85100.
2
Universidad de Sonora, Departamento de Agricultura
y Ganadería. Bulevar Luis Encinas. Colonia Centro.
Hermosillo, Sonora, México. CP 83000.
3
Universidad de Granma, Facultad de Ciencias Agropecuarias,
Departamento Agronomía, Bayamo. Carretera de Manzanillo
km 17 ½, Bayamo, Provincia de Granma, Cuba. CP 85100.
4
Universidad de Granma, Facultad de Ciencias Agropecuarias,
Departamento Agronomía, Bayamo. Carretera de Manzanillo
km 17 ½, Bayamo, Provincia de Granma, Cuba. CP 85100.
5
Centro de Investigaciones Biológicas del Noroeste,
Programa de Agricultura en Zonas Áridas. Km. 1 Carretera
a San Juan de La Costa, El Comitán. La Paz, B.C.S. México.
CP 23205.
Received: 23-06-2022
Accepted: 08-08-2022
Published: 25-08-2022
Abstract
Wilt caused by Fusarium oxysporum f. sp. nicotianae is one of the most
important fungal diseases in tobacco cultivation, being also one of the most
difcult to control. The in vitro effectiveness of two poisoning methods
was evaluated; sensi-disc (SD) and medium dilution (DMC) to determine
the inhibitory effect of four fungicides of different toxicological groups on
F. oxysporum isolated from tobacco in different regions of the province of
Granma, Cuba. A differential response was observed in the susceptibility
levels of all strains tested, regardless of the method of poisoning. The DMC
method was more efcient than the SD, observing increases from 4.37 % (S)
to 45.57 % (Ip+Pr) with respect to the SD. The highest inhibition values were
observed in DMC with mancozeb (100 %), Tz+Az (79.74 %), and Ip+Pr (96.9
%). The greatest effectiveness in sporulation inhibition was with mancozeb
by the SD method (0 %). The in vitro inhibitory effect of the fungicides
evaluated (alone or in combination) is indicative of the fungicidal effect on
the fungus under study and establishes the importance of inhibition methods
for the study of fungicides used in management programs of diseases caused
by Fusarium spp., in the tobacco growing areas of Cuba and other parts of
the world.
Wilson Geobel Ceiro-Catasú
1
Edgar Omar Rueda-Puente
2
Richard Rondón-Fonseca
3
Oandis Sosa-Sánchez
4
Ramón Jaime Holguín-Peña
5*
Rev. Fac. Agron. (LUZ). 2022, 39(3): e223944
ISSN 2477-9407
DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v39.n3.10
Crop Production
Associate editor: Dra. Lilia Urdaneta
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Rev. Fac. Agron. (LUZ). 2022, 39(3): e223944. July - September. ISSN 2477-9407.
2-6 |
Resumen
La marchitez causada por Fusarium oxysporum f. sp. nicotianae
es una de las enfermedades fúngicas más importantes en el cultivo
del tabaco siendo también una de las difíciles de control. Se evaluó la
efectividad in vitro de dos métodos de envenenamiento; sensi-disco
(SD) y dilución en el medio (DMC) para conocer el efecto inhibitorio
de cuatro fungicidas de diferentes grupos toxicológicos sobre F.
oxysporum aislado de tabaco en diferentes regiones de la provincia
de Granma, Cuba. Se observó una respuesta diferencial en los niveles
de sensibilidad de todas las cepas evaluadas, independientemente
del método de envenenamiento. El método DMC fue más eciente
que el SD, observándose incrementos del 4,37 % (S) al 45,57 %
(Ip+Pr) con respecto al SD. Los valores de inhibición más altos
observados fueron en DMC con el mancozeb (100 %), Tz+Az (79,74
%) y el Ip+Pr (96,9 %). La mayor efectividad en la inhibición de la
esporulación fue con mancozeb por el método SD (0 %). El efecto
inhibitorio in vitro de los ingredientes activos evaluados (solos o en
combinación) es un indicativo del efecto fungicida sobre el hongo y
establece la importancia de estos dos métodos de inhibición para el
estudio de los productos que mayormente se utilizan en los programas
de manejo de las enfermedades causadas por Fusarium spp., en las
zonas tabacaleras de Cuba y otras partes del mundo.
Palabras clave: Combate químico, fusariosis, tabaco, sensibilidad,
fungitoxicidad, marchitez.
Resumo
Murcha causada por Fusarium oxysporum f. sp. nicotianae é
uma das doenças fúngicas mais importantes na cultura do tabaco,
sendo também uma das mais difíceis de controlar. A ecácia in
vitro de dois métodos de envenenamento foi avaliada; sensi-disc
(SD) e diluição média (DMC) para determinar o efeito inibitório
de quatro fungicidas de diferentes grupos toxicológicos sobre F.
oxysporum isolados de tabaco em diferentes regiões da província de
Granma, Cuba. Uma resposta diferencial foi observada nos níveis
de suscetibilidade de todas as cepas testadas, independentemente do
método de intoxicação. O método DMC foi mais eciente que o SD,
com aumento da inibição de 4,37 % (S) para 45,57 % (Ip+Pr) em
relação ao SD. Os maiores valores de inibição observados foram no
DMC com mancozeb (100 %), Tz+Az (79,74 %) e Ip+Pr (96,9 %). A
maior ecácia na inibição de esporos foi mancozeb pelo método SD
(0 %). O efeito inibitório in vitro dos fungicidas avaliados (sozinhos
ou em combinação) é indicativo do efeito fungicida sobre o fungo
em estudo e estabelece a importância dos métodos de inibição para o
estudo de fungicidas utilizados em programas de manejo de doenças
causadas por Fusarium spp., nas áreas de cultivo de tabaco de Cuba e
outras partes do mundo.
Palabras-chave: Combate químico, fusarium, tabaco, sensibilidade,
fungitoxicidade, murcha.
Introduction
Tobacco (Nicotiana tabacum L.) is one of the main crops in Cuba,
being the essential raw material for the tobacco company, one of the
most iconic in the country. Of the total surface available for annual
crops, tobacco represents around 6 % with an average sown area of
~25,000 ha (Wikle, 2015), which has decreased by ~3,000 by 2020
(CubaNews, 2022). However, the estimated annual production is
26,000 t.leaf
-1
.
Cuba is the main exporter of cigars in the world, being
the tobacco industry with an estimated income of 267 million dollars,
the fourth most important sector in the generation of the country’s
gross domestic product (GDP) (Cosner, 2015).
One of the most important limitations in production and quality
is the presence of vascular diseases, mainly those caused by various
species of Fusarium oxysporum (Olivares et al., 2021; Martín et al.,
2021). In Cuba, 31 species of Fusarium associated with different
commercial crops have been reported (López, 2004), although
F. oxysporum f. sp. nicotinae is the one that has had the greatest
importance due to its high affectation in the Burley-type varieties,
which are the most productive and with highest commercial value
(García et al., 2015). These peculiarities combined with the climatic
conditions and the type of soils prevalent in the region (uvisols)
have been particularly difcult in the tobacco-growing regions of
Bayamo, province of Granma (López, 2004; Mariña de la Huerta et
al., 2005; Villa et al., 2015; Ceiro et al., 2021). In fact, the biological
complexity described for Fusarium spp. (Edel-Hermann and Lecomte,
2019; Lombard et al., 2019) is one of the main characteristics that
has made it difcult to control the disease in the eld, in addition
to the low availability of varieties tolerant to the fungus (García et
al., 2015). On the other hand, the range of fungicides for the control
of Fusarium spp. in Cuba, including broad-spectrum (Cu, S) and
specic (benzimidazoles, triazoles, strobirulins, and carbamates) have
shown some type of resistance in other fungi for which the product
was designed, thereby causing unwanted effects on agricultural
ecosystems (Silva-Marrufo and Marín-Tinoco, 2021). In this way, the
various methods of poisoning the culture medium are an important
tool to evaluate the biological effectiveness on the growth and/or
sporulation of the fungus, especially where it is necessary to evaluate
fungicides with native phytopathogenic strains (Ceiro et al., 2021).
The objective of the present work was to evaluate the in vitro
inhibitory effect of fungicides of different toxicological groups by
two poisoning methods on mycelial growth and sporulation of F.
oxysporum f. sp. nicotinae isolated from tobacco in Granma, Cuba.
Materials and methods
Collection of Fusarium spp. strains
The fungal strains were collected from infected stems of tobacco
crops (Nicotiana tabacum L.) with symptomatology according
to the reported for Fusarium spp. (Nelson et al., 1990; Blancard,
1998). The regions of collection were Buey Arriba (20°14’20.5”N
76°46’28.8”O), Monjará, Guisa (20°18’07.3”N 76°36’42.6”O) and
El Dorado, Bayamo (20°15’52.3”N 76°44’43.8”O) in the province
of Granma, Cuba. The nomenclatures of the strains under study were
assigned according to the region and the variety of tobacco where the
isolation was carried out, where; Fus= Fusarium, BA= Buey Arriba,
G= Guisa, B= Bayamo, Hab = CV of 92 Havana tobacco, Cor (CV
Corojo-2006). The other strains were isolated from SS-96 variety.
The strains were prepared as monosporic culture in the Agricultural
Microbiology Laboratory of the Faculty of Agricultural Sciences,
University of Granma for subsequent identication and evaluation.
Identication of Fusarium spp.
The identication was carried out according to the morphological
characters for the determination of genus and specie. The
morphometric tests for the characterization of the fruiting bodies
were according to the color, pigmentation, and mycelial growth
of the colonies. The characterization of conidia and microconidia
was carried out according to size, shape, and the number of septa,
the shape of apical cells, shape, and size of microconidia, presence
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3-6 |
and arrangement of chlamydospores, the color of sporodochia,
and observation of phialides according to Summerell et al. (2007).
Molecular identication was performed by PCR amplied with
primers Bik1 (5 ́-TTCAAGAGAGCTAAAGGTCC-3 ́) and Bik4
(5 ́-TTTTGACCAAGATAGATGCC-3’). The PCR conditions
were according to the species (Holguín-Peña, 2007). All F.
oxysporum strains were compared with those of reference from the
Phytopathology Laboratory of the Northwest Research Center. For
identication, each strain from each locality was morphologically
characterized.
Sensitivity studies
The sensitivity study of strains to fungicides of different
taxonomic groups was performed by two poisoning methods; 1) by
diffusion of the active ingredient (i.a) on a paper disc (sensi-disc)
(Clinical Laboratory and Standards Institute [CLSI], 2008) and 2) by
the method of serial dilutions in culture medium (DMC) (Webster et
al., 2008). The sensi-disc method was performed by immersing (5
minutes) a paper disc in a solution with the fungicide and depositing
four sensi-discs in the Petri dish with PDA medium. A 5 mm diameter
disc of PDA with 3-day-old fungal mycelium was placed in the center
of each one. After incubation time (28 °C/7 d), mycelial growth
was determined for the calculation of inhibition using the equation
[%inhibition = (CMC-CMT)/CMC*100], where: CMC is the mycelial
growth of the control (mm) and CMT is the mycelial growth of the
treatment (mm). For the DMC method, PDA medium (39 g.L
-1
) plus
streptomycin (50 mg.L
-1
) was prepared according to the methodology
described by Ceiro et al. (2015).
Fungicide selection
Fungicide treatments were assigned according to table 1,
considering six toxicological groups (alone or in combination). For
in vitro doses, they were calculated according to each method and as
reported in the literature (Nisa et al., 2011; González-Merino et al.,
2021).
Table 1. Fungicides of different toxicological groups used for in
vitro control of Fusarium oxysporum in tobacco.
Fung
1
Active
Ingredient (IA)
Concentration
of
IA (%)
2
Chemical
group
Trade name
Mz Mancozeb 0.8 Dithiocarbamate
Mancozeb
PH 80
(Limin
Chemical
Co. LTD)
Tz+Az
Tetraconazole+
Azoxystrobin
0.08/0.01
Triazole+
strobilurin
Galileo SC18
(Isagro S.
P. A.)
Ip+Pr
Iprovalicarp+
Propineb
0.09/0.6
Isopropyl
carbamate+
dithiocarbamate
Positron
Duo 69
WP, Bayer
CropScience
S Sulfur 0.8
Inorganic
element
Thiovit Jet
80 WG,
Sygenta
1
Fung = abbreviation according to the formulation of the active ingredient (IA).
2
Percentage (%) of active ingredient (i.a) in the commercial formulation. The
concentration of each fungicide is according to those reported on the trade name
label. The selection of fungicides was according to prior consultation of the
inhibition of the active ingredient; Mz (Gullino et al., 2010; Runkle et al., 2017),
Az (Wang et al., 2016), Tz, Ip, Pr and Az (Ceiro et al. 2015; Lucas et al., 2015;
Nisa et al., 2011).
Sporulation inhibition
For the quantication of macroconidia concentration, 10 mL of
distilled sterile water was added over the fungal growth on each plate
and homogenized with a Drigalsky spatula. The spore suspension
was collected in a 20 ml test tube. Dilutions up to 10
-2
were prepared
from these. A capillary tube was used to extract small portions of
the last dilution and a drop was deposited in a Neubaüer chamber
to determine the concentration of macroconidia with the help of a
Novel brand optical microscope at 400X. The methodology for
quantication was according to Savin-Molina (2021) and González-
Merino et al., (2021).
Statistical analysis
Statistical processing was performed using a 5x2x4 factorial
ANOVA where; factor I = Fusarium isolates (two from Buey Arriba,
two from Guisa, and one from Bayamo), factor II = poisoning methods
(two poisoning methods DMC, SD), and factor III = fungicides (four
fungicides; Mz, Tz+Az, Ip+Pr, S). From the combinations made, 40
treatments resulted in four replicates, under a completely randomized
design. The control consisted of puried water. When detecting
signicant differences, Tukey’s multiple comparisons of means test
was used (P≤0.05). The values of macroconidia concentration were
previously transformed to ANOVA by LOG (x+1) as well as the
percentage of inhibition by . Processing was performed
with InfoStat statistical package, 2008.
Results and discussion
The fungus associated with tobacco stem wilt disease
was identied as Fusarium oxysporum f. sp. nicotianae. The
morphological characteristics were in accordance with what was
reported (Blancard, 1998; Summerell et al., 2007; Rentería-Martínez
et al., 2019), observing a colonial growth (72 h) of salmon/purple-
colored hairy appearance at the bottom of the Petri dish. The observed
macroconidia were ≈18x140 µm, crescent-shaped in appearance, and
hyaline with three to four septa. The presence of microconidia of
≈2-3 x 5-12 µm with oval to reniform appearance without obvious
septa, was observed. Globose chlamydospores (single and double)
with short monophialides were observed. Sporodochium formation
was not observed. The specie was conrmed by molecular techniques
according to the amplication of the expected fragment 943 bp with
primers Bik 1 and Bik 4, which corresponds to that reported for F.
oxysporum (Holguín-Peña, 2007; Watanabe, 2013).
In sensitivity studies according to the inhibition of mycelial growth
(%) signicant differences (Tukey P≤0.05) were found between the
poisoning methods and the fungicides evaluated. A higher inhibition
was observed by the dilution method in culture medium (DMC),
compared to the sensi-discs method (SD) (gure 1).
The highest inhibition values were obtained with the DMC
method with mancozeb (100 %), followed by tetraconazole +
azoxystrobin (79.74 %) and iprovalicarp + propineb (51.7 %). The
lowest values with DMC were observed with sulfur (37.12 %). With
the SD method, inhibition ranges were from 6.13 % (Ip+Pr) to 85.55
% (Mz) (table 2). It has been documented that methods with serial
dilutions such as DMC are more sensitive for assessing biological
effectiveness and the homogenization of the medium with the active
ingredient is more consistent (Zgoda and Porter, 2001). In addition,
the log dilution series (2.5 log) can be interpreted in a probabilistic
trend curve (probit) for the calculation of the minimum inhibitory
concentration (CMI) and the effective concentration at 50 % (EC50)
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4-6 |
or 90 % (EC90) (Förster et al., 2004; Webster et al., 2008). In the
SD method, the fungicide only interacts with the phytopathogen on
the poisoned seed disc, causing a fungistatic effect in the rst hours
of interaction. However, if the mycelium of the fungus exceeds the
area of interaction with the fungicide, it can grow and multiply on
the non-poisoned growing medium.
Mancozeb is one of the most used active ingredients for the
control of this type of fungi, due to the fact that its mechanism of
action is aimed at inhibiting enzymatic processes and respiration; as
well as, it inactivates the sulfhydric groups (-SH), affects the Krebs
cycle, prevents the formation of ATP and denatures the lipids of
the cytoplasmic membrane (Runkle et al., 2017). This implies that
the different activation sites increase the spectrum of action and
decrease the selection pressure associated with the appearance of
resistant strains (Gullino et al., 2010).
In the present research, mancozeb proved the most effective
fungicide in both poisoning methods, coinciding with the reported
inhibition range of the CI
50
between 500 and 1000 ppm (Sultana and
Ghaffar, 2013). Regarding the effect of azoxystrobin, in our study,
the inhibition percentages at 168 h, were from 56.06 % with the
strain (Fus-BA1) to 87.74 % (Fus-BA2), both strains from Bayamo.
Table 2. Mycelial inhibition (%) at 168 h of fungicides of different toxicological groups on Fusarium oxysporum f. sp. nicotinae isolated
from tobacco in the province of Granma, Cuba.
Fungicides (% inhibition)
3
Strain
1
Method
2
Mancozeb
Tetraconazole+ azoxystrobin
(Tz + Az)
Iprovalicarp + propineb
(Ip + Pr)
Sulfur
(S)
Dilution in culture medium
Fus-BA1 DMC 100 a 56 de 26.9 h 36.8 fg
Fus-BA2 DMC 100 a 87.7 b 80.1 bc 34.8 g
Fus-G(Hab) DMC 100 a 84.5 b 35.4 fg 34.2 g
Fus-G(Cor) DMC 100 a 86.1 b 42.1 fg 34.7 g
Fus-By DMC 100 a 81.2 bc 73.9 c 45.1 f
Sensi-disc
Fus-BA1 SD 100 a 62.2 d 2.9 j 24.2 h
Fus-BA2 SD 45.4 f 60.7 d 9.7 i 26.4 h
Fus-G(Hab) SD 97.9 ab 39.7 fg 2.7 j 20.6 h
Fus-G(Cor) SD 84.5 b 53.6 e 8.2 i 51.1 e
Fus-By SD 100 a 61.9 d 7.2 ij 41.3f g
Averages
DMC 100 79.74 51.7 37.12
SD 85.55 55.66 6.13 32.75
1
Strain abbreviations; Fus (Fusarium), BA (Bayamo), G (Guisa), Hab (tobacco C.V. Havana 92), Cor (tobacco C.V. Corojo-2006), By (Bayamo). The BA and By isolates
were isolated from tobacco C.V. SS-96.
2
Poisoning method; SD = sensi-disc, DMC = dilution in culture medium.
3
Different lowercase letters in columns differ signicantly according to Tukey’s test (P≤0.05).
Method of poisoning by dilution in culture medium
Sensi-disc poisoning method
Figure 1. Inhibition at 168 h of mycelial growth of F. oxysporum
(Fus-By) isolated from tobacco (Nicotiana tabacum L.)
by two different poisoning methods. DMC method. a)
mancozeb, b) tetraconazole + azoxystrobin, c) iprovalicarp
+ propineb, d) sulfur. SD method; e) mancozeb, f)
tetraconazole + azoxystrobin, g) iprovalicarp + propineb,
h) sulfur.
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5-6 |
Azoxystrobin belongs to the methoxy-acrylates chemical group
with a translimiting action within leaves and petals and it is an
effective inhibitor of respiratory processes in mitochondria and also
affects the proper electron transfer between cytochromes, thereby
affecting the proper ow of ATPs and the adequate cellular energy
transfer (Wang et al., 2016).
In the study of the effects of fungicides on sporulation, signicant
differences were observed (Tukey P≤0.05) between all the treatments.
The highest inhibition values were observed with the DMC method
with 96.7 % (0.2x10
5
spores.mL
-1
), compared to the sensi-discs (SD)
where the highest concentrations (5.18x10
5
spores.mL
-1
) (S and
Tr+Az) were observed (table 3). The lowest sporulation was recorded
with Bayamo strains (Fus-BA2 and Fus-G/Hab) with mancozeb
(0.15x10
5
mL
-1
). The highest concentration of spores (5.9x10
5
mL
-1
)
was observed in sulfur with the SD method with the Fus-BA2 and Fus-
G(Cor)/Tr+Az strains. Castellanos-González et al. (2011), showed
that doses higher than 100 mg.L
-1
of mancozeb could totally inhibit
the sporulation of the entomopathogenic Beauveria bassiana, and in
other fungi such as Pochonia chlamydosporia var. catenulate can
also inhibit the production of chlamydospores (Ceiro et al., 2015).
On the other hand, in addition to the inhibition effect, it has been
observed that some mixtures such as udioxonil + metalaxyl could
affect the morphology of the hyphae and cause cell wall disorders
(Miguel et al., 2015).
According to the results obtained, all the fungicides evaluated can
efciently inhibit the mycelial growth of the fungus. The evaluated
methods DMC and SD showed to be a good criterion for the
evaluation of the in vitro activity of F. oxysporum. The widespread
use of fungicides with single-site mechanisms of action requires
a diverse set of chemical management tools to slow the evolution
of fungicide-resistant pathogens (Avenot and Michailides, 2010;
Lucas et al., 2015). In this sense, both methods (DMC and SD) can
be complementary and an excellent tool to establish phytosanitary
management programs where fungicides with different modes of
Macroconidia concentration (x10
5
mL
-1
)
Mancozeb Tetraconazole+azoxystrobin Iprovalicarp+propineb Sulfur
Xt
2,3
Xt Xt Xt
Fus-BA1 DMC 0.15 a 4.0 defgh 3.0 cd 5.2 fghi
Fus-BA2 DMC 0.15 a 4.7 fgh 3.7 def 5.3 ghi
Fus-G(Hab) DMC 0.2 a 3.3 cde 4.2 defgh 3.7 def
Fus-G(Cor) DMC 0.2 a 4.8 fgh 4.2 defgh 4.9 fghi
Fus-By DMC 0.3 a 3.7 def 3.0 cd 4.7 fgh
Fus-BA1 SD 0.2 a 5.3 ghi 4.2 defgh 55 ghi
Fus-BA2 SD 4.6 fgh 5.0 ghi 5.4 ghi 5.9 hi
Fus-G (Hab) SD 1.2 ab 5.3 ghi 4.0 defgh 4.7 fgh
Fus-G(Cor) SD 2.2 bc 5.9 hi 4.4 efghi 5.92 hi
Fus-By SD 0.1 a 4.4 fgh 4.2 defgh 3.9 defgh
1
MET = method; DMC (dilution in culture medium), SD (sensi-disc). 2Xt = transformed value LOG (x+1) of the average for each treatment. Control concentration (without
fungicide) = 6 x105 mL
-1
. 3 Different letters in the columns differ signicantly according to Tukey’s test. (P≤0.05).
action are involved. However, it is important to consider that the most
effective in vitro fungicides will only be a reection of the sensitivity
of the active ingredient in each strain evaluated, and the efcacy in
open-eld conditions will not necessarily coincide.
Conclusions
The results of the in vitro studies indicate that the greatest
inhibitory effect on mycelial growth in F. oxysporum f. sp. nicotinae
was with mancozeb and tetraconazole + azoxystrobin, regardless of
the poisoning method, although the highest efciency was observed
with the dilution method in the culture medium, compared to the
sensi-disc method.
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