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Antifungal activity of zinc oxide nanoparticles in Fusarium oxysporum‐

Solanum lycopersicum pathosystem under controlled conditions

Article in Journal of Phytopathology · June 2021

DOI: 10.1111/jph.13023

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DOI: 10.1111/jph.13023

ORIGINAL ARTICLE

Antifungal activity of zinc oxide nanoparticles in Fusarium

oxysporum-­Solanum lycopersicum pathosystem under controlled
conditions

Ana María González-­Merino1 | Agustín Hernández-­Juárez1 |

Rebeca Betancourt-­Galindo2 | Yisa María Ochoa-­Fuentes1 |
Luis Alonso Valdez-­Aguilar3 | Mónica Lorena Limón-­Corona1

1
Departamento de Parasitología,
Universidad Autónoma Agraria Antonio
Narro, Saltillo, México
2
Centro de Investigación en Química
Aplicada, Saltillo, México
3
Departamento de Horticultura, Universidad
Autónoma Agraria Antonio Narro, Saltillo,
México
Correspondence
Agustín Hernández-­Juárez, Departamento
de Parasitología, Universidad Autónoma
Agraria Antonio Narro, Calzada Antonio
Narro # 1923, C.P. 25315. Buenavista,
Saltillo, Coahuila, México.
Email: chinoahj14@hotmail.com
Abstract
Antifungal activity of zinc oxide (ZnO) and ZnO nanoparticles (ZnO NPs) was evalu-
ated on the control of Fusarium oxysporum Schltdl. (Nectriaceae) under laboratory
and greenhouse conditions. In vitro evaluation, poisoned culture media was pre-
pared, and an explant was placed in the centre of solid medium. The experimental
design was completely randomized with 18 treatments. Mycelial growth and co-
nidia concentration were evaluated. Subsequently, three treatments (3,000, 1,500,
100 ppm) of ZnO NPs and ZnO were chosen for their evaluation in the greenhouse in
tomato plants of Floradade variety under a randomized block design. Inoculation was
carried out with a 1x107 conidia per mL suspension of F. oxysporum, when the plants
presented the third pair of true leaves. Later, the application of different concentra-
tions of ZnO and ZnO NPs was carried out; in this investigation, the incidence and
severity and plant height were evaluated into account to determine the treatment ef-
fect on F. oxysporum. In vitro, the best treatments in mycelial growth inhibition were
the high concentrations of ZnO NPs from 1,600 to 3,000 ppm with 81%–­83%, and
in the sporulation of the fungus, they were also those that inhibited from 82.57% to
83.85%. In greenhouse, the treatments that reached the highest plant height were
ZnO NPs from 1,500 to 3,000 ppm, with a range of 166.0–­175.40 cm, with a severity
on the scale of 0.40–­0.80 and an incidence of 20%–­40%. ZnO NPs have a potential
application as an antifungal agent and can be used to control the spread of F. oxyspo-
rum in tomato plants, in addition to improving the promoter effect related to the Zinc
activity as a precursor in auxins synthesis, cytokinins and gibberellins biosynthesis,
as well as the induction of higher activity of antioxidant enzymes useful in response
to the pathogens attack.

KEYWORDS

disease, nanoparticles, tomato, toxicity

Journal of Phytopathology. 2021;00:1–12. wileyonlinelibrary.com/journal/jph © 2021 Wiley-VCH GmbH | 1

2 |
1 | I NTRO D U C TI O N
The tomato Solanum lycopersicum L. (Solanaceae) is the most
important vegetable worldwide. China is the most important
producer and tomato consumer, while the United States is the
main importer and Mexico is the main exporter of this vegetable
(FIRA, 2019). Mexico stands out with a production of 3,441,639.37
t and a production value of $ 29 874 007.50 (MXN) (SIAP, 2020),
in addition, whose cultivation, harvest and marketing generate
72 thousand direct jobs and approximately 10.7 million indirect
jobs. The production of this crop is affected by a large number
of phytopathogenic microorganisms that affect its performance,
such as bacteria, fungi and viruses. One of the main fungi that
most affects the crop are of the genus Fusarium Link (Nectriaceae)
that cause losses between 21% and 47% in protected and open-­
field crops (Cardona-­Piedrahíta & Castaño-­Z apata, 2019; Mejía &
Hernández, 2001).
Fusarium oxysporum Schltdl. (Nectriaceae) is the causal agent
of tomato wilt disease. The main symptoms of the disease include
chlorosis, progressive wilting of leaves, discoloration of vascular
tissue, growth retardation and up to causing the death of the plant
(Abdallah et al., 2016, 2019).
In conventional agriculture, chemical fungicides are the main tool
for the control of phytopathogenic fungi. The continuous and exces-
sive use of these products has resulted in the development of resis-
tance to the fungicides used (Coromoto & Reyes, 2018). Chemical
products enter the human body and remain in it, causing diseases
such as cancer, permanent damage in children born with genetic
damage with deterioration, environmental damage such as soil ster-
ilization and the desertification of large regions (Rivero et al., 2014).
Currently, the search for control alternatives has increased, such
as the use of nanotechnology in agriculture; whose applications in-
clude fertilizers to increase plant growth and yield, pesticides for
pest and disease management, and sensors to monitor soil quality
and plant health (Servin et al., 2015).
Nanoparticles (NPs) are materials between 1 and 100 nanome-
ters (nm) in size and exist as metalloids, metal oxides, non-­metals
and carbon nanomaterials, etc., whose small size, large surface area
and high reactivity have allowed their use in disease control and as
nanofertilizers (Elmer & White, 2018). Nanoparticles have gained im-
portance as novel antimicrobial agents due to their strong germicidal
properties, against a wide range of multi-­resistant microorganisms
(Singh et al., 2010).
Zinc oxide nanoparticles (ZnO NPs) in different crops of eco-
nomic interest have been reported to have properties such as
growth promoters, bactericides and fungicides (Esparza, 2016;
Méndez-­Argüello et al., 2016); in the latter, it has been pointed
out that their antifungal activity against Penicillium expansum
Link. (Eurotiales: Trichocomaceae), Botrytis cinerea Pers.: Fr.
(Helotiales: Sclerotinicaeae), Aspergillus flavus Link., Aspergillus
niger, P.E.L. van Tieghem, Aspergillus fumigatus Fresenius
(Eurotiales: Trichocomaceae), Fusarium culmorum (Wm.G. Sm.) Sacc.
(Hypocreales: Nectriaceae) y F. oxysporum (Jamdagni et al., 2018,
GONZÁLEZ-­MERINO et al.
2019; Rajiv et al., 2013) resulted in a reduction in the presence of
diseases.
There is little information on applications of ZnO NPs as fungi-
cides for F. oxysporum in tomato, which is why this research has the
objective of evaluating the antifungal activity of zinc oxide nanopar-
ticles for the control of F. oxysporum under controlled laboratory
conditions and greenhouse in tomato S. lycopersicum.
2 | M ATE R I A L S A N D M E TH O DS
2.1 | Experiment location
Research was conducted at the Entomologia Molecular Laboratory
and greenhouse of Universidad Autónoma Agraria Antonio Narro, in
Saltillo, Coahuila, Mexico.
2.2 | Obtaining zinc oxide
Zinc oxide powder was obtained commercially at Sanely Marketer.
2.3 | Synthesis and characterization of
nanoparticles
ZnO NPs were synthesized at Centro de Investigación en Química
Aplicada, in Saltillo, Coahuila. They were synthesized through
controlled precipitation according to Betancourt et al. (2010)
technique, by the chemical hydrolysis method as follow: 13.7 g
of Zn(O2CCH 3)2 and 600 ml of ethanol were placed in a ball flask
with three necks. This solution was constantly stirred at a tem-
perature of 75℃ under reflux for 2 hr. Then, an aqueous solu-
tion of 0.22 M NaOH and an additional 100 ml of distilled H2O
were added to complete the reaction mixture. Constant stir-
ring was continued for 24 hr. Subsequently, the ZnO NPs ob-
tained immersed in ethanol were recovered by centrifugation
at 15,000 rpm for 5 min for their recovery. The precipitate was
washed two times with ethanol and dried in an oven at 60℃ for
24 hr. The dried ZnO NPs were crushed in an agate mortar to ob-
tain a fine powder and stored at room temperature until use. The
size and morphology of nanoparticles were measured by means
of a high-­r esolution transmission electronic microscope (HRTEM)
Titan 80–­3 00 kV (FEI Company).
2.4 | Isolation and purification of F. oxysporum
To obtain the strains of the fungus, plant tissue infected by F. ox-
ysporum was collected from the tomato crop; for its isolation, the
tissue was sectioned with a scalpel, then it was disinfected with
a triple wash with 3% hypochlorite, distilled water sterile (dH2O),
70% alcohol and dH20 and later it was seeded in culture medium
GONZÁLEZ-­MERINO et al.
Potato Dextrose Agar (PDA) (Bioxon®) and incubated at 27 ± 2℃ in a
®
growth chamber (Yamato ), and from their growth, the strains were
purified by hypha point.
2.5 | Morphological and molecular identification
Morphological identification was carried out by mycelium struc-
tures and their respective conidia on slide with a lactophenol and
cotton blue solution. Its observation was performed at 40 and
100X in a compound microscope, and for identification at the genus
level, it was supported by the taxonomic keys for imperfect fun-
gal genera of Barnett and Hunter (1998) and at the species level
with the keys of Leslie and Summerell (2006). Molecular identifi-
cation was performed by extracting DNA from the fungus, using
the CTAB protocol (Lee & Taylor, 1990). DNA was resuspended in
30 µl of nuclease-­free water and stored at −20℃. The polymerase
chain reaction (PCR) was carried out in 25 µl containing 12.5 µl of
Gotaq® DNA Polymerase (Promega corporation), 1 µl of each oligo-
nucleotide, FOF1: (5'-­ACATACCACTTGTTGCCTCG-­3') and FOR1:
(5'-­CGCCAATCAATTTGAGGAACG-­3 ') at 0.35 µM and 20 ng of
genomic DNA, and the final volume was adjusted with nuclease-­free
water. The amplification conditions were as follows: an initial dena-
turation of 1 min at 95℃, followed by 25 cycles of 94℃ for 1 min,
52℃ for 30 s, 72℃ for 1 min and a final extension of 7 min at 72℃.
The length of the amplified fragments was 340 bp, which were visu-
alized on a 1% agarose gel (Mishra et al., 2003).
2.6 | Evaluation in vitro
2.6.1 | Culture medium poisoned with ZnO NPs
PDA culture medium added with 5g/L of malt extract was prepared.
It was placed in a pressure cooker (Presto Model 79,291, capacity
21 L) for 15 min at 121℃ and 15 pounds for sterilization.
ZnO NPs and ZnO were prepared in glass tubes with 10 ml of
deionized water, and the corresponding concentrations of nanopar-
ticles and ZnO were added. The ZnO NPs were dispersed by soni-
cation in three cycles of 15 min. After the sterilization, the culture
medium was allowed to cool, which was completed with the previ-
ously sonicated NPs solution, stirred for a period of 15–­20 min to
achieve homogenization of the solution with the medium and finally
each treatment was emptied into Petri dishes (90 × 15 mm). 5 mm
explant of F. oxysporum was placed in the centre. The Petri dishes
were incubated at a temperature of 27 ± 2ºC for 8 days in a growth
chamber (Yamato®).
2.6.2 | Experimental design
The in vitro effect of F. oxysporum by ZnO NPs and ZnO was evalu-
ated in 8 concentrations (3,000, 2000, 1,600, 1,200, 800, 400, 200,
| 3
100 ppm) and an absolute control (PDA = 0 ppm) with 4 replicates,
and it considered a Petri dish as one repetition.
2.6.3 | Mycelial growth evaluation and conidia
concentration
The evaluation consisted of measuring the mycelial growth, in two
axes (horizontal and vertical) of the phytopathogen, every 24 hr,
until the filling of the control (8 days), using as support a calliper
(vernier) with digital compound (Titan®). To count the conidia, the
mycelium from Petri dishes was collected in dH2O, the suspen-
sion was shaken to homogenize the concentration of conidia, and
then, 10 µl was taken and placed in the hemocytometer (Marienfeld
Germany), following the steps and formulas of the guide provided
by Celeromics (Total Cells Counted / Number of Frames × 10,000).
With the data obtained in the evaluation, the per cent of inhibition
of mycelial growth was calculated using the formula described by
Orberá et al. (2009), taking as 100% the mycelial growth of the con-
trol (0 ppm).
( )
R1 − R2
PIRG = × 100
R1
Where:
PIRG: is the per cent of inhibition of radial growth.
R1: represents the average value of the radius of the fungus
growth (control).
R2: is the average value of the radius of the inhibited colony
(treatment).
2.7 | Evaluation in vivo
2.7.1 | Vegetative material
Tomato seedlings of S. lycopersicum Floradade variety type ball
were used (Fax de Occidente S.A. de C.V.). Seeds were planted in
expanded polystyrene germination trays of 200 cavities in peat
moss-­perlite substrate in a 3:1 ratio, and the transplant was car-
ried out 4 weeks after planting in polyethylene containers with a
capacity of 8 L, in conditions of 25ºC ± 2ºC and relative humidity
of 60 ± 10% and automated ventilation to reduce heat and renewal
of carbon dioxide.
2.7.2 | Inoculation and application of treatments
When the seedlings presented the third pair of true leaves, the first
inoculation of F. oxysporum was performed with a suspension con-
taining 1 × 107 conidia. The pathogen was inoculated again 15 and
30 days after the first inoculation to clearly observe the manifesta-
tion of the disease. The different concentrations of ZnO NPs and
4 |
Class Intensity of disease
0 No visible symptoms of the disease
1 Necrotic spots in hypocotyl
2 Wilted leaves, darkening at the base of the hypocotyl, or decreased plant growth
3 Wilting, necrotic lesions 1–­5 cm and decreased plant growth
4 6–­10 cm necrotic lesion, defoliation and decreased growth
5 Death of the plant
ZnO were sprayed onto the foliage with a manual sprayer (RLFlo
Master) 1900 ml capacity, the next day after each F. oxysporum
inoculation.
2.7.3 | Experimental design
In vivo effect over F. oxysporum by ZnO NPs and ZnO was evalu-
ated in three concentrations chosen based on the in vitro evaluation
(3,000, 1,500, 100 ppm) and an absolute control (without inocula-
tion with F. oxysporum = TA) and a control inoculated with spore
suspension (=T0) with five repiclates each, considered a plant as a
repetition.
2.7.4 | Evaluation
The plant height (cm) was determined with the support of a Gripper
tape measure (Truper) at the same time as the damage evaluation by
F. oxysporum. The evaluation of the damage severity by F. oxysporum
was carried out 75 days after the first pathogen inoculation, when
the control plants (T0) presented characteristic symptoms, caused
by the pathogen, using the symptomatological scale of Apodaca-­
Sánchez et al. (2004) and Clavijo (2014) (Table 1) and the incidence
was calculated with the following formula: I = (IA∕N) × 100where I =
Incidence, is the measure of the per cent of individuals affected (IA)
and showing symptoms of the disease (%) in a population (N).
2.7.5 | Agronomic crop management
The crop nutritional part was covered with foliar fertiliizer Ferti plus
+2 L/ha (AGROformuladora DELTA® S.A. DE C.V.) applied to the sub-
strate four times during the crop development. An application of the
insecticide Imidacloprid Confidel 350SC, 1 L/ha (AGROformuladora
®
DELTA S.A. DE C.V) was made for the management of the whitefly
Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae).
2.7.6 | Phytopathogen recovery
At the end of the data collection of results, healthy tissue with symp-
toms of the disease was sectioned to determine the presence of the
GONZÁLEZ-­MERINO et al.
TA B L E 1 Escala para evaluar la
severidad por Fusarium oxysporum f. sp.
radicis-­lycopersici en tomate
phytopathogen. Diseased plant tissue was disinfected and placed on
PDA as previously described.
2.8 | Analysis of results
With the percentage of mycelial inhibition and conidia concentra-
tion, probit analysis was performed and the values of the inhibitris
concentration (IC50 and IC90) and their fiducial limits at 95% reliabil-
ity were determined. To determine the effect of the treatments on
mycelial inhibition, sporulation inhibition, incidence, severity and
plant height in the different treatments, an analysis of variance and
comparison of means of the treatments with a Tukey multiple range
test (p < .05) were used. The data of mycelial inhibition, sporulation
inhibition and incidence expressed in percentage were transformed
by arcsine square root for analysis. SAS/STAT software was used for
all analyses (SAS, 2002).
3 | R E S U LT S
High-­resolution transmission electronic microscope micrograph
of ZnO nanoparticles is shown in Figure 1, which displays semi-­
spherical. The ZnO exhibited a morphology semi-­spherical with a
size in the range of 10–­4 0 nm with average diameter of (23.44 nm).
In the inhibition of mycelial growth, significant differences were
found, whose treatment with the greatest effect on F. oxysporum
was the ZnO NPs at 1,600 and 3,000 ppm with 83.3% and 81.05%
inhibition. The ZnO concentrations of 1,600, 2000 and 3,000 ppm
had the greatest effect with 76.6%, 76% and 78.63% inhibition. In
the low dose treatments, ZnO NPs at 400, 200 and 100 ppm to-
gether with ZnO at 400 ppm inhibited from 30.78% to 47.28% of
mycelial growth. However, at low concentrations of ZnO (200 and
100 ppm) they did not show any significant difference compared
with the control (Table 2, Figures 2 and 3).
In the conidia count, the treatments with the greatest inhibition
effect with a percentage greater than 80% are the ZnO NPs of 1,600
and 3,000 ppm, and in the ZnO treatments with the greatest effect
are 1,200–­3,000 ppm with an inhibition of 65.59%–­69.87%, while the
low concentrations of ZnO NPs (200, 400 and 800 ppm) together with
ZnO at 800 ppm presented a percentage of inhibition between 55.73%
and 59.73% of sporulation. The lowest concentration of ZnO NPs and
ZnO at 100 ppm inhibited 41.71% and 28.08%, respectively (Table 2).
GONZÁLEZ-­MERINO et al.
F I G U R E 1 Transmission electron microscopy micrograph
showing the spherical morphology of ZnO NPs
For the 50% inhibition of mycelial growth, a lower concentration
of ZnO NPs is required compared with ZnO, which requires almost
twice as much to inhibit the same percentage of growth. Similarly,
for the 50% inhibition of spores a lower concentration of ZnO NPs
is required, while with zinc oxide almost three times as much is re-
quired to inhibit the same percentage of growth (Table 3).
From the greenhouse evaluation, at the end of the tests, the phy-
topathogen was recovered from healthy and diseased tissue and was
re-­isolated, again coinciding with the F. oxysporum species.
It was observed that ZnO NPs at 1,500 ppm presented signifi-
cantly the highest plant growth with 175.4 cm, followed by ZnO NPs
at 3,000 ppm with a plant height of 166 cm, the latter without signif-
icant differences with the absolute control that presented 157.4 cm
in plant height. The ZnO compared with the ZnO NPs showed signifi-
cantly less growth from 106 to 140 cm and with much less growth
the inoculated control without protection against F. oxysporum with
a height of 73 cm in plants that ended up dead (Table 4).
In terms of disease damage, there were significant differences,
with a higher incidence in the plants corresponding to the inoculated
control and ZnO, whereas the plants with application of NPs had
significantly lower incidence (Table 4). Regarding the severity, there
were significant differences between the treatments, without sever-
ity in the absolute control without inoculation, followed by the ZnO
NPs with less severity with 0.4–­1.2 in the symptomatological scale
of damage by F. oxysporum, whereas the ZnO presented the highest
severity (2.0–­4.8 on the scale) together with the inoculated control
without protection against the pathogen with the highest severity
(Figures 4 and 5).
In the present investigation, with respect to the inoculated plants
(T0), the plants treated with ZnO showed an increase in height by
| 5
61%, the absolute control (without inoculation) 115% and the treat-
ments with ZnO NPs up to 118%, even higher than the control with-
out inoculation (Figures 4 and 5).
4 | D I S CU S S I O N
In recent years, nanoparticles have been developed for use in
agriculture, with action in the improvement of physiological
processes, promoted as mineral stimulants, particularly essen-
tial micronutrients necessary for host defence, as resistance in-
ducers, reducing the presence of pathogenic organisms (Lamsal
et al., 2011; Servin et al., 2015). These characteristics highlight
the potential application of nanoparticles as antifungal agents to
control the spread and infection of several pathogens (Raskar &
Laware, 2014).
Among the nanoparticles used for crop protection, ZnO NPs are
most recurrent because of their excellent chemical and thermal sta-
bility, low production cost and are not harmful to the environment
(Rajiv et al., 2013; Sabri et al. 2013, taken from Kriti et al., 2020).
The role of ZnO release from NPs in antimicrobial activity was
tested using microparticles and nanoparticles, demonstrating that
the nano-­specific effects of ZnO play a contributing role in the
level of antifungal effects. Bioactivity was more effective with
NPs, confirming that nanometer size enhances the effectiveness of
ZnO, increasing its toxicity relative to larger microparticles, inhib-
iting mycelial growth of the fungus Fusarium graminearum (Dimkpa
et al., 2013).
The ability of metal oxide NPs through foliar application to pro-
vide disease resistance is a relatively new and underexplored con-
cept. Initially, it was attributed that the increase in crop growth and
yield was the result of inhibition or control of some phytopathogen
by ZnO NPs (Servin et al., 2015). On the other hand, it was consid-
ered that the growth-­promoting effect of ZnO NPs can be attributed
to the activity of Zinc, essential micronutrients for overall plant
growth and development and precursor of auxin production that
promote division, cell elongation, have influence on the reactivity
of indoleacetic acid, which functions as a hormonal phytostimulant,
associated with the biosynthesis of cytokinins and gibberellins; as
well as the induction of increased activity of antioxidant enzymes
useful in the response to pathogen attack (Kriti et al., 2020; Méndez-­
Argüello et al., 2016).
ZnO NPs (23.44 nm) demonstrated an effective control over
F. oxysporum growth and can be an alternative for the management
of the phytopathogen, as described by Esparza (2016) in their eval-
uation of the antifungal activity of ZnO NPs on F. oxysporum, who
observed a 91.13% inhibition of growth at 1,000 mg/L concentra-
tion. However, this observation may vary depending on the phyto-
pathogen to be controlled; in this sense, Esparza-­Rivera et al. (2014)
observed that ZnO NPs with a size of 200 nm at 2000 ppm against
Phytophthora capsici Leonian (Peronosporaceae) and Rhizoctonia
solani J. G. Kühn (Ceratobasidiaceae) inhibited mycelial growth be-
tween 14% and 16%.
6 | GONZÁLEZ-­MERINO et al.

TA B L E 2 Percent inhibition means,

Concentration Mycelial growth inhibition Inhibition of
sporulation inhibition per cent, ± standard
Treatment (ppm) (%)a,b sporulation (%)a,b
deviation of Fusarium oxysporum in PDA
Testigo 0 0 ± 0j j 0 ± 0.00 h medium at different concentrations of
ZnO 3,000 78.63 ± 1.72 bcd 69.87 ± 1.92 c ZnO and ZnO NPs at 8 days of evaluation

ZnO 2000 76.00 ± 0.91 de 66.81 ± 1.21 c

ZnO 1,600 76.60 ± 0.51 cd 68.06 ± 1.17 c
ZnO 1,200 65.85 ± 2.73 f 65.59 ± 0.49 c
ZnO 800 62.63 ± 0.57 g 56.83 ± 2.79 d
ZnO 400 46.32 ± 1.36 h 48.38 ± 1.88 e
ZnO 200 0 ± 0.00 j 28.81 ± 3.17 g
ZnO 100 0 ± 0.00 j 28.08 ± 2.32 g
ZnO NPs 3,000 81.05 ± 1.84 ab 83.85 ± 0.39 a
ZnO NPs 2000 79.35 ± 0.41 bc 76.38 ± 1.60 b
ZnO NPs 1,600 83.30 ± 0.91 a 82.57 ± 1.49 a
ZnO NPs 1,200 73.25 ± 1.12 e 75.56 ± 0.97 b
ZnO NPs 800 66.40 ± 1.98 f 59.72 ± 2.43 d
ZnO NPs 400 47.28 ± 0.74 h 57.42 ± 1.20 d
ZnO NPs 200 33.35 ± 0.83 i 55.73 ± 1.47 d
ZnO NPs 100 30.78 ± 1.09 i 41.71 ± 5.48 f
CV 2.32 3.79
R2 0.99 0.99
df 16,67 16,67
F 2,386.22c 417.49c
Pr>F <0.0001 <0.0001

Abbreviation: ppm, parts per million.

a
Means with the same letter in same column are not significantly different (Tukey; p < .05).
b
Data for analysis are transformed by arcsine square root.
c
Indicate significance contrast value F to p < .001.

Rajiv et al. (2013) synthesized ZnO nanoparticles from Parthenium
hysterophorus L. (Asteraceae) in different sizes and explored their an-
tifungal activity against A. flavus and A. niger, showing that ZnO NPs
are good antifungal agents, inhibiting these phytopathogens with a
size of 27 ± 5 nm, in addition to being respectful to the environment.
Dimkpa et al. (2013) ratified the antifungal effect of ZnO NPs on
Fusarium graminearum Schwabe (Nectriaceae), reducing the growth
of the phytopathogen, in addition to being supplemented with
the biological control agent Pseudomonas chlororaphis O6 Bergey
(Pseudomonadaceae), by not inhibiting the metabolites of the bac-
teria, though it inhibit the development of F. graminearum, opening
the possibility of using them as formulations to complement existing
strategies to improve crop health.
Recently, Pariona et al. (2020) studied the in vitro antifungal ac-
tivity of ZnO in three forms of particles, including NPs with a size
of 18 ± 2 nm, for three species of fungi: F. oxysporum f.sp. lycop-
ersici, Fusarium solani (Mart.) Sacc. (Nectriaceae) and Colletotrichum
gloeosporioides (Penz.) Penz & Sacc. (Glomerellaceae), finding very
similar control results with 1 mg/ml against F. oxysporum with 54%
and 53% inhibition by ZnO and ZnO NPs, respectively. At the same
concentration in F. solani, the inhibition was 65% and 55% and for
C. gloeosporioides 60% and 59% by ZnO and ZnO NPs, respectively.
Regarding the sporulation of the fungus, with the treatment with
ZnO NPs, its effect was correlated with the mycelial growth, inhib-
iting the conidia to a greater degree, compared with the control and
the eight treatments with ZnO, an effect that can be attributed to
the fact that the nanoparticles induce damage to the hypha of the
fungus and at the same time to the conidia, a characteristic observed
by Jo et al. (2009) and Lamsal et al. (2011) with Ag NPs (silver), who
indicated that NPs influence the formation of spore colonies, damag-
ing and penetrating the cell membrane, and the disease progression
of plant pathogenic fungi.
On the one hand, He et al. (2011) describe that ZnO NPs can
exhibit different antifungal activities. On the other hand, they ob-
served that P. expansum in PDA with ZnO NPs not only produced
conidia but also inhibited their development and distorted conidio-
phores, whereas the biomass of B. cinerea developed mainly hyphae,
with apparently more resistance to ZnO NPs, conserving the fine
structure of the mycelia, although the surface of the hyphae of the
fungi was deformed.
Wani and Shah (2012) reported a high rate of inhibition in
the germination of spores of the fungi Alternaria alternata (Fr.)
Keissl. (Pleosporales: Pleosporaceae), F. oxysporum, Rhizopus sto-
lonifer (Ehrenb.) Vuill. and Mucor plumbeus Bonord. (Mucorales:
GONZÁLEZ-­MERINO et al. | 7

F I G U R E 2 Antifungal activity of
(a) (b) (c)
ZnO NPs on Fusarium oxysporum in
vitro test: (a) 0 ppm, (b) 3,000 ppm, (c)
2000 ppm, (d) 1,600 ppm, (e) 1,200 ppm,
(f) 800 ppm, (g) 400 ppm, (h) 200 ppm and
(i) 100 ppm

(d) (e) (f)

(g) (h) (i)

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

F I G U R E 3 Antifungal activity of ZnO

on Fusarium oxysporum in vitro test: (a)
0 ppm, (b) 3,000 ppm, (c) 2000 ppm, (d)
1,600 ppm, (e) 1,200 ppm, (f) 800 ppm,
(g) 400 ppm, (h) 200 ppm and (i) 100
to ppm
8 | GONZÁLEZ-­MERINO et al.

TA B L E 3 Concentration inhibitory of
ppm
mycelial growth and sporulation inhibition
Fiducial limits 95% Fiducial limits 95% and fiducial limits of ZnO and ZnO NPs
applied to Fusarium oxysporum in PDA at
Treatments IC50 Lower Upper IC90 Lower Upper 8 days of evaluation
Mycelial growth inhibition (%)
ZnO NPs 364.40 286.54 446.37 5,017 3,537 8,081
ZnO 776.04 465.94 1,191 3,329 1943 11,401
Sporulation inhibition (%)
ZnO NPs 186.46 111.94 264.71 8,327 4,744 20,327
ZnO 545.68 416.40 694.56 17,072 9,245 43,671

Abbrevaitions: IC50, 50% growth inhibitory concentration; IC90, 90% growth inhibitory
concentration; ppm, parts per million.

TA B L E 4 Plant height, incidence and severity of damage by Fusarium oxysporum ± standard deviation at different concentrations of ZnO
NPs and ZnO at 75 days after the first inoculation

Concentration
Treatments (ppm) Plant height (cm)a Incidence (%)a,b Severitya,c

Absolute control = TA 0 157.4 ± 13.2 ab 0.0 ± 0.0 b 0 ± 0.0 c

Inoculated control = T0 0 73.0 ± 6.02 d 100.0 ± 0.0 a 5.0 ± 0.0 a
ZnO 3,000 140.0 ± 24.3 b 100.0 ± 0.0 a 2.0 ± 0.0 b
ZnO 1,500 108.4 ± 15.2 c 100.0 ± 0.0 a 2.0 ± 0.0 b
ZnO 100 105.6 ± 13.4 c 100.0 ± 0.0 a 4.8 ± 0.45 a
ZnO NPs 3,000 166.0 ± 18.1 ab 40.0 ± 54.7 ab 0.8 ± 1.10 bc
ZnO NPs 1,500 175.4 ± 12.8 a 20.0 ± 44.7 b 0.4 ± 0.89 c
ZnO NPs 100 136.6 ± 13.9 bc 60.0 ± 54.7 ab 1.2 ± 1.10 bc
CV 11.62 48.65 32.19
R2 0.85 0.65 0.90
df 7,39 7,39 7,39
F 25.50 d 8.43d 42.82d
Pr > F <0.0001 <0.0001 <0.0001

Abbreviation: ppm, parts per million.

a
Means with the same letter in same column are not significantly different (Tukey; p < .05).
b
Data for analysis are transformed by arcsine square root.
c
Symptomatic scale of Apodaca-­Sánchez et al. (2004) andClavijo (2014)
d
Indicate significance contrast value F to p < .001.

(a) (b)

F I G U R E 4 Tomato plants S.
lycopersicum (a) inoculated with Fusarium
oxysporum, (b) absolute control without
inoculation of the phytopathogen
GONZÁLEZ-­MERINO et al.
F I G U R E 5 Control of Fusarium (a)
oxysporum with ZnO NPs and ZnO in
Solanum lycopersicum tomato plants. (a)
ZnO NPs at 3,000 ppm, (b) ZnO NPs at
1,500 ppm, (c) ZnO NPs at 100 ppm, (d)
ZnO at 3,000 ppm, (e) ZnO at 1,500 ppm,
(f) ZnO at 100 ppm
(b)
(c)
Rhizpodaceae) to the exposure of ZnO NPs and NPs of magnesium
oxide (MgO) at concentrations of 100 mg/L.
Among the antifungal properties, attributed to the nanoscale
surface-­to-­volume ratio of ZnO NPs (Kriti et al., 2020), is the in-
duction of deformations in the structure of fungal hyphae, nota-
ble thinning of fibres in the hyphae and tendency to agglutinate
(Arciniegas-­Grijalba et al., 2017; Yehia & Ahmed, 2013); causing liq-
uefaction of the cytoplasmic content, making it less electron-­dense,
with a significant detachment of the cell wall, which causes cell
death of the pathogen, causing growth to stop (Arciniegas-­Grijalba
et al., 2017; Sawai & Yoshikawa, 2004; Yehia & Ahmed, 2013).
Additionally, ZnO NPs generate various other reactive oxygen spe-
cies, such as hydroxyl radicals and singlet oxygen, which in turn stim-
ulate cell death (Jamdagni et al., 2018).
| 9
(d)
(e)
(f)
The effect of ZnO NPs on plant growth is not fully distinguished.
In this regard, Navarro et al. (2008) suggest that nano-­sized materials
with larger surface areas could more efficiently absorb, translocate
and retain nutrients in plants.
In other crops such as Capsicum annuum L. (Solanaceae), Méndez-­
Argüello et al. (2015) evaluated ZnO NPs doped with Ag at 2.5% in
the growth and production of biomass in seedlings, finding only an
increase in height of 16.8% compared with control plants.
Most of the studies on NPs in plant pathology have examined
the direct antifungal and antibacterial activity against the pathogen
in question (Ocsoy et al., 2013; Saharan et al., 2015; Strayer-­Scherer
et al., 2018; Wani & Shah, 2012; Zabrieski et al., 2015).
In the present study, it was demonstrated that foliar application
of ZnO NPs inhibits the development of the disease and positively
10 | GONZÁLEZ-­MERINO et al.
affects plant growth, presumably through better mineral nutrition
and host defence. The effect of ZnO at the nanoscale (25 nm) on
plant growth and development was verified by Prasad et al. (2012)
in the peanut crop Arachis hypogaea L. (Fabaceae), observing that
at 1,000 ppm it promoted the germination of seeds, plant vigour,
as well as increased stem and root growth, early flowering and in-
creased chlorophyll content and pod yield, although the increase in
concentration (2000 ppm) presented adverse effects such as toxic-
ity in the growth and performance.
Stampoulis et al. (2009) investigated the effects of five nano-
materials including ZnO NPs in Cucurbita pepo L. (Cucurbitaceae)
in a hydroponic medium at 1,000 mg / L, finding that the NPs did
not affect germination, but did present toxicity on root length and
biomass.
In watermelon plants, Citrullus lanatus (Thunb.) Matsum. and
Nakai, Elmer et al. (2018) evaluated the efficiency of the ZnO NPs
and CuO on F. oxysporum and observed that the plants treated with
the nanoparticles had a range of disease classification values signifi-
cantly lower than the control plants that were not treated.
Recently, different antifungal activity of ZnO NPs effective in in-
terfering with the metabolism of phytopathogenic fungi was deter-
mined; it was observed that ZnO NPs at a concentration of 20 ppm
were effective in inhibiting spore germination on Bipolaris sorokin-
iana (Sorokin) Shoemaker (Pleosporaceae) and at 10 ppm effective
for Alternaria brassicicola (Schwein.) Wiltshire (Pleosporaceae) and
at 100 ppm significantly inhibit mycelial growth of both pathogens
(Kriti et al., 2020).
The lower mycelial growth in vitro and the reduction of the
disease in the crop suggest an effective action of ZnO NPs, on the
growth of the fungus F. oxysporum; in addition, enhancing the pro-
moter effect by the action of zinc as a precursor of various phys-
iological mechanisms in plants highlights the feasibility of these
nanoparticles as an alternative management of F. oxysporum in
tomato.
5 | CO N C LU S I O N
ZnO NPs showed antifungal activity inhibiting in vitro mycelial
growth and sporulation of Fusarium oxysporum. Foliar application
of ZnO NPs decreased the incidence and severity of the disease
caused by F. oxysporum, allowing the growth of tomato plants. ZnO
NPs have the potential as a biostimulant to promote plant growth
and to be used in the prevention and control of plant deterioration
by phytopathogenic microorganisms.
C O N FL I C T O F I N T E R E S T
The authors declare no conflict of interest.
AU T H O R C O N T R I B U T I O N S
Ana María González-­Merino designed the research and methodol-
ogy, and conducted samplings and experiments. Agustín Hernández-­
Juárez designed the research and methodology, conducted
samplings and experiments, revised the original draft and prepared
the final manuscript. Rebeca Betancourt-­Galindo designed the re-
search and methodology and conducted samplings and experiments,
and involved in writing—­review and editing the final version of the
manuscript. Yisa María Ochoa-­Fuentes analyzed data, revised the
original draft and prepared the final manuscript. Luis Alonso Valdez-­
Aguilar revised the original draft and prepared the final manuscript
and involved in writing—­review and editing the final version of the
manuscript; Mónica Lorena Limón-­Corona conducted samplings and
experiments, revised the original draft and prepared the final manu-
script. All authors have read and agreed to the published version of
the manuscript.
PEER REVIEW
The peer review history for this article is available at https://publo​
ns.com/publo​n/10.1111/jph.13023.
DATA AVA I L A B I L I T Y S TAT E M E N T
Data are available on request from the authors. All the data are in-
corporated in the manuscript.
ORCID
Agustín Hernández-­Juárez https://orcid.
org/0000-0001-7059-4471
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