USE SILICA NANOPARTICLES IN CONTROLLING LATE WILT DISEASE IN MAIZE CAUSED BY HARPOPHORA MAYDIS

USE SILICA NANOPARTICLES IN CONTROLLING
LATE WILT DISEASE IN MAIZE CAUSED BY
HARPOPHORA MAYDIS
El-Shabrawy, E.M.
Plant Pathology Research Institute
A.R.C. Giza, Egypt
Email - said_wsha@hotmail.com
Key Words: late wilt disease, Silica nanoparticles, Green synthesis.
ABSTRACT
Green synthesized silica nanoparticles (SiNPs) and their optimization
under different pH values i.e., (5, 7, 9, 11) giving sizes 20, 40.2, 70.2 and
95.5 nm, were evaluated for controlling Harpophora maydis the causal
agent of late wilt disease in Zea maize in vitro and in vivo. Under laboratory
conditions, PDA medium revealed that all tested SiNPs sizes, 20, 40.2, 70.2
and 95.5nm at different concentrations (0.5, 2.5, 5 and 10 ppm) significantly
inhibited the mycelia radial growth of Harpophora maydis. Reduction
increased by increasing concentration compared to control. The most
efficient treatment was SiNP- 20 nm followed by SiNP-40.2 nm.
Greenhouse experiment indicated that seed coated by SiNPs significantly
reduced the infection percentage of late wilt and enhanced the germination
percentage compared with check treatment (70.8%). The SiNP-20 nm
followed by SiNP-40.2 nm introduced superior reduction in disease
incidence (88.2 and 87.7% reduction, respectively) at concentration 10 ppm.
The lowest effect was SiNP-95.5 nm which gave 52.9% reduction. Results
of field trails during 2019-2020 growing season at Giza and Gemmeza
disease nurseries indicated that treated seeds with SiNPs showed significant
reduction in maize infected with late wilt compared with check plants
(78.3% and 81.7% at Giza 2019, Gemmeza 2020, respectively). Also, there
were significant differences between treatments in yield average of the two
seasons. The disease reduction and yield increased with increasing
concentrations. The SiNPs-20 nm and SiNPs-40.2 nm treatments were the
most efficient treatments in decreasing disease incidence and enhancing
yield when recorded 6.7% at 10 ppm at first season (Giza-2019) for both
treatments while gave 6.7% and 8.3% in the second season at Gemmeza-
2020, respectively. On the other hand, average yield were 29 ard/fd in cases
of SiNP-20 nm and 27.3 ard/fd in case of SiNP-40.2 nm. In contrary, the
lowest treatments were the concentration 0.5 ppm of treatments SiNPs 95.5,
70.2 nm. Where, the infection was 34% and average yield of 17.6 ard/fd for
SiNP-95.5 nm and it was 26.3% infection % which yielded 18 (ard/fd) in
treatment SiNP-70.2 nm at Gemmeza location. It could be concluded that
using the green synthesized SiNPs ecologically welcomed at sizes 20 and
Egypt. J. of Appl. Sci., 36 (3) 2021 1-19
40.2 nm, were more efficient than that with higher sizes in controlling maize
late wilt disease and enhancing maize yield productivity.
INTRODUCTION
Maize (Zea mays L.) is considered as one of the most important
cereal crops in Egypt. The late wilt disease of maize caused by
Harpophora maydis (Games, 2000) synonymous: Cephalosporium
maydis (Samra, et al., 1963) is one of the most important diseases on
maize in Egypt (Sabet et al., 1966b; Ali, 2000 and Saleh & Leslie,
2004). Moreover, Egyptian isolates of C. maydis vary in their
morphological characters and capability to cause infection as well as
their genetic structures (Saleh et al., 2003). Furthermore, the yield losses
may reach up to 40% in naturally infested fields with infection up to 80%
(El-Shafey and Clafline, 1999). Late wilt appears during tasseling as a
rapid wilting of the lower leaves and develops to hollow and shrunken
stalks with a dark yellow-to-brown or black-stained pith (El-Shafey and
Claflin 1999). The pathogen is mainly a soil-borne fungus whichable to
invade root tissue and colonizes the xylem (Sabet et al. 1970). Breeding
of resistant varieties of maize is the most effective method for controlling
this disease (El-Shafey et al. 1988). In recent years, using pesticide for
controlling plant disease resulted in many environmental hazards. Many
attempts were made to control the pathogen using chemical and
biological methods (El-Mehalowy et al. 2004; Ashour et al. 2013; El-
Moghazy et al., 2017 and Elshahawy and El-Sayed, 2018). Some
tested fungicides worked well in pots but failed in field experiments.
Therefore, many researchers are trying to find an alternative method for
pesticides as inorganic nanoparticles. Nanotechnology is characterized by
the formation of particles with variable sizes, shapes, chemical
compositions, depending on their applications. Although chemical and
physical methods may produce pure and well-defined nanoparticles,
these methods are just costly and critical to the habitat (Reddy et al.,
2012).
Green synthesis of nanoparticles with the help of plants as reducing
agents is considered an efficient, cost effective, fast and eco-friendly in
manner (Yugandhar and Savithramma, 2015a). In recent past, most of the
scientists adopted green synthesis methods for the production of narrowranged
particles, like calcium (Yugandhar and Savithramma, 2013),
copper (Shende et al. 2015), gold (Gopinath et al. 2014), iron (Naseem
and Farrukh, 2015), silica (Athinaranan et al. 2015), silver (Yugandhar
and Savithramma, 2016), and zinc (Bala et al. 2015) from different
medicinal plants including Nerium oleander among them, silica
nanoparticles (SiNPs) were recognized as important in the fields of
chemistry, physics and plant disease control due to their distinctive
properties.
2 Egypt. J. of Appl. Sci., 36 (3) 2021
Present study aims to investigate the antifungal activity of green
synthesized silica nanoparticles with different sizes against Harpophora
maydis, the causal agent of maize late wilt disease under in vitro and
invivo conditions.
MATERIAL AND METHODS
Synthesis of silica nanoparticles
Synthesis of silica nanoparticles (SiNPs) was achieved with slight
modifications of Adam et al. (2011) protocol. The well-ground (20 g) of
Nerium oleander plant leaf powder was subjected to acid treatment by
mixing it with 500 ml of 1 M HNO3 in a 1000-ml Erlenmeyer conical
flask and stirred for 24 h. this step was done before SiNPs synthesis to
purify the reaction mixture from plant impurities (Yugandhar et al.
2015a).Mixture was centrifuged at 14.000 rpm for 20 minutes, yielded
raw silicon dioxide (SiO2)in the form of a pellet at the bottom of
centrifuge tubes. Filtrate part was discarded and SiO2 pellet was collected
and washed several times with distilled water the pH up to (4.0–5.0). The
solution was dried in an oven between 100 and 110 ◦C for 12 h. The
evaporated turbid solution was stirred with 500 ml of 1 M (NaOH)
solution up to 24 h with a magnetic stirrer to reach the pH up to 12 to
form sodium silicate. The obtained reaction mixture was separated with a
suction pump and titrated with 3 M (HNO3) until the pH was attained up
to 8.5–9.0 to get the pure form of SiO2, sodium nitrate and water
molecules (Yugandhar and Savithramma, 2015 b; Yugandhar et al.,
2015). When 3M (HNO3) solutions was added drop wise to the purified
plant mixture, color pattern of the mixture was gradually changed from
brown to whitish precipitate. The contents were centrifuged at 5000 RPM
for 10 min to separate biological admixtures. The contents were washed
3 to 4 times with distilled water and dried in a hot air oven for 12 h at
80◦C. The preliminary indication of (SiNPs) formation can be confirmed
by its color change from brown to whitish precipitate. The obtained
powder was well-ground with a mortar and pestle, and was utilized for
characterization and antimicrobial studies.
Characterization and optimization of SiNPs
Ultraviolet–visible (UV–Vis) spectroscopic characterization of the
obtained nanoparticles before dryingwas analyzed by using UV–Vis
Spectrophotometer (Shimadzu UV-1800) at wavelengths ranged in the
200–800 nm compared with negative control (plant filtrate). Dynamic
light scattering (DLS) was carried out by using a Malvern Zetasizer Nano
ZS 90 (Worcestershire, UK) to determine SiNPs sizes. Microstructures
were recorded on a MSAL-XD2 X-ray di-ractometer (XRD, Bruker,
Karlsruhe, Germany) employing Cu target in the 2q range from 0o to 80
Egypt. J. of Appl. Sci., 36 (3) 2021 3
o(40 kV, 30 mA, 1.540513 =ג A). Transmission electron microscopic
(TEM) analysis of nanoparticles was performed by using JEOL JEM-
2010 (USA) with high-resolution transmission electron microscope
operated between 80 and 200kV accelerating voltages.
Optimization of silica nanoparticles sizes
Inan attempt to produce better size controlled silica nanoparticles,
the effect of the pH reaction was studied by varying it at a time, keeping
the other experimental conditions the same. In all, the reactions, the
concentrations of silica nanoparticles and the plant extract were set at 1
mM and 10 g (wet weight) of plant extract/100 ml. The mixtures were
incubated at different pH values (5, 7, 9 and 11) for various time periods
for one month with respect to SiNPs stability. Simply by varying the pH
value of the reaction system, size of the nanoparticles could be turned.
When the reaction is completed, products were collected and thoroughly
washed for several times with ethanol to obtain pure SiNPs without any
by-products and finally subjected to dry vacuum at 80 °C for 3 h.
optimum reaction parameters were then selected by measuring the
absorbance of resulting solutions spectrophotometric ally using a UV–
visible spectrophotometer (Shimadzu UV- 1800) at wavelengths ranged
in the 200–800 nm. For each condition, respective controls were
maintained. The hydrodynamic diameter of the formed SiNPs were
measured by dynamic light scattering (DLS) using a Malvern Zetasizer
Nano ZS 90 (Worcestershire, UK).
In vitro effect of nanoparticles on Harpophora maydis radial growth
A laboratory experiment was carried out to screening the inhibitory
effect of SiNPs against a highly virulent Harpophora maydis (No.9) liner
growth. In vitro assay was performed on Potato dextrose agar (Bilgrami
&Verma, 1981). The tested SiNPs sizes 20, 40.2, 70.2 and 95.5 nm at
different concentrations (0.5, 2.5, 5 and 10 ppm) were poured into growth
media prior to pouring in a Petri dish (9cm in diameter). Three replicates
were used for each concentration. Five mm indiameter agar plugs were
obtained from the actively growing Harpophora maydis (7 old day
cultures) inoculated in the center of plates supplemented with different
treatments. The plates were incubated at 28°C for 9 days. Colony
diameters were measured every 72h until full growth in control. Control
plates inoculated by Harpophora maydis in growth medium without
SiNPs. The percentage of inhibition zones were measured compared with
control using the following formula: Inhibition rate (%)= (R − r)/R×100
Where R is radial growth of fungi in control and r is the radial growth of
fungi in treated plates.
4 Egypt. J. of Appl. Sci., 36 (3) 2021
In vivo effect of nanoparticles on Harpophora maydis
-Seed coating technique
Seed coating was done following the method described by Bardin
et al. (2004). Seeds were soaked for 15min. in 1% methyl cellulose (MC)
solution at the rate of 3ml per 100 seeds. Thereafter, seeds were removed
and placed in plastic bags containing silica nanoparticles with four
different sizes (20, 40.2, 70.2, and 95.5nm) at the rate of 5 ml per 100
seeds from different four concentrations 0.5, 2.5, 5, and 10 ppm of each
SiNPs. Bags were inflated with air and shaken vigorously. Thereafter,
seeds were directly planted in the infested-potted soil. Seed coated with
sterile distilled water only acted as the control.
-Inoculum preparation
Substrate for fungal growth was prepared in 500 ml glass bottles
each contained 100 g of sorghum grains, 50 g of washed and 90 ml of tap
water. Bottles were autoclaved at 121ºC for 30 min then cooled down.
The fungus propagules of aggressive Harpophora maydis isolate No. 9
obtained from culture type collection of Maize and Sugar Crops Dis.
Dept., Plant Pathol. Res. Inst., ARC, taken from 7 day-old culture grown
on PDAY medium, were aseptically transferred into each bottle and
allowed to colonize on sorghum medium for 2-3 weeks at 27 ± 2ºC until
sufficient growth of the fungus was obtained (El-Shafey et al., 1979).
The incubated bottles were shack every three days to ensure uniform of
fungal growth. The content of the bottles of was poured out and mixed to
get homogenized, and then inoculum was used for soil infestation.
-Soil infestation
Soil infestation was carried out according to (Samra et al., 1966)
as follows: Batches of autoclaved clay loam soil were infested with
inoculum of Harpophora maydis with soil at the rate of 30 g/kg soiland
mixed thoroughly. Infested soil was dispensed into pottery pots (25-cmdiameter)
sterilized by 0.4 % formaldehyde. Maize grains (cv. Boushy)
were surface sterilized in 5% sodium hypochlorite solution for 3 min.
then washed in sterilized distilled water for 5 min. and air dried before
sowing. Ten grains were sowed in each pot. Four pots were used for each
treatment as replicates. On the other hand, autoclaved sterilized sorghum
grains, mixed thoroughly with soil at the rate of 30 g/kg soil and kept as
control (check) treatment. Pots were regularly watered every other day
for a week before sowing.
Field experiment
Silica nanoparticles with their four varied sizes at concentration
(0.5, 2.5, 5 and 10 ppm)were studied under field conditions in disease
nursery at Giza and Gemmeza Research Stations, ARC, during summer
of 2019-2020 growing seasons. Seed coating treatment, were used. Maize
seeds cv. Boushy, susceptible to Harpophora maydis, was used in this
Egypt. J. of Appl. Sci., 36 (3) 2021 5
study. Untreated seeds were used as check treatment. Four replicate plots,
20 maize plants were used in each plot.
Disease assessment
Disease incidence as percentage of infection was recorded 90 day
after sowing according to Sabet et al. (1966a).
Statistical analysis
Data were subjected to statistical analysis ofvariance (ANOVA)
test. A complete randomize design was applied and Duncan’s multiple
rangetests were used for comparing means (Gomez and Gomez, 1984).
RESULTS
Synthesis and optimization of silica nanoparticles
In this regard and before silica nanoparticles (SiNPs) synthesis, plant
material was subjected to acid treatment by using 1M (HNO3) in a way to
purify the reaction mixture from plant impurities. Finally, the preliminary
indication for the formation of (SiNPs) can be confirmed by its color change
from brown to whitish precipitate as mention before in material and
methods. Solutions after titration with 3 M (HNO3) were analyzed with the
help of UV–Vis spectrophotometer, which displayed a broad peak at 350nm
(Fig. 1) and (Fig.2A) due to the surface plasmon resonance nature of SiNPs
in the reaction solution. The obtained nanoparticles absorb light at different
wave lengths and are excited to give a broad peak.
Fig. (1): Plant synthesis of the formed silica nanoparticles: a UV– Vis spectra
of the formed SiNPs in comparison with the plant filtrate (negative
control).
The Morphology of the biosynthesized SiNPs were examined using
HR-TEM (Fig.2B). Measurements indicated the formation of polydispersed
spherical shaped SiNPs with 50 nm ± 5.5 average sizes and -
6 Egypt. J. of Appl. Sci., 36 (3) 2021
21.5 mV value of zeta potential as indicated by DLS analysis (Fig.2C),
which indicated ideal surface charge of the formed SiNPs, which prevent
the agglomeration and generate a strong repulsive force among the
particles that increase their stability. Also, crystallographic studies of the
synthesized SiNPs with XRD instrument displayed a broad and high
intensity peak at 22◦ of 2θ values of x-axis corresponding to the
amorphous nature of SiNPs which is correlated with JCPDS file No. 01-
0787 of the Joint Committee on Powder Diffraction Standards (Fig.3).
Fig.(2): Characterization of the formed silica nanoparticles:(A.) TEM
image of the formed SiNPs, (B) & (C) the particle and the zeta
potential of the formed SiNPs respectively.
B.
C.
A
Egypt. J. of Appl. Sci., 36 (3) 2021 7
Figure (3): X-ray Diffraction patterns of the formed SiNPs.
The absence of any other XRD peak indicating that the synthesized
nanoparticles were pure crystalline in nature. On the other hand, in a way
to obtain a smaller SiNPs size, the reaction medium of SiNPs was
changed over varied degrees of pH (5, 7, 9, 11). The color of the reaction
mixture and the intensity of the absorbance peaks were pH dependent.
Where, the results showed that SiNPs synthesized at pH of 5, 7, 9 and 11
presented absorption peaks at 300, 350, 360, and 380 nm respectively.
Figure (4): Plant mediated silica nanoparticles: a UV– vis spectra of the
formed SiNPs (20 nm) at different times as indicated during the
synthesis and after 4weeks of storage.
8 Egypt. J. of Appl. Sci., 36 (3) 2021
The absorption peaks shifted to shorter wavelength and became
narrower at alkaline pH values, possibly due to the decreased size or
anisotropy degree of SiNPs. DLS measurements were carried out to
observe the size of the produced SiNPs under pH values. The results
indicated that the smallest SiNPs produced were at pH=11 with 20 nm in
size. Particles obtained at pH= 9, 7 were larger size, (40.2, 70.5 nm)
respectively. At acidic condition (pH=5), the particle size was 95.5 nm
and cannot observe any characteristic absorbance band for SiNPs
formation at better size. Those results indicated that SiNPs were
produced in more small size at alkaline medium. Most importantly, the
UV spectra results showed that the produced silica nanoparticles (20 nm)
have a high stability over four weeks this was indicated by the absence of
red shifting in UV absorbance analysis over a course of time as indicated
in Fig. 4.
In vitro effect of nanoparticles on Harpophoramaydis radial growth
The inhibitory effect of SiNPs were evaluated against the linear
growth of the highly virulent Harpophora maydis isolate (No. 9) using
the culture technique on PDAY medium.
Table 1. Effect of silica nanparticals on Harpophora maydis radial
growth
Treatments Radial growth (cm)
8 days
Reduction
SiNPs sizes Concentration ppm
SiNP- 95.5 nm
0.5 3.8 55.2
2.5 1.5 82.3
5 1.1 87.0
10 0.5 94.1
mean 1.73 79.5
SiNP-70.2 nm
0.5 2.5 70.5
2.5 0.6 92.9
5 0.5 94.1
10 0.5 94.1
mean 1.03 88
SiNP-40.2 nm
0.5 0.7 91.7
2.5 0.7 91.7
5 0.6 92.9
10 0.6 92.9
mean 0.65 92.5
SiNP- 20 nm
0.5 0.6 92.9
2.5 0.5 94.1
5 0.5 94.1
10 0.5 94.1
mean 0.53 93.8
Control 8.5
L.S.D. 0.05 1.519
Results in Table (1) reveal that all tested nanoparticles, SiNP-20,
SiNP-40.2, SiNP-70.2 and SiNP-95.5 nm at the different tested
concentration (0.5, 2.5, 5 and 10 ppm) significantly inhibited radial
growth of Harpophora maydis on PDA compared to control. Generally,
growth inhibition (%) was increased with the increasing of the
Egypt. J. of Appl. Sci., 36 (3) 2021 9
concentrations of all substances. However, the highest effect was
recorded beginning from 2.5 ppm concentration being 94.1, 92.9, 91.7
and 82.3% reduction of SiNP-20 nm, SiNP-70.2 nm, SiNP-40.2 nm and
SiNP-95.5 nm, respectively. It was observed that on average, SiNP-20
nm (93.8%) and SiNP-40.2 nm (92.5%) where, the most efficient
treatment at any concentration followed by treatment SiNP-70.2 nm
(88%) and SiNP-95.5 nm achieved 79.5% reduction.
In vivo effect of SiNPs nanoparticles on maize late wilt:
Pot experiment was carried out to study the effect of seed coated
SiNPs treatments at the tested four concentrations (0.5, 2.5, 5 and 10
ppm) against Harpophora maydis the casual organism of maize late wilt.
Results in Table (2) show that, all seed treatments with SiNPs
significantly reduced the infection %of late wilt compared with check
treatment (70.8%) under artificial infestation.
Table. 2 Effect of SiNPs treatments on maize late wilt under
greenhouse conditions, Giza, 2019.
Treatment
Ger% Infection (%) Reduction (%)
SiNPs sizes Concentration
SiNP- 95.5 nm
0.5 85 33.3 52.9
2.5 85 27.8 60.7
5 85 25.0 64.6
10 81 20.1 71.6
SiNP- 70.2 nm
0.5 87.5 27.8 60.7
2.5 87.5 19.4 72.5
5 85 17.0 75.9
10 85 14.2 79.9
SiNP- 40.2 nm
0.5 90 19.4 72.5
2.5 90 16.7 76.4
5 89 11.8 83.3
10 86 8.7 87.7
SiNP- 20 nm
0.5 90 16.7 76.4
2.5 90 11.1 84.3
5 90 8.7 87.7
10 88 8.3 88.2
Control 85 70.8 -
L.S.D. 10.952
The most efficient treatments in reducing disease incidence were
SiNP-20 and SiNP- 40.2 nm at 10 and 5ppm concentrations gives
(76.4%, 72.5% reduction at 5 ppm, respectively) and at 10 ppm (88.2,
87.7% reduction, respectively) followed by SiNP-70.2 nm (79.9%). The
lowest treatment was SiNP-95.5 nm ranged between 52.9-71.6%
reductions. Germination percentages were not negatively affected and
sometimes slightly enhanced.
Field experiments:
The influence of seed coating with four SiNPson maize late wilt
and growth parameters was studied in disease nursery of Harpophora
maydis at Giza and Gemmeza Research Stations under field conditions.
Untreated plants were used as check. Data presented in Table (3) reveal
10 Egypt. J. of Appl. Sci., 36 (3) 2021
that seed treatment with SiNPs significantly reduced maize infection with
late wilt compared with check plants (78.3% infection at Giza 2019 and
81.7% at Gemmeza 2020). It was observed that disease incidence was
higher in Gemmeza location than Giza. There were significant
differences between treatments in yield average of the two seasons. The
disease reduction and yield were increase with increasing concentrations.
The SiNP-20, 40.2 nm treatments were the most efficient in decreasing
disease incidence and enhancing yield where infection percentage
recorded 6.7% at 10 ppm in first season resulted from both treatments
and 6.7% and 8.3% in second season in SiNP-20nm and SiNP-40.2nm,
respectively. On the other hand, average yield were 29 (ard/fd) in SiNP-
20 nm and 27.3 (ard/fd) in case of SiNP-40.2 nm. In contrary, the lowest
treatments were those at concentration 0.5 ppm for treatments SiNPs
95.5nm, 70.2 nm where infection was 34% and average yield 17.6
(ard/fd) for SiNP-95.5 nm and it was 26.3% infection which yielded 18
(ard/fd) in SiNP-70.2nm.
Table. 3 Effect of SiNPs on incidence of maize late wilt and yield
under field conditions at disease nursery, Giza and
Gemmeza, 2019-2020 growing seasons.
Treatment Giza Seasons 2019 GemmezaSeasons 2020
Yield*
SiNPs sizes (ard/fd)
Conc.
Infection%
Redaction
%
Infection%
Redaction
%
SiNP- 95.5 nm
0.5 26.7 65.9 34 58.3 17.6
2.5 18.3 76.6 24 70.6 20
5 15.0 80.8 20 75.5 20.2
10 13.3 83.0 18 77.9 21.7
SiNP- 70.2 nm
0.5 23.3 70.2 26.3 67.8 18
2.5 16.7 78.6 21.7 73.4 18
5 15.0 80.8 18.3 77.6 23.0
10 11.7 85.0 13.3 83.7 26.0
SiNP- 40.2 nm
0.5 20.0 74.4 23.3 71.4 19
2.5 13.3 83.0 11.9 85.4 23
5 10.0 87.2 11.7 85.6 25.6
10 6.7 91.4 8.3 89.8 27.3
SiNP- 20 nm
0.5 18.3 76.6 18.2 77.7 19.7
2.5 10.0 87.2 11.7 85.6 23.7
5 8.3 89.3 10.0 87.7 26
10 6.7 91.4 6.7 91.7 29
Control 78.3 81.7 14
L.S.D. 9.588 7.321 1.571
*: Grain yield per feddan (GYPF), in ardab by adjusting grain yield / plot to grain
yield per feddan(adjusted at 15.5% grain moisture).
DISCUSSION
Late wilt disease of maize caused by Harpophora maydis is one of
the most important diseases on maize in Egypt. Breeding of resistant
varieties of maize is the most effective method for controlling this
disease (El-Shafey et al., 1988). Many attempts were made to control the
disease using chemical and biological methods (Abdel-Hamid et al.,
Egypt. J. of Appl. Sci., 36 (3) 2021 11
1981; Singh and Siradhana, 1989; El-Mehalowy et al., 2004 and
Ashour et al., 2013). In recent years, using pesticide for controlling plant
disease resulted in many environmental hazards. Therefore, this work
demonstrates the use of safe management method that pose less danger to
humans and animals also controlling the disease. In this study, maize
seeds coated with silica nanoparticales (20, 40.2, 70.2 and 95.5 nm) were
used in vivo and in vitroto control late wilt disease. All tested SiNPs in
vitro inhibited the fungal radial growth at different tested concentrations
resulted in significant reduction of Harpophora maydis on PDA
compared to check treatment. Reduction (%) increased with increasing of
SiNPs concentrations. The reduction in SiNPs sizes 20, 40.2 nm were
higher at any concentration ranged between 94- 92% followed by SiNP-
70.2 nm then SiNP-95.5 nm. The results were in harmony with those
obtained by Suriyaprabha et al.(2014) who stated that maize nanosilicatreated
plants showed a higher expression of phenolic compounds and a
lower expression of stress-responsive enzymes against fungal infection.
Maize expressed more resistance to Aspergillus spp., than Fusarium spp.
These results showed significant higher resistance in maize treated with
silica nanoparticales than with bulk silicon. Hence, silica nanoparticles
can be used as an alternative potent antifungal agent against
phytopathogens. El-Gazzar and Rabie (2018) reported that using
AgNPs against Harpophora maydis alone was efficient more when
combined with chemical fungicides MaximXI and Vitavax reduced
fungal growth to 51%. The mechanisms responsible for inhibiting of
fungal growth by silicon are not well understood. In spite of that, some
hypotheses were made by some investigators to illustrate the mode of
action of Si in this respect. Bi et al, (2006) stated that Si resulted in a
collapse and shrinkage of fungal hyphae and spores, which consequently
causing the loose of fungal sporulation. Li et al, (2009) observed that
ultrastrucural alterations were happened by using transmission electron
microscopy, including thickening of the hyphal cell walls. Meanwhile,
the inhibitory effect of nanoparticles may be due to release of
extracellular enzymes and metabolities (Perez-de-Luque, and Rubiales,
2009) also some studies proposed that nanoparticles may cause structural
changes of microbial cell membrane, causing cytoplasm leakage and
eventually the cells (Sawai and Yoshikawa, 2004; Brayner et.
al.,2006).
Nanoscience and technology are enabling the development of a
wide range of materials for plant growth enhancement (Nair et al.,
2010). Nano-materials such as titanium and alumina penetrate the plants,
thereby improving or decreasing their growth characteristics (Carmen et
al., 2003; Yang and Watts, 2005). Silica (SiO2) is an essential element
for monocotyledon plants and it is known to confer biotic and abiotic
12 Egypt. J. of Appl. Sci., 36 (3) 2021
stress tolerance (Rains et al., 2006 and Epstein, 2001). The present
study raveled that, in vivo results supported the in vitro ones that all
SiNPs treatments, whether in the greenhouse or in field significantly
reduced the disease infection when compared to the control. Efficiency in
decreasing the disease incidence was increased by increasing the SiNPs
concentrations. Also the SiNPs enhanced the germination (%) than check
treatment. Similar results were found by Suriyaprabha et al.(2012) they
reported that maize treated with nanosilica showed the highest
germination (98.5%) while sodium silicate treatment gave 92.5%
germination (bulk silicon). They suggested that immediate uptake of
SiNPs through seeds and its role in biochemical induction. Current
results also showed that, under greenhouse conditions the SiNPs-20, 40.2
nm were the most significant treatments in reducing disease incidence
followed by SiNPs70.2, 95.5nm. The same trend was obtained in the
field. In previous study on bulk silicon, El-Shabrawyet al.(2014) reported
that using sodium silicate as an eco-friendly compound, in managing
stalk-rot disease complex of maize thatreduced significantly the linear
growth of Cephalosporium maydis, Rhizoctonia solani, Fusarium
verticillioides and Sclerotium rolfsii in Si amended-PDA medium.
Sodium silicate completely suppressed the tested fungi at concentration
3.0 %. Using sodium silicate as seed coating, seed soaking or soil
treatment, managed significantly late-wilt infection in greenhouse and
stalk-rot disease complex of maize in field trials of sick plots with
efficiency reached 76.9% under field conditions. In contrary in the
present investigation, reduction in H.maydis by SiNPs achieved 88.2%
under greenhouse and 91.7 % under field conditions. Moreover, average
yield of the two seasons were 29 (ard/fd) in SiNP-20nm the highest
efficient treatment and while it was 17.6 (ard/fd) in case of the lowest
ones SiNP-95.5 nm when compared to control treatment yielded 14
(ard/fd). These findings are in agreement with (Yuvakkumar et
al.,2011) who reported that SiNPs were important for maize growth
enhancement with respect to morphological parameters. However, it was
essential to soil microbial biomass and silica during maize cultivation.
Their results suggested the use of nanofertilizers in maize crop to
enhance yield due to its cost-effectiveness. In addition, the effect of silica
sources on the changes in the microbial biomass (C and N) components
in soil as well as the rhizosphere should also be considered. Soil
beneficial microorganisms such as nitrogen fixers and phosphatesolubilizing
bacteria (PSB) are potent plant growth promoters. On the
other hand, silica nanoparticlescould be easily synthesized with a
controlled size, shape, and structure, making them highly advantageous
delivery vehicles (Mody et al., 2014). They are commonly produced in a
spherical shape with pore-like holes; for example, porous hollow silica
Egypt. J. of Appl. Sci., 36 (3) 2021 13
nanoparticles (PHSNs) or mesoporous silica nanoparticles (MSNs).
PHSN and MSN commonly load the pesticide into the inner core to
protect the active molecules and, therefore, provide a sustained release.
The shell structure of PHSNs protects the active molecules inside the
nanoparticles against degradation by UV light. The available literatures
suggest that silicon has already been used to enhance plant tolerance
against various abiotic and biotic stresses and, therefore, silica
nanoparticles seem to be the natural choice for the development of agriproducts
against pests (Barik et al., 2008). Numbers of studies have
shown that SiNPs may directly interact with plants and help in improving
plant growth and yield (Strout et al., 2013 and Suriyaprabha, 2014). In
this regard, silica nanoparticles were observed to form a binary film at
the epidermal cell wall after absorption, which may add structural color
to plants (Strout et al., 2013). The impact was not limited to coloring;
SiNPs were also speculated to act as a strengthening material that may
act as an agent to prevent fungal, bacterial, and nematodes infections and
thus, may increase disease resistance.
In conclusion, the efficiency of green synthesis SiNPs in reducing
late wilt disease incidence in maize was higher. All tested treatments
showed similar trends in both vitro and vivo studies. These results
suggested the possibility of using SiNPs to control the disease and
enhancing maize yield productivity. Moreover, SiNPs may be considered
as a green method of controlling plant diseases as an environmentally
safe substitute of the synthetic fungicide.
REFERENCES
Abdel-Hamid, M. S. ; M.F. Abdel-Momeim ; H.A. El- Shafey and
S.T. El-Deeb (1981). Biological control of late wilt disease of
maize caused by Cephalosporium maydis. Agric. Res. Rev., 59:
pp. 253-260.
Adam, F. ; T.S. Chew and J. Andas (2011). A simple template-free
sol–gel synthesis of spherical nanosilica from agricultural
biomass. J. Sol–Gel Sci Technol, 59: 580–583.
Ali, M.A.Y. (2000). Diversity in isolates of Cephalosporium maydis, the
causal of late wilt of maize in egypt. M.Sc. Thesis, Fac. Agric.,
Cairo Univ., Egypt, 95pp.
Ashour, A.M.A. ; K.K.A. Sabet ; E.M. El-Shabrawy and A.M.
lhanshoul (2013). Control of maize late wilt and enhancing
plant growth Parameters using rhizobacteria and organic
compounds. Egypt. J. Phytopathol., 41 (2):187-207.
Athinaranan, J. ; V.S. Periasamy ; M. Alhazmi ; K.A. Alatiah and
A.A. Alshatwi (2015). Synthesis of biogenic silica nanoparticles
from rice husks for biomedical applications. Ceramics
International, 41(1 A): 275-281.
14 Egypt. J. of Appl. Sci., 36 (3) 2021
Bala, N. ; S. Saha ; M. Chakraborty ; M. Maiti ; S. Das ; R. Basu and
P. Nandy (2015). Green synthesis of zinc oxide nanoparticles
using Hibiscus subdariffa leaf extract: effect of temperature on
synthesis, anti-bacterial activity and anti-diabetic activity. The
Royal Soc. of Chemis., 5: 4993–5003.
Bardin, S.D.; H.C. Huang and J.R. Moyer (2004). Control of pythium
damping-off of sugar beet by seed treatment with crop straw
powders and a biocontrol agent. Biol. Control, 29:453-460.
Barik, T. ; B. Sahu and V. Swain (2008). Nanosilica from medicine to
pest control. Parasitol. Res., 103: 253–258.
Bi, Y. ; S.P. Tian ; Y.R. Gue ; Y.H. Ge and G.Z. Qin (2006). Sodium
silicate reduces postharvest decay on homi melons: Induced
resistance and fungistatic effects. Plant Dis., 90 (4): 279-283.
Bilgrami, K.S. and R.N. Verma (1981). Physiological of Fungi. 2nd
ed., pp. 23-27. Vikas Publishing, PTV.,LTd Indian.
Brayner, R. ; R. Ferrari-Iliou ; N. Brivois ; S. Djediat ; M.F.
Benedetti and M. Fievet (2006). Toxicological impact studies
based on Escherichia coli bacteria in ultrafine ZnO nanoparticles
colloidal medium. Nano Lett., 6:866–870.
Carmen, I.U. ; P. Chithra ; Q. Huang ; P. Takhistov ; S. Liu and J.L.
Kokini (2003). Nanotechnology: a new frontier in food science.
Food Technol.,57: 24–29.
EL-Gazzar, N.S. and G.H. Rabie (2018). Application of silver
Nanoparticles on Cephalosporium maydis in vitro and in vivo.
Egypt. J. Microbiol., 53: 69 – 81.
El-Mehalowy, A.A. ; N.M. Hassanein ; H.M. Khater ; E.A. Daram
El-Din and Y.A. Youssef (2004). Influence of maize root
colonization by Rhizosphere actinomycetes and yeast fungi on
plant growth and on the biological control of late wilt disease.
Int. J. Agric. Biol., 6:599-605.
El-Moghazy, S.M. ; M.E. Shalaby ; Ahlam A. Mehesen and M.H. Elbagory
(2017). Fungicidal effect of some promising agents in controlling
maize late wilt disease and their potentials in developing yield
productivity. Env.Biodiv.Soil Security,1: 129 – 143.
El-Shafey, H.A.; M.F. Abd-El-Rahim and M.M. Refaat (1979). A
new Cephalosporium wilt disease of grain sorghum in Egypt.
Proc. 3 rd Egypt, Phytopathology Congress, pp. 513-532.
El-Shafey, H.A. and L.E. Claflin (1999). Late Wilt. In: Compendium of
Corn Diseases. (ed. D.G. White), 3rd. St. Paul: APS Press, pp.
43-44.
El-Shafey, H.A.; F.A. El-Shorbagy ; I.I. Khalil and E.M. El-Assiuty
(1988). Additional sources of resistance to the late wilt disease
Egypt. J. of Appl. Sci., 36 (3) 2021 15
of maize caused by Cephalosporium maydis. Agric. Res. Rev.
Egypt, 66: 221–230.
Elshahawy, E.I. and B.A. El-Sayed (2018). Maximizing the efficacy of
Trichoderma to control Cephalosporium maydis, causing maize
late wilt disease, using fresh water microalgae extracts. Egyptian
J. of Biological Pest Control., 28(48):1-11.
Epstein, E. (2001). Silicon in plants: facts vs. concepts, in Dant off, L.E.,
Snyder, G.H., Korndoper, G.H. (eds.): Silicon in agriculture.
(Elsevier Science, Amsterdam), pp. 1–15.
Games, W. (2000). Phialophora and some similar morphologically littledifferentiated
anamorphs of divergent ascomycetes. Stud.
Mycol., 45:187–199.
Gomez, K.A. and A.A. Gomez (1984). Statistical Procedure For
Agricultural Research. 2 nd (ed.), John Wily & Sons, New York.
Gopinath, K. ; S. Gowri ; V. Karthika and A. Arumugam (2014).
Green synthesis of gold nanoparticles from fruit extract of
Terminalia arjuna, for the enhanced seed germination activity of
Gloriosa superb. J Nanostruct Chem., 4:115.
Li, Y.C.; Y. Bi ; Y.H. Ge ; X.J. Sun and Y.J. Wang (2009). Antifungal
activity of sodium silicate on Fusarium sulphureum and its
effect on dry rot of potato tubers. J. Food Sci., 74 (5): 213-218.
Mody, V.V. ; A. Cox ; S. Shah ; A. Singh ; W. Bevins and H. Parihar
(2014). Magnetic nanoparticle drug delivery systemsfor
targeting tumor. Appl. Nanosci., 4: 385–392.
Nair, R. ; S.H. Varghese ; B.G. Nair ; T. Maekawa ; Y. Yoshida and
D.S. Kumar (2010). Nanoparticulate material delivery to
plants.Plant Sci., 179: 154–163.
Naseem, T. and M.A. Farrukh (2015). Antibacterial Activity of Green
Synthesis of Iron Nanoparticles Using Lawsoniainermis and
Gardenia jasminoides Leaves Extract. Journal of Chemistry,
Article https://doi.org/10.1155/2015/912342.
Perez-de-Luque, A. and D. Rubiales (2009). Nanotechnology for
parasitic plant control. Pest Manage. Sci., 65: 540-545.
Rains, D.W. ; E. Epstein ; R.J. Zasoski and M. Aslam (2006). Active
silicon uptake by wheat, Plant Soil, 280:223–228.
Reddy, K.A.G. ; M.J. ; Joy ; T. Mitra ; S. Shabnam and T. Shilp
(2012). Nanosilver-A Review. International Journal of
Advanced Pharmaceutics, 2(1):9-15.
Sabet, K.A. ; A.S. Samra and N.A. Dawood (1966a). Combined
Infection with Stalk-Rot Fungi. In: A. S. Samra and K. A. Sabet,
Eds., Investigations on Stalk-Rot Disease of Maize in U.A.R.,
Ministry of Agriculture, Government Printing Offices, Cairo,
pp. 195-204.
16 Egypt. J. of Appl. Sci., 36 (3) 2021
Sabet, K.A. ; A.S. Samra and I.M. Mansour (1966b). Late-wilt of
maize and a study of the causal organism. In: Investigations on
Stalk-Rot Diseases of Maize in U.A.R. Ministry of Agric.,
Egypt, Tech. Bull., pp. 8-45.
Sabet, K.A. ; A.M. Zaher ; A.S. Samra and I.M. Mansour (1970).
Pathogenicbehaviour of Cephalosporium maydis and C.
acremonium. Annu. Appl. Biol., 66(2):257-263.
Saleh, A.A. and J.F. Leslie (2004).CephalosporiummaydisIs a Distinct
Species in the Gaeumannomyces-Harpophora Species Complex.
Mycologia, 96: 1294- 1305.
Saleh, A.A. ; K.A. Zeller ; A.S.M. Ismael ; Zeinab M. Fahmy ; E.M.
El- Assiuty and J.F. Leslie (2003). Amplified fragment length
polymorphism diversity in Cephalosporium maydisfrom Egypt.
Phytopathology, 93:853–859.
Samra, A.S. ; K.A. Sabet and M.K. Hingorani (1963). Late Wilt
Disease of Maize Caused by Cephalosporium maydis.
Phytopathology, 53: 402-406.
Samra, A.S. ; K.A. Sabet ; M.F. Abdel-Rahim; H.A. El-Shafey ; I.M.
Mansour ; F.A. Fadl ; N.A. Dawood and Ikbal H.I. Khalil
(1966). Investigations on Stalk-Rot Diseases of Maize in U.A.R.
Ministry of Agric., Egypt, Tech. Bull., 204 p.
Sawai, J. and T. Yoshikawa (2004). Quantitative evaluation of
antifungal activity of metallic oxide powders (MgO, CaO and
ZnO) by an indirect conductimetric assay. J. Applied Microbiol.,
96: 803-809.
Shende, S. ; A.P. Ingle ; A. Gade and M. Rai (2015). Green synthesis
of copper nanoparticles by Citrus medica Linn. (Idilimbu) juice
and its antimicrobial activity. World J Microbiol Biotechnol,
31:865–873.
Singh, S.D. and B.S. Siradhana (1989). Chemical control of late wilt of
maize induced by Cephalosporium maydis. Indian Journal of
Mycology and Plant Pathology, 19:121-122.
Strout, G. ; D.S. Russell ; P.D. Pulsifer ; S. Erten ; A. Lakhtakia and
W.D. Lee (2013). Silica nanoparticles aid in structural leaf
coloration in the Malaysian tropical rainforest under storey herb
Mapania caudate. Ann. of Botny.112(6): 1141–1148.
Suriyaprabha, R. ; G. Karunakaran ; R. Yuvakkumar ; P. Prabu ;
V. Rajendran and N. Kannan (2014). Effect of silica
nanoparticles on microbial biomass and silica availability in
maize rhizosphere. Biotechnology and Applied Biochemistry,
61(6):668-75.
Suriyaprabha, R. ; G. Karunakaran ; R. Yuvakkumar ; V.
Rajendran ; P. Prabu and N. Kannan (2012). Growth and
Egypt. J. of Appl. Sci., 36 (3) 2021 17
physiological responses of maize (Zea mays. L) treated with
silica nanoparticles in soil. J. Nanopart. Res., 14:1–14.
Yang, L. and D.J. Watts (2005). Particle surface characteristics may
play an important role in phytotoxicity of alumina nanoparticles.
Toxicol. Lett., 158: 122–132.
Yugandhar, P. and N. Savithramma (2015a). Leaf Assisted Green
Synthesis of Silver Nanoparticles from Syzygium alternifolium
(Wt.) Walp. Characterization and antimicrobial studies an
endemic, endangered medicinal tree taxon. Journal Nano
Biomedicine and Engineering, 7(2):29-37.
Yugandhar, P. and N. Savithramma (2015b). Biosynthesis,
characterization and antimicrobial studies of green synthesized
silver nanoparticles from fruit extract of Syzygium alternifolium
(Wt.). Walp. an endemic, endangered medicinal tree taxon. Appl.
Nanosci. http://www.springer.com/materials/nanotechnology/journal/13204
Yugandhar, P. and N. Savithramma (2016). Biological synthesis of
silver nanoparticles from Adansonia digitata L. fruit pulp
extract, characterization, and its antimicrobial properties. J
intercult Ethnopharmacol, 5(1): 79–85
Yugandhar, P. and N. Savithramma (2013). Green synthesis of
calcium carbonate nanoparticles and their effects on seed
germination and seedling growth of Vignamungo (L.). Hepper.,
1(8):89-103.
Yugandhar, P. ; R. Haribabu and N. Savithramma (2015). Synthesis,
characterization and antimicrobial properties of greensynthesised
silver nanoparticles from stem bark extract of
Syzygium alternifolium (Wt.) Walp. 3 Biotech, 5(6): 1031–1039.
Yuvakkumar, R. ; V. Elango ; V. Rajendran ; N. Kannan and P. Prabu
(2011). Influence of Nanosilica Powder on the Growth of Maize
Crop (Zea mays L.). Int. J. Green Nanotechnol., 3: 180–190.
استخدام النانوسيميکا فى مکافحة مرض الذبول المتأخر فى الذرة الشامية
Harpophora maydis المتسبب عن
السعيد محمد الشب ا روى
معيد بحوث ام ا رض النباتات- مرکز البحوث الز ا رعية
تم استخدام جزيئات النانوسيميکا المخمقة نباتيا والمحسنة بالتحضين تحت ارقام مختمفو
2 و 9505 النانومترية لتقييميا ،، 5،7،9 و 11 لانتاج الاحجام 4،02،7،02 pH من الحموضة
المسبب لمرض الذبول المتأخر فى الذرة الشامية Harpophora maydis فى مکافحة الفطر
تحت ظروف المعمل، الصوبة اولحقل 0 باستخدام بيئة الدکستروز اجار، اظيرت النتائج ان
جميع احجام النانوسيميکا تحت الاربع ترکي ا زت المستخدمو خفضت معنويا النمو الخطى لفطر
18 Egypt. J. of Appl. Sci., 36 (3) 2021
وانخفض النمو اکثر بزيادة الترکي زبالمقارنو بالکنترول 0 وکانت نانوسيميکا-، 2 H. maydis
نانوميتر افضل المعاملات تبعيا نانوسيميکا- 4202 نانوميتر 0 اظيرت نتائج تجربة الصوبة ان
التقاوى المعاممة بالنانوسيميکا خفضت معنويا نسبة الاصابة بالذبول المتاخر وحسنت من درجة
0 وکانت المعاممتين نانوسيميکا-، 2 % الانبات بالمقارنة بنسبة الاصابة فى الکنترول 7،08
نانوميت ر اولنانوسيميکا- 4،02 نانوميتر اکثر المعاملات خفضا لدرجة الاصابة
0 بينما کانت المعاممو نانوسيميکا- ppm %8707 عمى الترتيب( تحت ترکيز ، 1 ،%8802(
0 اظيرت تجارب % 9505 نانوميتر اقل المعاملات تاثي ا ر حيث کانت نسبة خفض الاصابة 5209
2،2 ان ،- الحقل المنفذه فى حقول العدوى بالذبول بمحطتى الجيزه والجميزة مواسم 2،19
التقاوى المعاممة بجزيئات النانوسيميکا اظيرت خفضا معنويا فى درجة الاصابة بالذبول المتأخر
بالمقارنة بالکنترول 7803 % اصابة بحقل الجيزة 2،19 و 8107 % بحقل الجميزة ، 02،2 ايضا
کما کانت ىناک اختلافات فى متوسط المحصول لموسمى الز ا رعة باختلاف المعاممة حيث ا زد
المحصول بارتفاع الترکيز 0 وکانت المعاملات نانوسيميکا- ، 2 نانوميتر ونانوسيميکا- 4،02
نانوميتر من افضل المعاملات فى خفض الاصابة وزيادة المحصول، حيث کانت نسبة الاصابة
وکانت فى الموسم الثانى ppm فى المعاممتيين 607 % فى الموسم الاول بالجيزة عن ترکيز ، 1
بالجميزة 607 % نانوسيميکا- ، 2 نانوميتر و 803 % فى نانوسيميکا- 4،02 نانوميتر فى الموسم
الثانى بالجميزه 0 من ناحية اخرى کان متوسط المحصول 29 اردب/فدان فى معاممة نانوسيميکا-
2 نانوميتر و 2703 اردب/فدان فى نانوسيميکا - 4،02 نانوميتر 0 فى المقابل کانت اقل ،
المعاملات ىى نانوسيميکا- 9505 نانوميتر ونانوسيميک ا- 7،02 نانوميتر حيث کانت الاصابة
%34 ومحصول 1706 اردب/فدان بالمعاممة حجم ) 9505 نانوميتر( و بينما کانت نسبة
الاصابة بالمعاممة حجم ) 7،02 نانوميتر( 2603 % ومحصول 18 اردب /فدان فى حقل
الجميزة 0 من النتائج المتحصل عمييا يمکن استخلاص ان استخدام جزيئات النانوسيميکا المخمقو
نباتيا والامنو لمبيئة ومنخفضة التکمفةفى الاحجام الصغيرة ، 2 و 4،02 نانوميتر عن الاحجام
الکبيرة، کانت فعالة فى مکافحة مرض الذبول المتأخر ورفع انتاجية محصول الذرة 0
Egypt. J. of Appl. Sci., 36 (3) 2021 19