Biosynthesis of Chaetomium globosum-made
ZnO@Ag@Chitosan Nanoparticles and their Potential as an Eco-Friendly Bio-Fungicide
against Fungal Root Diseases of Cucumber and Tomato
1Department of Plant Pathology, Agricultural and
Biological Research Institute, National Research Centre, Cairo, Dokki, Egypt
2Department of
Spectroscopy Department, Physics Institute, National Research Centre, Cairo,
Dokki, Egypt
*For correspondence:
wafaa_haggag@yahoo.com
Received 17 June 2022; Accepted 16 August
2022; Published 12 December 2022
Abstract
In vitro and in vivo studies were conducted to
investigate the biosynthesis of ZnO@Ag@Chitosan nanoparticles (NPs) from Chaetomium
globosum as an eco-friendly biofungicide against fungal root rot diseases
of cucumber and tomato caused by Rhizoctonia solani, Sclerotinia sclerotiorum,
Fusarium oxysporum, F. solani, Macrophomina phaseolina, Phytophthora
parasitica, Pythium ultimum, Alternaria alternata and Colletotrichum
gloeosporioides. The physico-chemical characterization using UV–visible
spectroscopy, FTIR and HRTEM proved the formation ZnO@Ag@Chitosan NPs. The
distinct absorbance peak was discovered at 430 nm. Using Fourier transform
infrared (ATR-FTIR) spectroscopy, the extent vibrations of chitosan at 3431 cm-1.
In comparison to the crude metabolites of C. globosum, ZnO@Ag@Chitosan
nanoparticles from C. globosum inhibited the fungal growth of all tested
pathogens at low concentrations. All antioxidant activities of ZnO@Ag@Chitosan,
i.e., reducing power,
scavenging of DPPH [1,1-Diphenyl-2-picrylhydrazyl] radical, scavenging of
nitric oxide and ABTS free radical scavenging activities were increased. The
hematological alanine aminotransferase, serum aspartate aminotransferase, and
creatinine concentrations of white albino ZnO@Ag@Chitosan NPs were
investigated, showing that the NPs were extremely safe. In the greenhouse,
ZnO@Ag@Chitosan nanoparticles reduced the incidence of root diseases in tomato
and cucumber roots after treating them with 500 mg L-1 suspension of
ZnO@Ag@Chitosan NPs and increased crop yield. At the same time, higher levels of
peroxidase, polyphenoloxidase and chitinase were detected. In addition,
photosynthetic pigments such as chlorophyll a and b and carotenoids were also
increased. This study concludes that nanoparticle ZnO@Ag@Chitosan synthesized
from C. globosum metabolite is effective as an eco-friendly biofungicide
against soil-borne fungal pathogens. © 2022 Friends Science Publishers
Keywords: Antioxidant activities; Biofungicides; Chaetomium
globosum; Chitosan;
Cucumber; Nanoparticles;
Root diseases; Tomato
Introduction
In Egypt and many
other nations around the world, major soil and seeds borne plant pathogens such
as Fusarium oxysporum, F. solani,
Sclerotinia sclerotiorum, Phytopthora parasitica, Pythium ultimum, Rhizoctonia solani, Colletotrichum gloeosporioides (El-Kaed et
al. 2021), Macrophomina phaseolina (Javed et al. 2021),
Alternaria alternata (Javaid and Samad 2012) and Sclerotium rolfsii (Javaid
and Khan 2016) cause various deadly and destructive diseases.
Plant diseases are being controlled by beneficial microorganisms like
plant growth promoting rhizobacteria (PGPR), Trhichoderma spp. and Penicillium
spp. as a secure strategy for agricultural and environmental sustainability
(Haggag et al. 2015; Sharf et al. 2021; Khan and Javaid 2020,
2022). C. globosum can control Fusarium species, Phytophthora species,
C. gloeosporioides, and other plant diseases (Moya et al. 2016).
Compared to commercial agrochemicals, agriculture uses of nanotechnology are
expanding across a variety of fields, including nutrition, pathogen detection
and disease control (Haggag et al. 2018). Nanomaterials are used to
create biofungicides, which have been shown to have antibacterial, antifungal,
and antiviral properties against phytopathogenic microbes in vitro, in a
greenhouse, and in the field (Farhat et al. 2018; Haggag et al.
2018; Um-e-Aiman et al. 2021). Scientists are interested in surface
plasmon (SP), which is produced when a metal nanostructure and a dielectric
interact. Among its many uses are plasmon lasers and enhancing the light
absorption and Raman scattering intensity of materials close to its surface (Okamoto
et al. 2019a). Recent significant advancements in the luminescence
intensity and efficiency of light-emitting materials and systems have made
SP-mediated emission a popular research topic. By capping the Ag layer, Okamoto
et al. (2019b) showed a significant PL enhancement of InGaN/GaN quantum
wells, demonstrating the significance of SPs-mediated emission in raising LED
efficiency (Lu et al. 2011). Zinc oxide nanoparticle production methods
have been described by a number of organizations. These methods include
hydrothermal synthesis, alkali precipitation, thermal breakdown, organo-zinc
hydrolysis and more. In a 120°C autoclave, Yang et al. (2002) used a
CTAB-aided hydro-thermal ZnO nano-wire. ZnO nanorods and prisms were produced
by Wang et al. (2006) using zinc foil, NaOH and CTAB at a temperature of
160°C. According to Fageria et al. (2014), gold nanoparticles on the
surface of ZnO can be used to achieve photocatalysis. Zheng et al.
(2007) used a solvothermal method to create Ag/ZnO heterostructures with
variable silver content for use in photocatalysis. Zhou et al. (2011)
conducted research on ZnO's morphology and application in photocatalysis. The
polymer-based methodology is one of the many synthetic techniques used to
create metallic nanoparticles, and it has garnered a lot of interest. There are
several simple processes for creating silver nanoparticles that use chitosan as
a mediator (Hajji et al. 2019; Haggag and Eid 2022). A polycationic
amino polysaccharide, chitosan (CS) is derived from the chitin present in fungi
and crustacean shells. Due to the appealing properties brought on by the
presence of functional groups (amino and hydroxyl), this biocompatible and
biodegradable polymer has a wide range of applications in biology (Eid et
al. 2019; Kutawa et al. 2021). In this
study, ZnO@Ag@Chitosan nanoparticles were biosynthesized from an active
metabolite of C. globosum, and their antifungal, antioxidant, and
biosafety activities were assessed. Additionally, their potential as an environmentally
friendly biofungicide to lessen diseases brought on by soil-borne pathogens of
tomato and cucumber in greenhouse conditions was assessed.
Materials and
Methods
Soil-borne fungal
pathogens i.e., F. oxysporum,
F. solani, S. sclerotiorum, R. solani, M. phaseolena,
P. parasitica, P. ultimum, A. alternata and C. gloeosporioides were
isolated from tomato and cucumber diseased plants grown in greenhouses in
Boheria Governorate, Egypt and identified in department of Plant Pathology,
National Research Centre, Egypt. We used Chaetomium globosum,
which had previously been isolated from healthy tomato plants and identified in
the Plant Pathology Department of the National Research Centre. For 10 days,
fungal cultures were grown in potato dextrose agar (PDA) plates at 25°C. The
culture was identified according to Arx et al. (1986).
Green synthesis of
Ag@cs by C. globosum
For 7 days, C.
globosum was grown on potato dextrose medium at 28°C and pH 7. The broth
medium was filtered with filter paper and a 100 mL solution (79 mL filtrate
plus 21 mL distilled water) was mixed at room temperature with Silver Nitrate
(0.1) and adjusted to pH 9 with 1 N HCl and 10 M NaOH. The silver formation was detected using UV-vis absorption
analysis at 400 nm (Osman et al. 2016).
Zinc
oxide preparation
At 60°C, 5 g of zinc
acetate were dissolved in 100 mL of distilled water. In a separate pot, 4 g
NaOH was dissolved in 100 mL distilled water and dropped onto the zinc acetate
solution dropwise. Then, the pellet was washed with filtrated and distilled water,
dried at 60°C for 24 h, calcined at 400°C for 4 h and centrifugation at 10000
rpm at 10°C for 20 min (Zhang et al. 2009).
Preparation of
Ag@zno@cs (core/shell)
Ag@CS (2 g) was
mixed with zinc oxide (2 g), ground for 15 min to get a fine powder, dispersed
in distilled water, then centrifuged at 10000 rpm for 20 min at 10°C and the
pellet was dried at 50°C for 24 h (Vijayakumar et al. 2013).
Characterization of
ZnO NPs, Ag@CS NPs, Ag@ZnO@CS NPs
A UV–vis
spectrophotometer (JASCO, V-530) with a 400 nm resolution was used. ATR-FTIR
spectra were measured using a Perkin Elmer FTIR spectrophotometer equipped with
a TGS detector and 2 mg of each sample to depict the functional groups
responsible for metallic ion reduction. The spectra were captured at a resolution
of 4.0 cm-1 and 64 scans were combined to achieve a reasonable
signal-to-noise ratio. The dried extract powders were tested in parallel. The
crystalline structure of the developed phases was examined. The Cu K radiation
(1.5418) and a scanning speed of 0.3 S were used on the Bruker XRD X-ray powder
diffractometer. The operating voltage and current were both set to 40 kV and 40
mA.
The zeta
potential of core-shell nanoparticles was measured using a Zetasizer 1000 at
633 nm at a fixed refractive index of the respective formulation to assess
their stability. At pH 7.0, the size of Ag@ZnO@CS nanoparticles (NPs) was
measured using a particle size analyzer (PSA: Malvern Zeta Sizer Nano ZS).
In vitro bioassay test
Agar well diffusion assay: The antifungal activity of ZnO@Ag@Chitosan NPs using agar well
diffusion assay at concentrations of 50, 250 and 500 mg L-1 were
tested against fungal growth of F. solani, F. oxysporum, R.
solani, M. phaseolena, S. sclerotiorum, P. parasitica,
P. ultimum, A. alternata and C. gloeosporioides
(Hongtao et al. 2013).
Crude metabolite of Chaetomium was prepared by
grown on PDB medium for 21 days at room temperature (25–30°C). Biomass was
filtrate, dried at room temperature for 7 days and crushed into fine powder using
grinder. Metabolite was extracted using successive solvents of ethyl acetate
(1:1 v/v) and methanol (1:1 v/v) (Bhardwaj et al. 2015). Solvent was
evaporated and the residues compound was dried in rotator vacuum evaporator to
harvest the crude metabolite.
Crude
extract metabolite of C. globosum was tested against pathogens using
concentrations of 0, 50, 250 and 500 mg L-1. Crude metabolite was
dissolved in dimethyl sulfoxide (2%). Colony diameter (mm) was measured after
five days using five replicates for each treatment. As a control, carbendazim
50% wettable powder (WP) was used at
recommended dose.
Antioxidant assay
Reducing
power: The percentage
inhibition was determined using an Oyaizu (1986). The Ag@ZnO@CS NPs and
metabolite (0.2–1.0 mg mL-1) were mixed with 2.5 mL of water and
methanol, 2.5 mL of sodium phosphate buffer (200 mM; pH 6.6) and 1% potassium ferricynide (2.5 mL) and incubated for
20 min at 50°C. Following that, 2.5 mL of 10% trichloroacetic acid was added
and centrifuged at 200 g for 10 min. The upper layer (2.5 mL) was mixed with
deionized water and 0.5 mL of 0.1% ferric chloride. A spectrophotometer set to
700 nm was used to measure absorbance against a blank. Assay for
diphenyl-scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH). Hu et
al. (2014) described a method for measuring the activity of produced
Diphenyl 1 picrylhydrazyl (DPPH) via
ZnO@Ag@Chitosan NPs. The sample solution (10 mg mL-1) was combined
with DPPH in ethanol (180 mol L-1) and incubated with water at 25°C
for 30 min. The absorbance was evaluated with a UV–visible spectrometer at 517
nm, with three duplicates for each sample (the control was distilled water
instead of DPPH). The scavenging effect was calculated by the following
equation: DPPH+ scavenging activity (%) = [(Abs Control – Abs Sample)]/(Abs
Control)]x100 where Abs Control was the absorbance of DPPH+ + methanol; Abs
Sample is the absorbance of DPPH+ + sample extract /standard.
Scavenging activity for nitric oxide: The ability of nitric oxide to scavenge free radicals was investigated
(Marcocci et al. 1994). 5.0 mL of reaction mixture including or not
containing ZnO@Ag@Chitosan NPs and crude metabolite was combined with sodium nitroprusside
(SNP) (5 mM), phosphate buffered
saline at pH 7.3 and incubated at 25°C for 180 min. Three replicates were used
to test nitric oxide scavenging at 540 nm using conventional sodium nitrite
salt solutions.
Free radical scavenging activity (ABTS•+) assay: The ABTS free radical scavenging assay was performed using the Zhishen et
al. (1999) method. The reaction mixture of 2.5 mM ABTS (2,2'-azino-bis [3-ethylbenzthiazoline-6-sulfonic acid]
diammonium salt) and 1.0 mM AAPH
(2,2'-azobis-[2-amidinopropane] HCl) in 100 mL phosphate-buffered saline
solution (100 mM potassium phosphate
buffer at pH 7.4 containing 150 mM
(Whatman Inc., Florham Park, NJ). At 734 nm and 37°C, the absorbance was
measured using a microplate reader.
Biosafety and toxicity studies
Tested animals: Female albino rats Rattus
norvegicus var. albinos (weighing 110–120 g) were employed in this experiment.
The rats were kept at a constant temperature of 25°C and were subjected to a
daily dark/light cycle. After two days, rats were randomly separated into five
groups and treated with ZnO@Ag@Chitosan NPs and crude metabolites for a month.
The final group was given carbendazim 50% WP (Anonymous 2012). The animals were
slaughtered after 30 days of therapy, and blood samples were taken to assess
the parameters of interest. For the purpose of evaluating toxicological
consequences, rats were monitored on a daily basis.
Hematological tests
Blood samples were collected in anticoagulant tubes
after 45 days to measure hematological parameters; Red blood cells (RBCs),
white blood (WBC) counts and hemoglobin values were determined using the Schalm
(1986) method.
Biochemical analysis
After 45 days of recuperation, blood samples were
obtained in sterile tubes and centrifuged at 3500 rpm for 20 min to separate
the serum. A spectrophotometer set to 400 nm was used to assess the activities
of blood serum transaminases such as aspartate transaminase (AST), alanine
transaminase (ALT), urea and creatinine. For each sample, five replicates were
employed.
Biofungicidal effect of Zno@Ag@chitosan nanoparticles
under greenhouses
During the 2020–2021
seasons, the effects of ZnO@Ag@Chitosan NPs as bio fungicides on lowering root
rot and wilt disease severity in tomato and cucumber were studied in commercial
greenhouses under naturally infected conditions in El Bahera Governorate,
Egypt. We employed 500 mg L-1 concentrations of ZnO@Ag@Chitosan NPs
and crude metabolite. Tomato (Lycopirsecon esculentum cv. Bigdena F1)
and cucumber (Cucumis sativus cv. Rada F1 hybrid) transplanting were
coated for 4 h with ZnO@Ag@Chitosan NPs and surfactant. Control treatments included crude C. globosum metabolite at an effective
concentration, sterile distilled water, and fungicide (carbendazim 50% WP).
During the 2020 and 2021 growing seasons in Giza governorate, experiments were
conducted using a randomized full block design with twenty replications.
Agricultural practices were followed to the letter.
Disease assay and
total yield
During growth
periods, the symptoms of illnesses such as root rot, stem cankers and wilt were
measured. The total accumulated yield of tomato and cucumber for each treatment
was reported at the harvest stage. Twenty plants were taken from each treatment
to determine the overall yield per plant (kg plant-1).
The determination of
the activities of oxidative enzymes
The effects of
ZnO@Ag@Chitosan NPs and crude metabolite treatments on the activities of
oxidative defense enzymes, such as peroxidase, polyphenoloxidase and chitinase,
in tomato and cucumber plants grown in greenhouses were measured 60 days after
transplanting. Leaf samples (g) were homogenized in 0.2 M Tris HCl buffer (pH 7.8) containing 14 mM mercaptoethanol at a rate of 1/3 w/v at 0°C. A UV
spectrophotometer was used to measure the enzyme activities. According to Lee's
method, peroxidase activity was measured as an increase in absorbance at 470 nm/g fresh
weight/minute (Lee 1973). Polyphenoloxidase
activity was measured as an increase in absorbance at 475 nm g-1 fresh weight min-1 using
the Bashan et al. (1985) method. Chitinase activity was measured as mM N-acetyl
glucose amine equivalent released/g fresh weight/60 min at 540 nm
(Monreal and Reese 1969).
Estimation of photosynthetic
pigments
The amount of
photosynthetic pigments, such as chlorophyll a (chl a), chlorophyll b (chl b),
and carotenoids, was measured in fresh leaves (mg g 1 of fresh weight) (Königer
and Winter1993).
Statistical analysis
Duncan's multiple
range test was performed to compare means at P 0.05 levels and ANOVA was
utilized to analyze the efficiency of all trials (Duncan 1955). P =0.05 was
used as the significant level. The least significant difference (LSD) test was
used to compare the means of all five treatments. The STATISTICA-SP statistical
software application was used to create similarity coefficients based on
pairwise comparisons of treatments based on the existence (1) or absence (0) of
unique and shared polymorphism products.
Results
Synthesis and
characterization of ZnO@Ag@Chitosan nanoparticles
The biogenesis of
ZnO@Ag@Chitosan NPs was studied using UV-visible absorption. The biosynthesis
was carried out in two steps: first, the reduction of Ag+ to atoms
in the presence of the C. globosum fungus in one pot, then the formation
of Zn-OH and subsequently calcination to ZnO in another pot, and finally, the solid-state
approach to make ZnO@Ag@Chitosan NPs in a third pot. The density of the
yellowish-brown color increases as the surface plasmon resonance of Ag NP is
activated. UV-vis spectroscopy in aqueous solution could be used to analyze
size-and shape-controlled NPs. The unique absorbance peak in the UV-vis
spectrum of ZnO@Ag@Chitosan NPs produced by Fungus was discovered at 430 nm
(Fig. 1). Using ATR-FTIR, the stretch vibrations of OH groups were
discovered to correspond with the peaks of chitosan at 3431 cm-1.
The band at 1635 cm-1 has been linked to amide I because proteins
have carbonyl extensions. The peak at 1390 cm-1 indicates the symmetric
deformation vibration mode of CH3, while the peak at 1274 cm-1 shows
carboxylic acid C-O expansion vibration. The peak of Zn-O of zinc oxide (ZnO)
occurs around 490 cm-1 in Fig. 2. The zeta potentials of ZnO and
Ag@ZnO are -4.85 and 22.27 mV, respectively, as shown in Fig. 2. As illustrated
in Fig. 3, the HRTEM was utilized to create ZnO, Ag, and ZnO@Ag@Chitosan NPs.
In vitro bioassay
Agar well
diffusion assay: In vitro antifungal activity of ZnO@Ag@Chitosan NPs and crude
metabolite against Fusarium oxysporum, Fusarium solani, Sclerotiorum
sclerotiorum, R. solani, M. phaseolena, P. parasitica, P. ultimum, A. alternate
and C. gloeosporioides is shown in Table 1. The results revealed a
distinction between the regimens. ZnO@Ag@Chitosan NPs showed the highest
activity when compared to crude metabolite and untreated control. When compared
to Carbendazim 50% WP at a concentration of 150 ppm, R. solani (33.6
& 34.7 mm) and F. oxysporum (30.6 & 35.7 mm) showed the greatest
growth inhibition at 250 and 500 mg/l, respectively. ZnO@Ag@Chitosan NPs are
also effective against M. phaseolena (32.6 & 33.7 mm) and C.
gloeosporioides at 250 and 500 mg L-1 (29.7 & 32.7 mm).
Antioxidant
assay of ZnO@Ag@Chitosan NPs by C. globosum
Reducing
power activity: Activity of ZnO@Ag@Chitosan NPs obtained reducing power
which significant increased from (1.5) at 50 mg L-1, (2.8) at 250 mg
L-1 and to (3.9) at 500 mg L-1concentrations when
compared to crude metabolite of C. globosum at 500 mg L-1 (0.87)
(Table 2).
Table 1: Agar well diffusion assay of test plant pathogens with ZnO@Ag@Chitosan
NPs and crude metabolite of C. globosum
Plant
Pathogens |
Inhibition
(mm) |
||||||
Carbendazim
50% WP (150 ppm) |
Crude
metabolite |
ZnO@Ag@Cs
NPs |
|||||
50 mg L |
250 mg L |
500 mg L |
50 mg L-1 |
250 mg L |
500 mg L |
||
F.
oxysporum |
36.2 a |
05.2 c |
10.2bc |
22.7a |
25.2 a |
30.6 c |
35.7 a |
F. solani |
33.2 bc |
04.6d |
09.5d |
20.6c |
23.6 b |
25.6 e |
31.1 bc |
S.
sclerotiorum |
27.2 ef |
05.6b |
09.6d |
20.7c |
24.5 ab |
28.6 d |
32.6 b |
R. solani |
30.1 d |
06.6 a |
11.6a |
21.6 b |
25.6 a |
33.6 a |
34.7 a |
M. phaseolena |
31.7 d |
06.3 ab |
09.4d |
21.4 b |
24.8 ab |
32.6 b |
33.7 ab |
P. parasitica |
34.7 b |
05.5bc |
09.4d |
19.6 cd |
23.6 b |
29.7 c |
31.7 bc |
P. ultimum |
28.7 e |
04.4de |
10.4b |
18.6 d |
22.7 c |
27.8 d |
29.7 d |
A. alternata |
25.6 g |
04.5d |
10.5 b |
18.9 d |
23.7 b |
26.9 de |
28.6 d |
C.
gloeosporioides |
30.2 d |
05.5bc |
10.5b |
21.7 b |
25.8 a |
29.7 c |
32.7 b |
Table 2: Antioxidant activity
of ZnO@Ag@Chitosan nanoparticles produced from C. globosum
Antioxidant
activity |
Crude
metabolite |
ZnO@Ag@Cs
NPs |
||||
50 (mg L) |
250 (mg L) |
500 (mg L) |
50 (mg L) |
250 (mg L) |
500 (mg L) |
|
Reducing
power assay |
0.04 d |
0.21 d |
0.87 d |
1.5 d |
2.8 d |
3.9 c |
DPPH
radical scavenging activity (%) |
0.05 b |
0.67 c |
2.20 c |
11.0 c |
32.8 c |
77.5 b |
Nitric
oxide scavenging assay |
0.09 a |
0.98 a |
32.2 a |
71.9 a |
86.0 a |
89.4 a |
ABTS free
radical scavenging assay % |
0.08 c |
0.86 b |
13.5 b |
36.5 b |
55.2 b |
75.1 b |
Values are
means of five replications. (c) ANOVA was used to analyses the efficiency of
all trials, and Duncan's multiple range test was used to compare means at P = 0.05 levels
Fig. 1: UV-visible spectra of a) ZnO@Ag@CS and ATR-FTIR b) for C. globosum
Fig. 2: Zeta potential of
ZnO and ZnO@Ag synthesized from C. globosum
Diphenyl‑1‑picryl hydrazyl (DPPH) activity: DPPH radical was utilized to evaluate the scavenging activity of
ZnO@Ag@Chitosan NPs. percentage of (11.0) at 50 mg L-1 (32.8) at 250
mg L-1 and (77.5) at 500 mg L-1when compared to crude
metabolite of C. globosum at 500 mg L-1 (2.20) (Table 2).
Nitric oxide
scavenging assay: When compared
to crude
metabolite of C. globosum, the ZnO@Ag@Chitosan
NPs demonstrated scavenging activity that
reach to (71.9) at 50 mg L-1 (86.0) at 250 mg L-1
and (89.4) at 500 mg L-1 when compared
to crude
metabolite of C. globosum at 500 mg L-1 (32.2)
concentrations (Table 2).
ABTS free radical scavenging activity: ZnO@Ag@Chitosan NPs demonstrated scavenging activity with increasing concentrations,
reaching (36.5) at 50 mg L-1 (55.2) at 250 mg L-1 and (75.1) at 500 mg L-1when compared to crude metabolite of C. globosum at 500 mg L-1 (13.5) (Table 2).
There is no difference in rats body weight treated with ZnO@Ag@Cs NPs
at 500 mG/L (188.9 G) compared to crude metabolite at 500 mg L-1 (186.2g)
and fungicide, according to Fig. 4. When compared to the untreated control,
fungicide produced a decrease in rat body weight (176.7 g) after 45 days (185.0
g). Haemoglobin levels are also significantly lower in rats treated with a
chemical fungicide (11.13) compared to ZnO@Ag@Cs NPs (11.84) crude metabolite (11.81)
and untreated animals (11.89). The most effective concentration (500 mg L-1)
of both crude extract metabolite and ZnO@Ag@Cs NPs was employed in the bioassay
test and antioxidant bioassay.
There is no difference in rats body weight treated with ZnO@Ag@Cs NPs at 500 mg/L (188.9 G) compared to crude metabolite at 500
mg L-1 (186.2G) and fungicide, according to Fig. 4. When compared to
the untreated control, fungicide produced a decrease in rat body weight (176.7
g) after 45 days (185.0 g). Haemoglobin levels are also significantly lower in
rats treated with a chemical fungicide (11.13) compared to ZnO@Ag@Cs NPs
(11.84) crude metabolite (11.81) and untreated animals (11.89). When compared
to crude metabolites of Chaetomium and ZnO@Ag@Cs NPs,
fungicide, and control group (27.5, 28.0, 36.6, and 26.6 mg dL-1,
respectively), fungicide significantly increased aspartate transaminase enzyme (AST)
activity in treated rats.
Fig. 3: HRTEM of a) ZnO NPs,
b) Ag NPs,c) ZnO@Ag@CS NPs by C. globosum
There were no significant changes in creatinine concentrations of crude
metabolite and ZnO@Ag@Cs NPs (0.90 and 0.89 mg dL-1, respectively)
as compared to chemical fungicide (0.93 mg dL-1), which caused a
significant increase in creatinine concentration, and untreated control (0.87
mg dL-1). The same results were obtained for urea concentration,
with no significant difference between crude metabolite and ZnO@Ag@Cs NPs (33.9
and 33.3 mg dL-1, respectively) and the fungicide (34.8 mg dL-1),
which caused a large increase in urea, and the untreated control (31.7 mg dL-1).
Field application
Disease assay: Under natural infection condition of greenhouses, treated tomato and
cucumber transplanting with ZnO@Ag@Chitosan NPs biosynthesis from C.
globosum and its crude metabolite at 500 mg L-1 significantly
reduced the root-rot, stem canker and wilt diseases in compared with chemical
fungicide and untreated control (Table 3). The most effective treatment was
ZnO@Ag@Chitosan NPs at 500 mg L-1, which reduced root rot, stem
canker, and wilt illnesses by 2.3, 1.0, and 2.7% in tomato plants and 3.0, 0.3
and 2.1% in cucumber plants, respectively, when compared to untreated controls.
Tomato plants had 12.7, 9.5 and 25.6% yields, while Table 3: In greenhouses, potential effect of ZnO@Ag@Chitosan NPs biosynthesis from C. globosum and
its crude metabolite on root rot and wilt
diseases of tomato (A) and cucumber (B) transplanting’s
Cucumber |
Tomato |
Treatment |
||||||
yield (kg
Plant-1) |
Wilt |
Stem canker |
Root rot |
Yield (kg
Plant-1) |
Wilt |
Stem
cankers |
Root rot |
|
7.7 b |
8.8 a |
1.8 b |
6.6 b |
6.4 ab |
6.4 b |
3.1 b |
6.5 ab |
Crude
metabolite |
8.6 a |
2.1 c |
0.3 d |
3.0 c |
7.7 a |
2.7 d |
1.0 c |
2.3 d |
ZnO@Ag@Ch NPs |
6.7 bc |
3.0 c |
0.9 c |
4.0 c |
5.6 b |
5.6 c |
1.4 c |
5.0 bc |
Fungicide |
5.1 d |
24.7 a |
5.6 a |
15.0 a |
4.6 c |
25.6 a |
9.5 a |
12.7 a |
Untreated
control |
Values are means of twenty replications. (c) ANOVA was
used to analyses the efficiency of all trials, and DMR test was used to compare
means at P = 0.05 levels
Fig. 4: Histological study,
white and red blood cell counts, ALT and AST activities and Creatinine
concentrations of white albino treated with ZnO@Ag@Chitosan NPs and crude cell
free culture of C. globosum. Different letters indicate significant
differences among treatments according to the least significant difference test
(P = 0.05); means of standard
deviations for five animals per treatment are shown
cucumber plants had
15.0, 5.6 and 24.7% yields. The disease incidence in treated tomato and
cucumber plants was 6.5, 3.1, and 6.4 in tomato and 6.6, 1.8, and 8,8 in
cucumber plants, respectively, and the fungicide was 5.0, 1.4 and 5.6 in tomato
and 4.0, 0.9 and 3.0 in cucumber. The other treatments were only somewhat
successful. Treated plants with ZnO@Ag@Chitosan NPs significant increased yield
being (7.7 kg plant-1) of tomato and (8.6 kg plant-1) of
cucumber in compared with the crude metabolite at 500 mg L-1 by (6.4
and 7.7 kg plant-1) and untreated control (4.6 and 5.1 kg plant-1),
respectively (Table 3).
Determination of the
activities of oxidative enzymes
The impact of ZnO@Ag@Chitosan NPs at 500 mg L-1 treatments on
the activities of oxidative defense enzymes such as peroxidase,
polyphenoloxidase, and chitinase in tomato and cucumber plants cultivated in
greenhouses was
Fig. 5: In greenhouses, potential effect of ZnO@Ag@Chitosan NPs biosynthesis from C. globosum and its crude
metabolites on oxidative defense enzymes, i.e.,
peroxidase, polyphenoloxidase and chitinase of
(A) tomato and (B) cucumber. Different letters indicate significant
differences among treatments according to the least significant difference test
(P = 0.05); means of standard
deviations for twenty plants per treatment are shown
assessed 60 days
after transplanting (Fig. 5). When compared to the control and fungicide, the
activities of oxidative defense enzymes, such as peroxidase, polyphenoloxidase,
and chitinase, were considerably elevated in all treatments of two crops,
cucumber and tomato. When cucumber was treated with ZnO@Ag@Chitosan NPs, the
activities of peroxidase, polyphenoloxidase and chitinase were 0.8, 0.97, and
1.8, respectively and in tomato, 1.6 mg of total protein, compared to untreated
control (0.35, 0.4 and 0.6 mg of total protein) and (0.3, 0.4 and 0.5 mg of
total protein). Other treatments recoded moderate activities of all enzymes
activities.
Estimated pigment
contents
ZnO@Ag@Chitosan NPs
at 500 mg/l significantly increased chlorophyll a, b, and carotenoids in tomato
and cucumber plants, respectively, with 4.3, 3.9 and 2.7 (FW) (mg g−1 FW) for tomato
and 4.4, 3.0 and 2.8 (FW) (mg g−1
FW) for cucumber, compared to the untreated control. Cucumbers have 3.3,
1.5 and 1.9 (FW) (mg g−1 FW),
while tomatoes have 3.0, 1.2 and 1.8 (FW) (mg g−1 FW) (Fig. 6).
The treatment of crude metabolite at 500 mg L-1 was relatively
effective in raising chlorophyll a, b and c when compared to carotenoids, which
were 3.8, 2.0 and 2.1(FW) (mg g−1 FW) in tomato
and 3.9, 2.9 and 2.4 (FW) (mg g−1 FW) in
cucumber. The outcomes were less successful in the fungicide and untreated
control groups.
Discussion
This study suggested
an eco-friendly biofungicide to compact and manage fungal root rot diseases of
cucumber and tomato caused by F. oxysporum, F. solani, S. sclerotiorum, R.
solani and M. phaseolena. The finding was confirmed in vitro using green biosynthesised
ZnO@Ag@Chitosan NPs from C. globosum. Green nanotechnology is a novel
pesticide alternative that is based on the natural synthesis of nanoparticles
from plant and microorganisms (yeast, bacteria, algae, fungi and so on) using
environmentally friendly procedures (Karn and Wong 2013; Shah et al.
2015).
UV–visible spectroscopy was used to
investigate the biosynthesis of ZnO@Ag@Chitosan NPs generated by C. globosum.
The peak of zinc oxide (ZnO) is around 490 nm. ZnO and Ag@ZnO zeta potentials
were found to be -4.85
Fig.
6: In greenhouses, potential effect of ZnO@Ag@Chitosan NPs biosynthesis from C. globosum and its crude
metabolites on chlorophyll a and b and carotenoids
of (A)
tomato and (B) cucumber
Different letters indicate significant differences among
treatments according to the least significant difference test (P = 0.05); means of standard deviations
for twenty plants per treatment are shown
and 22.27 mV, respectively (Samuel et
al. 2018). The size of spherical nanoparticles in Ag@ZnO@Cs drops
considerably due to quantum size confinement and the presence of chitosan in
the core-shell (Eid et al. 2019).
Due
to a wide range of many secondary metabolite contents such as polysaccharides,
polyphenols, chitosan, alkaloids, antioxidants, and flavonoids, green
nanotechnology has the ability to set bio-nanoparticles (Manikandan et al.
2020). The interactions between plants, nanomaterials, microbes, endophytes,
and pathogenic fungus have been studied extensively (Farhat et al. 2018;
Haggag et al. 2018; Kalaivani et
al. 2018; Haggag and Eid 2022). To attain food security, boost crop yield, and reduce
pesticide use, Bahrulolum et al.
(2021) employed microorganisms in the green manufacture of metal nanoparticles.
The
antioxidant activity of ZnO@Ag@Chitosan NPs was determined using the reducing
power, DPPH, nitric oxide scavenging, and ABTS assays, which are commonly used
to investigate the radical scavenging ability of green produced NPs. The
antioxidant activity of ZnO@Ag@Chitosan NPs was determined using DPPH, nitric
oxide scavenging and the ABTS assay. Biomaterials comprising Ag@Cs NPs and
chitosan, according to a prior study, have a high potential for suppressing
harmful bacteria (Haggag and Eid 2022).
This
suggests that ZnO@Ag@Chitosan NPs have an inhibitory effect on fungal pathogens
in plants. The MtNPs’ small size and unique physical and chemical properties
make them an excellent material in this scenario. Since harmful bacteria are
highly inhibited by biomaterials containing ZnO@Ag and chitosan NP.
The
potential health benefit of ZnO NPs, which have significant antibacterial and
antioxidant activity (Safawo et al. 2018), has been a crucial factor.
Biopolymer chitosan has a lot of potential in biomedical sectors because of its
biocompatibility, biodegradability, non-toxicity, high permeability,
antioxidant, and antibacterial characteristics (Kalaivani et al. 2018;
Hajji et al. 2019). Mousa et al. (2015), who researched the
toxicity of nanopesticide on mice and found it to be less harmful than chemical
pesticide, confirm that ZnO@Ag@Cs NPs is a non-toxic pesticide when compared to
chemical pesticide. Nanosized silica-silver particles completely controlled
powdery mildew disease in cucurbits growing in the field after 21 days of
treatment (Park et al. 2006). In general, nano fungicides can be
utilized to lessen chemical pesticides' environmental and human impacts (Farhat
et al. 2018; Haggag et al. 2018;
Haggag and Eid 2022). One of the most often utilized metal oxide nanoparticles
is zinc oxide nanoparticles (ZnO-NPs) (Haggag and Eid 2022).
The
qualities of NPs, such as size, surface properties and characteristics,
solubility, chemical reactivity, physical properties and interactions between
nanoparticles and biomolecules in vivo,
may affect their safety (Bahrulolum et al.
2021). The cytotoxicity of NPs was linked to the surface charge of metals,
according to Hu et al. (2009). The majority of prior research has found
that the cytotoxic and genotoxic effects of Ag NPs are proportional to their
size and dosage. Field testing revealed that Chetomium's biosynthesis of
ZnO@Ag@Chitosan NPs has biofungicidal capability against soil-borne fungal
infections.
The prevention of growth and development was related to the antifungal
impact of ZnO@Ag@Chitosan NPs. In addition, when compared to fungicide and
control plants, NP application resulted in improved antimycotic effects and
plant development. When the chemical fungicide was administered under the same
conditions, the outcomes were virtually invariably the same. Farhat et al.
(2018) discovered that biocontrol agents were more successful in controlling
powdery mildew disease and wheat plant growth when they produced silicon and
titanium nanoparticles. Chitosan and metal nanoparticles (ZnO@Ag@Chitosan) are
more effective at inhibiting the growth of numerous microorganisms than
chitosan and metal nanoparticles alone due to their small size, high
permeability, biocompatibility, and biodegradability.
As a result, under greenhouse conditions, the ZnO@Ag@Chitosan NPs are
likely to be effective in pathogen growth. Similarly, Haggag and Eid (2022)
discovered that Streptomyces aureofaciens synthesis of
Ag@FeO-NPs@Chitosan was effective against several soil-borne fungi, including F.
oxysporum, F. solani, R. solani and air-borne pathogenic fungi, including Botrytis
cinerea, C. gloeosporioides, Alternaria solani, More than 20
pathogenesis genes have been reported to be suppressed by chitosan, including
defense enzymes peroxide, polyphenol oxidase, -1,3-glucanase, lignification,
chitinase, -gluconase, plant metabolism-related genes, antioxidant, antifungal
and antimicrobial (Kalaivani et al. 2018; Farhat et al. 2018;
Hajji et al. 2019).
AgNPs were used by Jo et al. (2009) to protect tomato plants
from fungal pathogens while also increasing plant growth. These in vivo studies revealed that the
biofungicidal potency of ZnO@Ag@Chitosan NPs could result in the formation of
shielding effects of ZnO@Ag@Chitosan NPs around the seedlings' root, acting as
a barrier that prevented pathogens from entering the root, colonizing it, and
causing disease symptoms. Simultaneously, chitosan can increase the amount of
chlorophyll in the body.
Conclusion
Endophytes fungi C.
globosum have the potential to be used for the green biosynthesis of
ZnO@Ag@Chitosan NPs. The synthesize ZnO@Ag@Chitosan NPs proved antifungal
activity against plant pathogens i.e., R. solani, F. oxysporum, F. solani, S. sclerotiorum, M.
phaseolena, P. parasitica, P. ultimum and have antioxidant activity. White
albino haematological aspartate transaminase (AST), alanine transaminase (ALT) and
creatinine concentrations demonstrated that ZnO@Ag@Chitosan NPs are safe when
compared to fungicide. Furthermore, ZnO@Ag@Chitosan NPs nanoparticles are
effective and environmentally friendly as nano-biofungicides in natural
greenhouses and can help by reducing root rot, stem canker, and wilt diseases
of tomato and cucumber, significantly increasing the activities of oxidative
defense enzymes such as peroxidase, polyphenoloxidase and chitinase and
photosynthesis concentrations i.e.,
chlorophyll a, b and carotenoids.
This research was funded
by the National Research Centre Fund, Egypt, Grant No.12050131 under title: Development
and Large-Scale Fermentation Manufacturing of Microbial Biofungicides for
Control of some Plant Diseases, from 2019–2022; PI. Wafaa M.
Haggag.
Author Contribution
WH and ME planned
the experiments and interpreted the results, WH applied of nanoparticles as
bio-fungicides and statistically analyzed, and ME prepared and characterize of
biosynthesized nanoparticles
Conflict of Interest
The authors declare
no conflict of interest.
Data Availability
The reported data
can be made available upon requesting to the corresponding author Ethics
Approval Not applicable in this research work.
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