Indian Journal of Microbiology

https://doi.org/10.1007/s12088-024-01363-x

ORIGINAL RESEARCH ARTICLE

Mycogenic Fabrication of Zinc Nanoparticles for Their Antifungal

Activity Against Alternaria mali
Shahnaz Anjum1,3 · Ashish Vyas2 · Tariq Ahmad Sofi3

Received: 14 February 2024 / Accepted: 24 July 2024

© Association of Microbiologists of India 2024

Abstract were assessed for their potential antifungal activity against

Synthesis of nanoparticles through environmentally friendly Alternaria mali, a notorious phytopathogen responsible for
and biologically mediated routes is the study upon which causing leaf spot in Apple plant. The antifungal activity was
lot of research attention has been focused in recent times. assessed through poison food technique and results indicated
In the present investigation, fungal-mediated synthesis of a significant inhibition of Alternaria growth by the zinc oxide
zinc oxide nanoparticles was explored using an economical nanoparticles particularly the ones synthesized from Pleuro-
and nature friendly approach. Synthesis of the extracellular tus, with a dose-dependent response. The mycogenic synthe-
zinc oxide nanoparticles was achieved using fungal cultures sis of zinc oxide nanoparticles is an eco-friendly and sustain-
of Pleurotus, Lentinus and Agaricus spp. The synthesized able approach, minimizing the need for hazardous chemicals
nanomaterial was thoroughly characterized using physical and reducing environmental impact. This study showcases
techniques including UV–Vis Spectroscopy, SEM, XRD, and the potential of zinc oxide nanoparticles as a novel and effec-
FTIR. UV–Vis Spectroscopy exposed a distinct absorption tive biofungicide against Alternaria mali and contributes to
peak in the range of 300–400 nm, validating the formation the growing body of knowledge on sustainable methods of
of nanoparticles. SEM images revealed the ZnNPs being nanoparticle synthesis and their applications in agriculture
agglomerated and irregular in shape. The size of the par- (disease management) and environmental management.
ticles is in the average range of 10–100 nm. XRD analysis
demonstrated the nature of the nanoparticles as crystalline, Keywords Nanoparticles · Extracellular synthesis · UV–vis
with prominent diffraction peaks corresponding to the face- spectroscopy · SEM · XRD · FTIR · Antifungal activity
centered cubic (fcc) crystal structure of zinc oxide. FTIR
analysis provided insights into the functional groups present
on the nanoparticle surface. This suggests that there is some Introduction
involvement of fungal biomolecules in the reduction and sta-
bilization of zinc oxide nanoparticles. The presence of zinc Nanotechnology is the developing field of science dealing
oxide nanoparticles was confirmed by Energy Dispersive with the use of materials and structures at the nanoscale,
X-ray Analysis. Furthermore, the synthesized nanoparticles which are typically measured in nanometres (nm) between
1 and 100. The manipulation of material at the near-atomic
* Ashish Vyas
level using a combination of biological, biochemical, and
ashish.vyas@lpu.co.in technical techniques is known as nanotechnology. The most
* Tariq Ahmad Sofi
extensively studied scientific issue nowadays is nanotechnol-
tariqsofi1@gmail.com ogy [28]. The Latin word Nanas, which means “dwarf,” is
where the term “nano” originates. The idea of nanotechnol-
1
Department of Botany, Lovely Professional University, ogy was first put out by Richard Feynman [8]. Nanoparticles
Phagwara 144411, Punjab, India
of both organic and inorganic substances exist. Scientists
2
Department of Microbiology and Biochemistry, Lovely from a variety of fields have also been interested in nano-
Professional University, Phagwara 144411, Punjab, India
particles [18]. There are two approaches for creating metal
3
Division of Plant Pathology, FoH, Sher-E-Kashmir nanoparticles: top-down and bottom-up (self-assembly)
University of Agricultural Sciences and Technology,
Shalimar 190025, J&K, India
[30]. Gradually, atom by atom, molecule by molecule, or

Vol.:(0123456789)
 Indian Journal of Microbiology

by spontaneous order, a structure is built in the bottom up have been identified worldwide. There are many different
approach, while a suitable starting material is shrunk using kinds of mushrooms on leaves, fallen wood, and dead plants
chemical or physical methods in the top down process. since some of them can cause wood rot in both live and dead
These two procedures are utilised to create nanoparticle plants. The use of mushrooms in medicine is rather wide-
and the process is included under "green technology" or the spread. The oyster mushroom, or Pleurotus spp., is a well-
biological technique. The convergence of nanotechnology known example of a fungus proven to have antimicrobial and
in biological science has been a great backbone to various antioxidant properties, and has been declared as a potential
research fields at large [32]. Nanoparticles can interact with pharmaceutical candidate in a variety of biological realms.
other biomolecules of the pathogens or target organisms High photosensitivity, high thermal stability, and environ-
inside or on the surface of cells since they are smaller than mental sustainability make it a desirable photocatalyst [58,
traditional biomolecules like metallic nanoparticles (gold, 69, 71, 86]. Moreover, ZnO nanoparticles show excellent
magnetic, and quantum dots), polymeric nanoparticles antibacterial action against gram-positive and gram-negative
(micelles and hydrogels), nanospheres, nanotubes, nanocap- bacteria, even in the absence of light, highlighting their huge
sules, cell ghosts, and nanocarriers including dendrimers, potential in the battle against microbial diseases [73]. Zinc
liposomes etc. Additionally, the increased surface area of oxide nanoparticles (ZnO NPs) may also be used in high-
nanoparticles improves their solubility and bioavailability. density broadcasting, sensor and solar technology, hydrogen
Different techniques are used in biological synthesis to cre- storage, nanoelectronics, photocatalysis, magnetic materials,
ate nanoparticles from bacteria, fungi, viruses, actinomyces, quantum machines in biomedical and environmental science,
and microalgae. The process of creating nanoparticles from and information storage [60]. Other nanoparticles like that
fungi is referred to as the “fungal mediated approach,” and of gold, silver, platinum also hold great applications, such
as a consequence, the produced particles are metal-tolerant, as, iron nanoparticles being significant to the aerospace and
extremely stable, safe for the environment, and beneficial industrial sector and having wide range of uses in catalysts,
from an economic standpoint [53, 94]. Because of their pigments, and gas sensors [65], but due to the unique and
perceptible metal tolerance, strong absorption capacity, and widespread anti-microbial properties, ZNO NPs hold prom-
ability to aggregate metals, these fungi are preferred over ising applications [50, 73]. Applications for ZnO NPs in
other micro-organisms and utilised as stabilising and reduc- medicine have also been widely documented [52]. ZnO NPs
ing agents [92]. Fungi can be used in top-down and bot- are also included in a number of different skin care products,
tom-up approaches because they release proteins and active including antidandruff shampoo, calamine cream, baby pow-
extracellular and intracellular enzymes that lower metal ions. der, barrier cream and antiseptic ointment, which are used to
By employing fungus as a capping agent for nanoparticles, treat diaper rashes. The surface oxygen species of ZnO NPs
fungal-mediated nanoparticles can also exhibit strong anti- enhance their biocidal characteristics [66].
bacterial and antifungal properties and be effective against Plant diseases are seen to be one of the main issues fac-
harmful microbes [12, 19, 26]. Because the nanoparticles ing agriculture globally, having an impact on both agricul-
may easily pierce biofilms, there is a way to prevent biofilm tural output and product quality [76]. Certain plant diseases,
formation. Because of their incredibly tiny size, nanopar- particularly those that impact economic crop plants, have a
ticles are able to compete with intracellular bacteria and detrimental effect on the plant at various phases of growth
carry out antibacterial activities with ease. To restrict its and have a major impact on crop output [9, 36]. A class of
fungicidal effects, nanoparticles can harm the fungal cell fungi that are dormant in the soil is the source of root rot
membrane; the resulting damaged cell membrane helps and wilting illnesses [57, 84]. One of these illnesses is Leaf
separate nucleic acids and other remaining ions into cell Spot, which has a noticeable effect on the autoimmunity
free filtrate [56]. of the plant and, as a result, impacts crop productivity and
A seasonal crop that grows during wet seasons, mush- quantity during planting. The Alternaria leaf and fruit spot
rooms are edible fruiting structures. Typically, species thrive disease, caused by Alternaria spp., stances a major danger
in wet environments with damp soil close to other food to apple productivity and output. Over the past 10 years,
sources. The fruiting body of multicellular fungus found Alternaria disease has significantly hurt the apple trade in
in mushrooms may be seen with the unaided eye and can most of the world's apple-growing regions [29]. The states
be either epigeous or hypogeous [49]. Understanding the of Himachal Pradesh, Uttarakhand, as well as the union ter-
fundamentals of variety at the species or community level ritory of Jammu and Kashmir (J&K), produce around 90%
helps assess the effects of man-made or natural disturbances. of the apples used in the country. The prevalence of illness
There are two major phyla of macrofungi that are distin- Alternaria mali has been reported in India in the Kash-
guished: Ascomycetes and Basidiomycota [51]. On Earth, mir valley [79]. It has been demonstrated that the major-
roughly 10% of the overall number of mushroom species ity of cultivars exhibit the ALFS; still, the Delicious-group
are believed to be represented by the 14,000 species that is more susceptible [16]. It is possible to handle diseases
Indian Journal of Microbiology

effectively by using resistant cultivars. To limit the ALFS decreased toxicity and bioactivity of metal oxide nano-
pandemic, a mix of mite treatments, fungicidal spray, and particles, including ZnO, make them an attractive tool in
suitable cultural practises might be employed. The chief the biological sciences. The synthesised carboxymethyl
source of inoculums is the leaf litter on the orchard base; cellulose-ZnO NP films, for instance, may be successfully
spores are detected on buds and twigs during winter. It is used as food product coating films that provide protection
therefore imperative to reduce the amount of leaf litter on [13]. Additionally, adding 40 or 60 mg/kg of ZnO NPs to
the orchard floor, keep the orchard clean, cut down on falling the broiler diet enhanced the physiological state and pro-
apple leaves and other rubbish, and prevent mite eggs from ductive performance of the birds. Moreover, a lower dose
hibernating in the debris to lessen the chance of Alternaria of Zn (40 mg/kg food) showed encouraging results and
leaf blotch fungus. Applying urea to fallen leaves, mulch- might be a useful feed additive for broilers [45]. The ZnO
ing, uprooting weeds, clearing apple litter from orchards, nanocomposites provide sustainable solution and exhibit
covering plastic foil, using lime sulphur, and physically promising potentials like wound healing, anti-inflamma-
removing leaf residues can all help reduce the number of tory [51], food packaging [39], antimicrobial activity [6,
inoculums in orchards. Pruning the canopy selectively also 24, 37, 40, 41], and acetylcholinesterase inhibition, which
lessens the inoculum found in the orchard's twigs and buds. contribute to achieving the Sustainable Development Goal
When applied at the proper timing, fungicide sprays may 3 by reducing disease burden, and promoting global health
help reduce infections. Leaf blotch incidence should be mon- and well-being.
itored; fungicide therapy may be necessary to avoid fruit In this study, cell-free extracts of Pleurotus sajor caju,
spot infection if, by 70–90 DAB (2 months before harvest), Agaricus bisporus, and Lentinus edodes were used to
the incidence is more over 15% [20, 34, 47, 87]. synthesize fungal-mediated zinc nanoparticles utilising
Plant diseases are spreading quickly as a result of the the proper nanotechnology tools and protocols, and then
recent escalation of the phenomena of climate changes thoroughly characterized and assessed in vitro against the
[72]. As safe substitutes for chemical pesticides, research- fungal pathogen of the apple plant—Alternaria mali to
ers are focusing on developing eco-friendly ways to stop determine the mycelial growth/inhibition.
the spread of plant diseases [1, 3–5, 14, 15]. The applica-
tion of nano-biotechnology to resist infections and stimu-
late plant physiological immunity has demonstrated nota- Materials and Methods
ble and robust effects in the battle against fungal diseases
because it aids the plant in improving disease resistance, Screening of Fungi
systemic immunity, and production [21, 31, 70]. Complete
biological particles have been included in the scope of bio- Three fungi—Lentinus edodes, Agaricus bisporus, and
logical methods to nanoparticles and nanocrystal fabrica- Pleurotus sajor caju were acquired from the Mushroom
tion [2, 89]. The biological manufacturing of metal nano- Research Training Centre (MRTC), SKUAST-Kashmir.
particles using plants, bacteria, and fungus has garnered a The process of purifying these fungi involved re-culturing
lot of attention lately since it is safe and environmentally each one on the PDA plates, which were then incubated
friendly [10, 42, 43, 54, 74, 75]. Recent applications that at 26 ± 2 °C for one week [44, 55, 62]. The Erlenmeyer
highlight the potential of using fungi as podiums for the flasks containing potato dextrose broth (PDB) were seeded
synthesis of nanomaterials have also been reported [22, with fungal mycelia and incubated for 7 days at 26 ± 2 °C.
23, 25, 48]. This is because the fungal extract contains dif- Mycelia were removed from the culture broth after 24/48 h
ferent secondary metabolites that are highly concentrated of incubation by filtering the contents with sterile What-
and offer hope for the environmentally friendly synthesis man filter Paper No. 1. The biomass collected on the filter
of various nanocomposites. The synthesis of metal nano- paper was rinsed three times with sterile distilled water
particles as safe reducing and stabilising agents is depend- to remove any media components from the mycelia [17].
ent on macromolecules contained in fungal extracts, such The washed fungal biomass was then kept in 50 mL mil-
as flavonoids, alkaloid polysaccharides, phenolics, and liQ water and further left for incubation at 26 ± 2 °C, for
tannins [63]. In order for plants to develop and oxidise 5 days. Following incubation, the cell-free filtrate was
their carbohydrates, zinc is a necessary element. It also obtained by filtering the mycelia-containing solution
plays a significant part in photosynthesis and the synthesis through sterile Whatman filter Paper No. 1 for extracellu-
of chlorophyll [35, 38, 64, 67]. It is important to note that lar ZnNP production. Duplicate experiments were carried
a number of research [25, 49] have shown how nanozinc out. For the production of ZnNPs of three different types,
helps to induce plant defence against fungal disease. The all three fungal filtrates were utilized.
 Indian Journal of Microbiology

Fig. 1  UV–Vis spectroscopic analysis of A ZnP; B ZnA; C ZnL nanoparticles

Preparation of ZnO NPs from the three fungal extracts. After preparing the com-
bined solutions, the pH of the mixture was measured and
The above-mentioned procedure was employed to obtain brought up to 8.0 using sodium hydroxide solution (1 mL
the cell filtrate, which was subsequently used for bio reduc- of 1 M NaOH per 100 mL solution). To achieve the most
tion. The myco-synthesis of ZnO NPs employs the spe- effective synthesis of ZnO NPs, the reaction conditions
cific quantity of metal salt. More specifically, 100 mL of were set at 60 °C under agitation (500 rpm) for roughly
(5.0 mM) Zn ­(CH3COO)2⋅2H2O was added to each 100 mL 24 h on a magnetic stirrer. Following the full incubation
of the fungal filtrate and thus making three suspensions period, slight colour change was noticed and documented
Indian Journal of Microbiology

Fig. 2  SEM Images of A ZnP NPs (1 μm scale); B ZnP NPs (500 nm scale); C ZnA NPs (1 μm scale); D ZnA NPs (500 nm scale); E ZnL NPs
(1 μm scale); F ZnL NPs (500 nm scale)
 Indian Journal of Microbiology

A Element Weight % Atomic %

OK 23.2 55.2
Zn K 76.8 44.8

B Element Weight % Atomic %

OK 36.8 70.4
Zn K 63.3 29.6

C Element Weight % Atomic %

OK 38.5 71.9
Zn K 61.5 28.1

Fig. 3  EDX spectrum showing the composition of A ZnP NPs; B ZnA NPs; C ZnL NPs

as a faint transparent, confirming the bio-formation of ZnO Material Characterization

NPs. A negative control comprising solely metal salt solu-
tion and a control consisting of cell filtrate without the UV–Vis Spectroscopy
metal salt were also kept.
Spectrophotometer analysis was used to do primary analy-
sis and optical property assessment after the colour change
indication for nanoparticle synthesis. To track changes in
absorption, the UV–Visible spectra was collected at regular
intervals in the 200–800 nm region using a double beam
UV–Vis Spectrophotometer (Labtronics).
Indian Journal of Microbiology

Fig. 4  XRD pattern of A ZnP NPs; B ZnA NPs; C ZnL NPs

Table 1  Values of ZnP NPs obtained through XRD analysis Table 2  Values of ZnA NPs obtained through XRD analysis
2 theta (2θ) Theta (θ) d-spacing (Å) 2 theta (2θ) Theta (θ) d-spacing (Å)

23.37 11.69 3.80 13.87 6.93 6.38

26.95 13.47 3.31 14.40 7.20 6.15
27.44 13.72 3.25 14.41 7.20 6.14
31.11 15.56 2.87 31.65 15.83 2.82
37.68 18.84 2.38 31.65 15.83 2.82
37.68 18.84 2.38 34.29 17.14 2.61
59.36 29.68 1.55 36.24 18.12 2.48
61.47 30.73 1.51 39.10 19.55 2.30
69.08 34.54 1.36 53.79 26.89 1.70
59.32 29.66 1.56
62.14 31.07 1.49
75.37 37.69 1.26
 Indian Journal of Microbiology

Table 3  Values of ZnL NPs obtained through XRD analysis where λ = 1.5406 Å (wavelength of incident X-ray), θ = Peak
2 theta (2θ) Theta (θ) d-spacing (Å) position (in radians), n = 1 (Order of diffraction), d = inter-
planar spacing or d-spacing (in Å).
16.15 8.08 5.48
16.15 8.08 5.48
Fourier Transform Infra‑Red Spectroscopy
21.84 10.92 4.07
30.71 15.36 2.91
Fourier Transform Infra-Red (FTIR) Spectroscopy helps
30.71 15.36 2.91
profile the chemical groups present in sample to then under-
33.05 16.52 2.71
stand the key agents involved in the interactions occurring
36.15 18.07 2.48
during bioconversion. An Infrared Spectrophotometer was
47.37 23.68 1.92
used to perform FTIR on ZnO NPs, and the spectra were
58.01 29.00 1.59
recorded in the 4000–400 ­cm−1 region using a Perkin Elmer
59.59 29.80 1.55
FT-IR spectrophotometer.
60.54 30.27 1.53
60.54 30.27 1.53
Control of Alternaria by ZnO NPs

Pathogen, Inoculum, and Culture Conditions

Scanning Electron Microscopy
Potato Dextrose Agar was used as the medium of growth
Scanning electron microscopy (SEM) is a valuable tool for
for Alternaria mali. One-week old fungus was inoculated
examining the surface morphology because the size and
on to the agar plates. ZnO NPs synthesized using the fun-
form of the synthesised nanoparticles greatly influence their
gal extract dissolved during media preparation. 3 mm discs
biological function [19]. To investigate the accurate pro-
of the Alternaria cultures were taken using cork borer and
duced shape of the synthesized ZnO NPs and to establish
inoculated on to the plates containing poisoned media to
their formation at the nanomaterial scale, FE-SEM, Gemin-
examine the antifungal activity of the ZnO NPs. In all three
iSEM 500, Carl Zeiss, Germany, was used.
of the nanoparticles, same procedure was followed.

X‑Ray Diffraction Analysis In Vitro Evaluation of Growth Inhibition and Antifungal

Activity
The crystal-like nature of the synthesised nanoparticles
stood assessed using X-ray diffraction (XRD) study (Smart- Poison Food Technique Using the poison food approach
Lab 3kW, RIGAKU, Japan). Working by a Cu anode at [80], the antifungal action of the fungal-mediated ZnO NPs
30.0 mA and 40.0 kV in the state of 2θ value inside 20° and against Alternaria mali was assessed. To test the antifun-
100° with a flow of 2°/min, the intensity of the diffracted gal activity, various quantities of ZnO NPs (0.10 mg/mL,
X-rays was estimated as a function of the diffracted angle 0.20 mg/mL, and 0.30 mg/mL) were prepared and put to
(2θ). The d-spacing and 2θ values will be estimated using PDA medium independently. For every concentration, three
Bragg’s law: duplicates were created. The isolated fungus from diseased
Apple leaves was grown on PDA medium in Petri plates for
n𝜆 = 2d Sin𝜃 ten days prior to setting the experiment. Petri plates with
n𝜆 (1) a diameter of nine centimetres were filled with the PDA
or d =
2Sin𝜃 medium that had been autoclaved with the three types of
ZnO NPs at three different concentrations of each of the NP
solution. The PDA medium was solidified before the fun-
Indian Journal of Microbiology

Fig. 5  FTIR Spectrum of A ZnP NPs; B ZnA NPs; C ZnL NPs

 Indian Journal of Microbiology

Table 4  Mycelial growth percentage of Alternaria mali Statistical Examination

Treatment Concentration Values SD
The data of various experiments conducted for charac-
F.E (Pleurotus) 100% 15.67 0.76 terization of nanoparticles was analyzed with the help of
F.E (Agaricus) 100% 18.42 1.81 Origin 6.1 and Image J software and statistical data analy-
F.E (Lentinus) 100% 17.75 0.66 sis with the help of standard statistical software. The sta-
ZnP NPs 0.10% 12.33 0.52 tistical data was subjected to appropriate transformations,
ZnP NPs 0.20% 7.92 1.18 wherever needed as suggested by Gomez and Gomez [33]
ZnP NPs 0.30% 1.33 0.63 before analysis.
ZnA NPs 0.10% 14.5 1.25
ZnA NPs 0.20% 8.58 3.11
ZnA NPs 0.30% 4.92 0.76
ZnL NPs 0.10% 14.33 0.52 Results
ZnL NPs 0.20% 9.5 0.25
ZnL NPs 0.30% 4.25 0.66 Since the metal salt solution was colourless, it gradually
Zn(CH3COO)2 300 ppm 22.25 0.66 became brackish which was the indication of formation of
Zn(CH3COO)2 500 ppm 14.5 0.75 ZnO NPs in solution. The reaction mixture color altera-
Control 100% 24.5 0.25 tion owing to surface plasmon resonance substantiates the
occurrence of ZnO NPs [81].

gus Alternaria was introduced. In the centre of each petri

plate, mycelial discs of 5 mm were taken from the periphery UV–Vis Spectrum of ZnO NPs
of ten day old culture. After that, the inoculum-containing
Petri plates were incubated at 26 °C. A PDA media plate UV–Vis spectrophotometry was used to gather data on
devoid of any treatment functioned as a positive control. the visual characteristics of the ZnO NPs that were syn-
Three replications were maintained for each treatment. thesised throughout the process. UV–Vis spectra of the
After being incubated for a week, fungi displayed varying three types of ZnO NPs were obtained in the wavelength
degrees of growth inhibition at varying nanoparticle doses. range of 200–800 nm. The UV–Vis spectrum indicated a
When growth of the fungus in control plate was complete Surface Plasmon Resonance (SPR) wavelength at 310 nm
(30 mm) the diameter of the colony was measured in four in ZnO NPs synthesised from Pleurotus. The UV–Vis
directions and average was worked out. The per cent inhibi- spectra of ZnO NPs synthesized from Agaricus revealed
tion of growth was calculated by using the formula given by absorption peak at the wavelength of 212 nm. Spectros-
Vincent [91]. copy verified the presence of an absorption peak at 328 nm
in the UV–Vis spectra of the ZnO NPs synthesised from
C−T
Growth inhibition (%) = × 100 Lentinus (Fig. 1). These results are in agreement with a
C
previous study [7].
where C = Average fungal growth, T = Average fungal
growth in treated/poisoned plates. SEM Analysis of ZnO NPs

The synthetic ZnO NPs were expected to have an uneven

form based on the SEM micrograph analysis and they
appeared aggregated and irregular in shape. SEM pictures
Indian Journal of Microbiology

Fig. 6  Antifungal activity of

ZnP NPs against Alternaria
R1 R2 R3
mali at concentrations: (1–3)
0.30; (4–6) 0.20; (7–9) 0.10; 1 2 3
(10–12) Pleurotus extract;
(13–15) Control
C1

4 5 6

C2

7 8 9

C3

10 11 12

F.E

13 14 15

Control
 Indian Journal of Microbiology

Fig. 7  Antifungal activity of

ZnA NPs against Alternaria R1 R2 R3
mali at concentrations: (1–3)
0.30; (4–6) 0.20; (7–9) 0.10; 1 2 3
(10–12) Agaricus extract;
(13–15) Control

C1

4 5 6

C2

7 8 9

C3

10 11 12

F.E

13 14 15

Control
Indian Journal of Microbiology

Fig. 8  Antifungal activity of

ZnL NPs against Alternaria R1 R2 R3
mali at concentrations: (1–3)
0.30; (4–6) 0.20; (7–9) 0.10; 1 2 3
(10–12) Lentinus extract;
(13–15) Control

C1

4 5 6

C2

7 8 9

C3

10 11 12

F.E

13 14 15

Control
 Indian Journal of Microbiology

also show that there was an attractive force between the par- extracts of nanoparticles have been used extensively to study
ticles, which caused them to clump together. Figure 2 shows the chemical groups that participate in the nanoparticle for-
a SEM micrograph of the synthesised NPs taken at diverse mation and stabilisation.
magnifications. In case of ZnP NPs, the production of nanoparticles
was majorly indicated by the peak at 1043.52 ­cm–1 in the
EDX Analysis ZnO NPs FTIR spectra. A peak at 1393.69 ­cm−1 can be attributed
to the N–H bond stretching of aliphatic primary amine.
EDX analysis may be used to determine if zinc oxide is Furthermore, the robust O–H bonds of alcohol and water
present in the nanoparticles. The myco-synthesized ZnO were identified as the source of peaks at 1490.14 ­cm−1
NPs had relative zinc and oxygen percentages of 76.8% and 3298.16 ­cm−1 respectively which is in agreement with
and 23.2%, respectively, which are comparable to the stoi- some studies [11, 27, 68]. The ZnP NP absorption peaks at
chiometric multitudes predicted theoretically. The relative 2979.88 ­cm−1 and 2197.17 ­cm−1 correspond to the vibra-
Zn and O percentages (63.3% of zinc, 36.8% of oxygen tional modes of the strong C–H medium band of alkane com-
and 61.5% of zinc, 38.5% of oxygen) of the ZnA NPs and pounds [11] and C=C stretching, respectively. The C–O–H
ZnL NPs likewise resemble the anticipated stoichiometric bond is responsible for the peak at 826.14 ­cm−1. In ZnA
masses. In each of the EDX spectrums, Zn showed three dis- NPs, absorption peaks at 1031.65 ­cm−1 and 677.76 ­cm−1
tinct signals, and O showed one. Figure 3 displays the peaks direct to the production of nanoparticles. Also, the C–H
shown while analysing the EDX spectrums of ZnO NPs. and ­CO2 bonds were recognized as the source of peaks at
2959.85 ­cm−1 and 2197.17 ­cm−1 respectively. Moreover,
XRD of ZnO NPs sharp peaks at 1550.98 ­cm−1 and 1385.53 ­cm−1 justify the
presence of strong O–H and N–H bonding thereby. In ZnL
The XRD patterns of ZnO NPs that were myco-synthesized NPs, peaks 1053.16 ­cm−1 and 821.69 ­cm−1 confirmed the
from three distinct extracts of mushrooms are displayed in formation of nanoparticles. The steep peaks at 3302.61 ­cm−1
Fig. 4. The produced diffraction patterns of the nanoparticles and 1499.97 ­cm−1 could be attributed to the O–H bonds of
were noted between 20° and 70°. The diffraction peaks of water/alcohol [11, 27, 68]. Furthermore, the bonds of C–H,
the synthesized material have a standard structure with sev- C–O, and a strong N–H bond made sources for the peaks of
eral peaks, indicating Zn-P at 2θ = 23.37°, 26.95°, 27.44°, 2970.24 ­cm−1, 2281.01 ­cm−1 and 1389.98 ­cm−1 respectively.
31.11°, 37.68°, 37.68°, 59.36°, 61.47° and 69.08°; Zn-A at
2θ = 13.87°. 14.40°, 14.41°, 31.65°, 31.65°, 34.29°, 36.24°,
39.06°, 53.79°, 59.32°, 62.13° and 75.37°; and Zn–L at Antifungal Activity of ZnO NPs
2θ = 16.15°, 16.15°, 21.84°, 30.71°, 30.71°, 33.05°, 36.15°,
47.37°, 58.01°, 59.59°, 60.54° and 60.54° which is highly To ascertain the antifungal effectiveness of ZnO NPs, Alter-
matched with the provided data of pure hexagonal Zinc naria mali, a harmful fungus, was extracted from the dis-
oxide crystals available in our library supplied with an X-ray eased apple plant leaves and subcultured on PDA media in
diffractometer [83, 93]. Applying Bragg’s law to the XRD the dark at 26 °C.
patterns, the d-spacing between the planes of the crystal was Efficacies of three types of ZnO NPs were tested at dif-
also evaluated. All the values estimated for ZnP, ZnA, and ferent concentrations against the growth of pathogen by Poi-
ZnL NPs are shown in Tables 1, 2 and 3. soned food technique. The per cent inhibition of growth of
the test fungus at different concentrations over the fungal
FTIR Analysis of ZnO NPs extracts and control was calculated. The results related to
the effect of different ZnO NPs on inhibition of Alternaria
The FTIR Spectroscopy profile of the ZnO NPs which mali are presented in Table 4.
revealed potential biomolecules in the fungal extracts of The poisoned food technique was used to conduct the
Pleurotus, Agaricus and Lentinus is shown in Fig. 5. antifungal test. ZnO NPs (0.10 mg/mL, 0.20 mg/mL, and
The FTIR profile of the ZnO NPs provides a profile 0.30 mg/mL) showed varying levels of suppression of fungal
of chemical groups present in the sample. This profile of growth. Regarding ZnP NPs, the fungal mycelial growth was
groups could help provide information regarding presence 12.33 mm at higher concentration (0.30 mg/mL), 7.92 mm
of proteins and other molecules that help stabilizing the and 1.33 mm at lower concentrations (0.20 mg/mL and
nanoparticles in solution. It also indicates the presence of 0.30 mg/mL) [88]. Additionally, our results demonstrate
metabolites that could have participated in the reduction of that the pathogenic fungus was inhibited by ZnA NPs and
Zinc acetate to ZnO NPs thus helping throw light on the syn- ZnL NPs at the nanoparticle concentrations of 0.30 mg/mL
thesis process which is largely unexplored in case of biologi- in each one respectively. Similarly, in ZnA treated plates,
cal synthesis of nanoparticles. The IR profiles of biological the mycelial growth was 14.5 mm, 8.58 mm and 4.92 mm
Indian Journal of Microbiology

at 0.10 mg/mL, 0.20 mg/mL, and 0.30 mg/mL of concen- no distinctive peak for much impurities ensuring the purity
trations. At the similar concentrations, ZnL NPs showed of hexagonal nanocrystals composed only from ZnO [85].
the mycelial growth of 14.33 mm, 9.50 mm and 4.25 mm The XRD pattern was in line with research, where P. djamor
respectively. Plates showing the visible inhibition of Alter- mushrooms were used for the synthesis of ZnO NPs [59].
naria mali by the nanoparticle concentrations are shown in Alternaria mali is a dominant phytopathogen of apple
Figs. 6, 7 and 8. The findings indicate that the eco-friendly (Malus domestica Borkh.) prominently damaging the leaves
role of nanoparticles in disease management is noteworthy. by causing leaf blotch in apple. In this regard, nanoparticles
were evaluated at three different concentrations for their
In vitro efficacy in inhibiting mycelial growth of A. mali.
Discussion Among nanoparticles with the increase in concentration of
nanoparticles from 0.10 to 0.30%, mycelial growth of test
The affirmation of ZnO NP formation was done by various fungus decreased significantly with increase in concentra-
characterization techniques as discussed earlier. Absorption tion. Concentrations used were; 0.10 (10:90), 0.20 (20:80)
peaks round 300 nm (Fig. 1) are the indication of ZnO NP and 0.30 (30:70) mg/mL. All the ZnO NPs showed a dif-
formation as seen previously [81]. The absorption wave- ferentiated activity against the pathogenic fungus tested.
length at about 300 nm of ZnO suggested the excitonic Among all three nanoparticles synthesized, ZnP NPs were
character of Zinc at room temperature. The UV emission more active against the test fungus but the activity varied on
is attributed to the radioactive recombination between the the basis of concentrations. The effectiveness of ZnO NPs
electrons in the conduction band and the holes in the valence against A. mali has been validated [77, 78]. A well-defined
band while working on ZnO phosphor powders [90]. The antifungal activity of bulk ZnO solution was seen in a study
SEM images of the ZnO NPs samples by using three differ- that was less than other ZnO NPs used in their study [46].
ent fungal extracts of Pleurotus sajor caju, Agaricus bispo- Among the various metal oxides studied for their antimicro-
rus and Lentinus edodes are shown in Fig. 2. The clustered bial activity, ZnO NPs have been found to be highly toxic.
structure in the images substantiate the presence of biologi-
cal debris in the sample. The synthesis of ZnO NPs is clearly
seen through EDX analysis of NPs. In case of ZnP NPs,
oxygen’s atomic weight was 55.2%, while its current weight Conclusion
was 23.2%. At the same time, the atomic weight of zinc
was 44.8%, while the current weight was 76.8%. In ZnA Fungi can be used for the synthesis of ZnO NPs. This paper
NPs, atomic and current weight of oxygen was 70.4% and presents the successful synthesis of ZnO NPs from Pleuro-
36.8%, whereas the atomic and current weight of zinc was tus sajor caju, Agaricus bisporus and Lentinus edodes. The
29.6% and 63.3% respectively. Similarly, the atomic weight aqueous extracts of the three fungal species, which contain
of oxygen in ZnL NPs was 71.9%, current weight was 38.5%, large amounts of carbohydrates, polysaccharides, proteins,
and the atomic and current weight of zinc was 28.1% and and phenolic compounds, were responsible for the forma-
61.5% correspondingly. Peaks shown in the EDX spectrum tion, and stabilization, and acted as a capping agent, of the
demonstrate that the main constituents of synthesized NPs particles. All these major constituents might have played role
are Zinc and oxygen [27]. Figure 5 shows the FT-IR spectra in the formation and stabilization of stable elemental zinc
of ZnO NPs prepared by using aqueous fungal extracts. The at a nanoscale. During the reaction, the formation of ZnO
different functional groups of ZnO NPs were found to be NPs was observed by the change in the mixture color (Zinc
O–H, N–H, C-O–H, and C = C, which made the three types Solution + fungal extract) from transparent to brackish. The
of nanoparticles potent antifungal agents. Our findings are differentiation of nanoparticles was found to be variable.
in line with a study which confirmed the binding of proteins Each of the three nanoparticles had its way with differences.
to metallic nanoparticles through amines as well as electro- UV–vis spectroscopy, SEM, XRD, FTIR, and other charac-
static interactions [82]. The interaction between the chemical terization processes were used study the morphology, com-
moieties of fungal extracts and the metallic scaffold may be position, crystallinity and other features of the nanoparticles.
the cause of the relative changes seen in the FTIR spectra. All three types of nanoparticles were found to be irregular
Furthermore, it is likely that the amides, carboxylic acids, in shape, which was confirmed by SEM analysis. The sharp
and phenolic acids found in enzymatic proteins of the fungal and strong diffraction peaks observed in the XRD patterns
extracts are crucial for the stability and bio-reduction of ZnO confirmed the crystallinity of the myco-synthesized ZnO
NPs. The results here agreed with previous reports of Asper- NPs. The FTIR analysis confirmed the functional groups
gillus species mediated-nanoparticle synthesis, describing present in the ZnO NPs. The different functional groups of
the involvement of fungal proteins in the bio-stabilization ZnO NPs were responsible for making the synthesized ZnO
and the capping of the Zinc [61]. XRD spectrum showed NPs effective antimicrobial agents too. The ZnP NPs are
 Indian Journal of Microbiology

the most potent antifungal agents against Alternaria mali. imaging. Compr Rev Food Sci Food Saf 18:1563–1621. https://​
Overall, all three ZnO NPs can be used as effective antifun- doi.​org/​10.​1111/​1541-​4337.​12490
9. Ali O, Ramsubhag A, Jayaraman J (2021) Biostimulant proper-
gal agents based upon the inhibition caused on the pathogen ties of seaweed extracts in plants: implications towards sustain-
as confirmed by poisoned food technique. able crop production. Plants (Basel) 10:531. https://​doi.​org/​10.​
Further studies should investigate the toxicity of the 3390/​plant​s1003​0531
myco-synthesized ZnO NPs since the further application 10. Ali OM, Hasanin MS, Suleiman WB, Helal EEH, Hashem AH
(2022) Green biosynthesis of titanium dioxide quantum dots
of these nanoparticles might have some toxic effects when using watermelon peel waste: antimicrobial, antioxidant, and
applied in the field of agriculture or medical sciences. Thus, anticancer activities. Biomass Conv Bioref. https://​doi.​org/​10.​
further investigation of potential toxic effects of the fungal 1007/​s13399-​022-​02772-y
extract-mediated ZnO NPs is warranted and recommended. 11. Alotaibi B, Negm WA, Elekhnawy E, El-Masry TA, Elharty
ME, Saleh A, Abdelkader DH, Mokhtar FA (2022) Antibacterial
Acknowledgements We acknowledge the Central Research Facility activity of nano zinc oxide green-synthesised from Gardenia
Centre (CRFC) and Department of Physics, NIT, Srinagar, J&K, India thailandica Triveng. leaves against Pseudomonas aeruginosa
for helping in UV–Vis, SEM, EDX, XRD and FTIR analysis. We also clinical isolates in vitro and in vivo study. Artif Cells Nanomed
extend our sincere thanks to Department of Plant Pathology, SKUAST- Biotechnol 50:96–106. https://​doi.​org/​10.​1080/​21691​401.​2022.​
Kashmir for making this research work possible. 20561​91
12. Anjum S, Vyas A, Sofi T (2023) Fungi-mediated synthesis of
nanoparticles: characterization process and agricultural applica-
Declarations tions. J Sci Food Agric 103:4727–4741. https://​doi.​org/​10.​1002/​
jsfa.​12496
Conflict of interest The authors declare that they have no conflict of 13. Anwar MM, Aly SSH, Nasr EH, El-Sayed ESR (2022) Improving
interest. carboxymethyl cellulose edible coating using ZnO nanoparticles
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