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Summer Research Fellowship Programme of India's Science Academies

Screening the antifungal activity of graphenized Au/ ZnO plasmonic nanocomposite

 Priyadharsini, S.

Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology ,Tamil Nadu Agricultural University, Coimbatore-641003

Dr. Jagreet Kaur

Department of Genetics,University of Delhi, South campus-110021

Abstract

Nano-technology has been widely studied for its greatest impact on Agriculture, Medicine, Pharma industries, food processing and drug delivery system. In agriculture, these particles are extremely beneficial towards precision farming, focusing on the use of less input and more yield. Main applications include Nano-fertilizers, Nano-biosensors, E-noses, Nanobots and Nano-pesticides. These particles are reported to have high level of antibacterial and antifungal activity, therefore these particles can be employed in agriculture to control antagonistic bacteria and fungi. This study is mainly focused to check the efficacy of using the graphenized Au/ZnO plasmonic nanocomposite as fungicide to control soil fungal pathogens. Most of the fungicides in the market are extremely toxic to beneficial microbes in the soil and also causes severe pollution to the environment. These toxic chemical fungicides can be replaced by nanocomposites, since they are less toxic and required only in small quantity. This work was carried out to test the antifungal activity of an available NC against Sclerotinia sclerotiorum.

Keywords: nanocomposite, antifungal activity, Sclerotinia, ROS

Abbreviations

Table of expansions 
NC Nanocomposite 
 ROSReactive oxygen species 
 nmnanometre 
AZG  Gold, Zinc, Graphenized NC
 UVUltra violet 
 PDAPotato Dextrose Agar medium 
C Control 
 CSControl-Sclerotia 
 T Treatment
 TS Treatment-Sclerotia
 UNUnactivated NC 
UNS  Unactivated NC-Sclerotia

INTRODUCTION

Nanoparticles have attracted the scientists of all over the world because of their efficient usage and minimal requirement of the particles without reduction in their efficiency. These particles have been reported to have more impact on agriculture where they have been extremely benefiting to solve most of the problems on pest and diseases. The main focus on the use of nanotechnology in agriculture is to reduce the cost of input through nanofertilizers and nanopesticides. Sustainable growth of agriculture requires new and innovative technology like nanotechnology. In modern agriculture, sustainable production and efficiency are unimaginable without the use of agrochemicals such as pesticides, fertilizers, etc. However, every agrochemical has some potential issues including contamination of water or residues on food products that threat the human being and environmental health, thus the precise management and control of inputs could allow to reduce these risks. The development of the high-tech agricultural system with use of engineered smart nanotools could be excellent strategy to make a revolution in agricultural practices, and thus reduce and/or eliminate the influence of modern agriculture on the environment as well as to enhance both the quality and quantity of yields.

Nanoparticles and their classifications

Nanoparticles are ultrafine units with dimensions measured in nanometres (nm; 1 nm = 10−9 metre). Nanotechnology is the application of these nanostructures into useful nanoscale devices. Nanoparticles exist in the natural world and are also synthesized by some physical, chemical and biological means. These nanoparticles are classified based on their available source and size.

Classification

Based on the available source

1)      Naturally occurring

Eg. Sea spray, Mineral composites, Volcanic ash, Viruses Fine sand and dust

2)      Incidental They are produced as a result of by product of a process like combustion, industrial manufacturing, and other human activities. They have poorly controlled size and shape. Eg. By products of Welding fumes, Large-scale mining – Coal fly ash, Industrial effluents.

3)      Engineered An Engineered Nanomaterial is the one intentionally produced material that has a size in 1, 2, or 3-dimensions of typically between 1-100 nm. They have definite shape, size and composition. Eg. Quantum dots, Buckyballs/nanotubes, Sunscreen pigments, Nanocapsules and carbon nano tubes.

 Based on the Size

This is classified based on the number of dimensions which are not confined to nanoscale range <100nm.

1)      Zero dimensional nanoparticles or clusters

Materials wherein all the dimensions are measured within the nanoscale i.e., less than 100nm. (no dimensions, or 0-D, are larger than 100 nm). The most common representation of zero-dimensional nanomaterials are nanoparticles.

Eg. Particles, quantum dots, nanoshells, microcapsules and hollow spheres.

2)      One-dimensional nanomaterials

Only one dimension is outside the nanoscale. This leads to needle like-shaped nanomaterials.

Eg. nanotubes, nanorods and nanowires.

3)      Two-dimensional nanomaterials

Two of the dimensions are not confined to the nanoscale. 2-D nanomaterials exhibit plate-like shapes.

Eg. nanofilms, nanolayers and nanocoatings.

4)      Three-dimensional materials

Bulk nanomaterials are materials that are not confined to the nanoscale in any dimension. These materials are thus characterized by having three arbitrarily dimensions above 100 nm. Materials possess a nanocrystalline structure or involve the presence of features at the nanoscale. In terms of nanocrystalline structure, bulk nanomaterials can be composed of a multiple arrangement of nanosize crystals, most typically in different orientations. With respect to the presence of features at the nanoscale, 3-D nanomaterials can contain dispersions of nanoparticles, bundles of nanowires, and nanotubes as well as multi-nanolayers.

 Synthesis of Nanoparticles

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Top down and bottom up approach of synthesizing nanomaterials

 1) Physical methods

Physical methods involve the use of mechanical energy, high energy radiations, thermal energy or electrical energy to effect material abrasion, melting, evaporation or condensation there by resulting in the synthesis of nanoparticles. The physical methods admire top-down strategy and are advantageous as the methods are free of solvent contamination. The physical method are simple method to produce uniform monodispersed nanoparticles. The cross contamination and production of abundant waste materials are limitations of physical synthesis of nanoparticles. Laser ablation, electro spraying, inert gas condensation, physical vapour deposition, laser pyrolysis, flash spray pyrolysis, molecular beam epitaxy, high energy ball milling, melt mixing are some of the commonly used physical methods for synthesizing nano particles.

2) Chemical methods

Chemical methods such as the reduction of transition metal salts are the most convenient ways to control the size of the particles. In most of the chemical synthesis process, the nanoparticles have been formed by the reduction and decomposition of precursors. There are two important processes are involved in the growth of nanocrystals from solution. First is the nucleation and second is the growth of the nanocrystals. In a typical synthesis of nanocrystals, precipitation reaction is important to form the nanocrystals. The precipitation process involves nucleation step followed by crystal growth stages. Nucleation plays an important role in controlling the size and shape of the final product. Chemical vapour Deposition, Electro depositions and Sol-Gel Processing are most commonly used chemical methods for synthesizing nano particles.

3) Biological synthesis

Biological synthesis of nanoparticles is based on green chemistry approach that make use of unicellular and multicellular biological entities like plants, bacteria, fungus, viruses, actinomycetes, and yeast. Synthesizing nanoparticles through biological entities as biological factories offers a safe, non-toxic and environment-friendly method of synthesizing nanoparticles with a wide range of sizes, shapes, compositions, and physicochemical properties. Generally, biological entities with a potential to accumulate heavy metals are suited for nanoparticle synthesis. Both, microorganisms and plants have the ability to uptake and accumulate inorganic metallic ions from polluted environment. The inherent ability of a biological entity to transform inorganic metallic ions into metal nanoparticles intracellularly and extracellularly is the major advantage of biological synthesis. Organisms used in nanoparticle formation vary widely, from simple prokaryotic bacterial cells to complex eukaryotes.

 Nanotechnology and sustainable agriculture

The nanotechnology takes an important part in the productivity through control of nutrients supply (Gruère, 2012; Mukhopadhyay, 2014) as well as it can also be participated in the monitoring of water quality and pesticides for sustainable development of agriculture (Prasad et al., 2014). 

The implication of the nanotechnology research in the agricultural sector is becoming to be necessary, even key factor for the sustainable developments. In the agri-food areas pertinent applications of nanotubes, fullerenes, biosensors, controlled delivery systems, nanofiltration, etc. were observed (Ion et al., 2010; Sabir et al., 2014). This technology has been proved to be as good in resources management of agricultural field, drug delivery mechanisms in plants and helps to maintain the soils fertility. Moreover, it is being also evaluated steadily in the use of biomass and agricultural waste as well as in food processing and food packaging system as well as risk assessment (Floros et al., 2010). Recently, nanosensors are widely applied in the agriculture due to their strengths and fast for environmental monitoring of contamination in the soils and in the water (Ion et al., 2010).

Background/Rationale

Graphenized Au/ ZnO plasmonic nanocomposite

Nanocomposite is a multiphase solid material where one of the phases has one, two or three dimensions of less than 100 nanometres (nm) or structures having nano-scale repeat distances between the different phases that make up the material. These particles will have high surface to volume ratio.

One such composite used in this study is graphenized Au/Zno plasmonic nanocomposite which is mainly composed of gold, Zinc and reduced graphene oxide. These particles aggregate to form macro sized particles and requires deep sonication to convert them into nanoparticles. Only nanosized particles can be efficiently used to target the antagonistic microbes because of their unique property to generate free radicals.

This composite is in gray color and can be stored for long period at normal room temperature. This can be dissolved in sterile water but at lower concentration, so that the 100% dissolution is achieved. Higher concentration will cause improper dissolution of the particles. They require deep sonication in the water bath sonicator for minimum of 10 minutes to break the aggregation and effect their dissolution.

Photoactivation of nanocomposite

The carbonaceous-metal-zinc oxide hybrid NC, AZG have been utilized for photocatalytic degradation under the influence of simple sunlight compared to other external stimulus like electric potential, specific UV exposure, thermal energy, etc.

On exposture to 30 mins of sunlight the particle gets activated at the intensity of 900±30Wm-2 which can be visually observed by the quick degradation of methylene blue as an indicator of NC activation. The gold particles absorb UV and zinc particles absorb Visible light which generates electron-hole pair resulting in the free radical production. These radicals will react with water molecules to produce reactive oxygen species including hydrogen peroxide and superoxide ions.

These reactive oxygen species attacks the cell membrane of the pathogens and causes mortality.Thus this composite can be well studied to use it as an antifungal agent. Antibacterial activity has been reported, 100% microbial killing of Gram positive S.aureus strain has been achieved with 60μg/ml of NC using sunlight as an activator (Juneja et al., 2017).

Nanoparticles have attracted the scientists of all over the world because of their efficient usage and minimal requirement of the particles without reduction in their efficiency. These particles have been reported to have more impact on agriculture where they have been extremely benefiting to solve most of the problems on pest and diseases. The main focus on the use of nanotechnology in agriculture is to reduce the cost of input through nanofertilizers and nanopesticides. Sustainable growth of agriculture requires new and innovative technology like nanotechnology. In modern agriculture, sustainable production and efficiency are unimaginable without the use of agrochemicals such as pesticides, fertilizers, etc. However, every agrochemical has some potential issues including contamination of water or residues on food products that threat the human being and environmental health, thus the precise management and control of inputs could allow to reduce these risks. The development of the high-tech agricultural system with use of engineered smart nanotools could be excellent strategy to make a revolution in agricultural practices, and thus reduce and/or eliminate the influence of modern agriculture on the environment as well as to enhance both the quality and quantity of yields.

Plant protection in general and the protection of crops against plant diseases in particular, have an obvious role to play in meeting the growing demand for food quality and quantity. Plant protection then primarily focused on protecting crops from yield losses due to biological and non-biological causes. The problem remains as challenging today with additional complexity generated by the chemical fungicides and pesticides causing severe pollution to soil, water and air, Yet, the control on spread of pathogen has not come to an end. Therefore, the new technologies have to be adopted which should be eco-friendly and promising to control the spread of pathogens.

Sclerotinia sclerotiorum

Sclerotinia sclerotiorum, a necrotrophic ascomycetous fungus, is the causal agent for the common stem rot disease in various dicot plants. The disease is manifested as a white cottony fungal growth on the infected plant, thus popularly known as “white mold”. The fungus causes huge yield losses every year globally. It has a huge host range of 600 plant species (Derbyshire et.al 2019) including almost all dicots and some monocot plants, most of them are important crops like soybean, sunflower , cotton and various vegetables (Bolton et.al 2006). In India the fungus is posing serious threat to the crop yield of Brassica juncea which is an important oilseed crop for the country. The pathogen produces brown or black colored small resting structures called sclerotia, on the substratum when the nutrient content of the environment is scarce, which actually help the fungus to survive under unfavorable conditions. These structures are aggregates of mycelia covered with a hard melanised outer covering. The sclerotia can remain viable in soil for several years. It can germinate both by sexual or asexual reproduction. The sexual reproduction takes place by the carpogenic germination in which the apothecia is formed and releases the ascospores, whereas the myceliogenic germination is asexual type of reproduction in which mycelia arises from the sclerotia that generates the hyphae. To overcome the high yield loss caused by the fungi, some effective fungicide has to be employed to control this pathogen. The main objective of this study is to standardize the effective concentration of nanocomposite and suitable parameters to achieve 100% killing.

Objective of the research

1)      The main objective of the study is to standardize the effective concentration of nanocomposite to use it as the environmentally safe and effective fungicide to control the spread of Sclerotinia sclerotiorum.

2)      This NC can also be tested against broad range of bacteria and fungi and it can be recommended for the farmers to use in the field to control the spread of various diseases across several agricultural crops.

LITERATURE REVIEW

In agriculture, annual crop losses due to pre and post harvest fungal diseases exceed 200 billion euros, and, in the United Stated alone, over $600 million are annually spent on fungicides. Nearly one quarter of food crops worldwide are affected by mycotoxins such as aflatoxins, ergot toxins, Fusarium toxins, patulin and tenuazonic acid.

Fungal plant diseases are generally managed with the applications of chemical fungicides. Chemical control has been found very effective for some fungal diseases, but it leaves several non-specific effects that destroy beneficial organisms along with pathogens. Such ecological disturbances open the route to undesirable health, safety, and several environmental risk. A promising method for protecting plants against diseases is constructing and employing pathogen-resistant cultivars. Although a number of resistant cultivars have been developed through breeding programs, these cultivars become obsolete in a short time due to the rapid evolution of the phytopathogens and the emergence of virulent forms capable to overcome the plant resistance. Breeders are often confronted with the issue of using a limited number of plants in their breeding programs, undesirable traits transferred together with the valuable resistance genes, and, in recent years, also with the depletion of potential gene sources.

Some of the nano particles that have entered into the arena of controlling plant diseases are nano forms of carbon, silver, silica and alumino-silicates.

The nanocomposite used in the study has reported to have high antibacterial activity (Juneja et al., 2017). Once the detrimental effect of NC against fungi is achieved, this would provide the promising solution against broad range of fungal pathogens.

Screening the antifungal activity against several plant pathogens will give the broad idea to use it against specific diseases and the optimum condition can be recommended for its application in the field as effective fungicide

METHODOLOGY

Concepts

Activation of the nanocomposite

These particles are in powdered form and needs to be dissolved in the sterile water. Less concentration of stock has to be prepared for complete dissolution after deep sonication of 10 minutes in water bath sonicator.

Standardization of stock concentration

Stock concentration at 1mg/ml resulted in the re-aggregation of particles even after sonication of 30 minutes, which resulted in inconsistant results in the fungal inhibition assays. Therefore stock concentration of 0.4mg/ml were tried which resulted in complete dissolution of the particles. Each time prior to the start of the experiment the stock had to be sonicated to disperse the aggregated particles. After sonication, the particles were stable in the solution for 3-4 hours.

Standardization of working concentration

Based on the literature survey, the optimum working concentration to use it as an antifungal agent is optimized. The significant growth inhibition of Sclerotinia isolates were observed at 200–400 μg/ml of ZnO NPs (60%-70% inhibition) (Junli Li et al., 2017). An antifungal assay was performed with the working standard of 0.05mg/ml, 0.1mg/ml, 0.2 mg/ml and 0.4 mg/ml. The working standards of different concentration was prepared from the 0.4 mg/ml stock using sterile distilled water.

Standardization of light intensity

The ternary NC have shown improved dye degradation capacity with 100 % efficiency (5μM Methylene Blue solution) as an indicator of activation of nanocomposite and average adsorption degradation capacity (Qo) of 83.34 mg/g within 30 mins. of sunlight exposure (900±30Wm-2) (Juneja et al., 2017). Light intensity around 11 AM to 1 PM would be around 700 to 800 Wm-2 so the optimum time for activating the nanocomposite is late morning at the bright sunlight. After 1 PM the sunlight intensity will decrease to 300-400 Wm-2 and it will not be sufficient to activate the nanocomposite at that time. Therefore the experiment conducted late in the afternoon showed poor inhibition and the one activated in the bright sunlight in the forenoon showed increased inhibition.

Methods

Pathogen

Sclerotinia sclerotiorum is allowed to grow on the potato dextrose agar medium. The actively growing mycelia after two days of subculturing is used in the study. The dark resting bodies sclerotia were also collected from the over grown plates, sterilized and used in the experiment.

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Fig.1.a

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Fig.1.b

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Fig.1.c

Fig. 1.a- A representative picture of Subcultured plates grown for two days at 22OC.

Fig. 1.b- Sclerotia inoculated in the PDA and grown for four days at 22OC.

Fig. 1.c- Subcultured plates grown for one week at 22OC.

Preparation of culture media

For the media, 3.9 gm Himedia potato dextrose agar (PDA) powder was dissolved in 100mL of distilled water. Media was Autoclaved at 121OC under 15psi of pressure for 15 minutes. After autoclaving media was poured in petri plates and can be stored at 4OC for longer usage.

 Sclerotia sterilization and inoculation

Sclerotia were first sterilized before inoculating them on the PDA petri dishes. The surface sterilization of the sclerotia was done by using 70% ethanol for 1 minute followed by 2-3 minutes in 0.1% mercuric chloride. After the chemical treatment they are washed in the distilled water for at least 4-5 times to remove the chemicals and then sclerotia were cut into two pieces with the flame sterilized blades and transferred to the petri dishes containing 15 ml of PDA and then allowed to grow at 22oC for three to four days.

 Subculturing the fungal mycelia

After 3 days of the inoculation, when mycelium was grown from sclerotia, primary subculturing was made by puncturing out the mycelia plugs (5mm) from the edge of the actively growing mycelia and placing them inversely (the fungal inoculum touching the fresh plate) at the center of the freshly prepared PDA plates and then maintained at 22oC. The isolates were sub-cultured regularly at an interval of 1-2 week to maintain their vigor.

RESULTS AND DISCUSSION

Screening the antifungal activity

Method-1

Initially, the stock of 0.4 mg/ml of NC is prepared using sterile water in the laminar air flow chamber and is sealed with the parafilm. Then it is subjected for deep sonication for 10 minutes in the water bath sonicator.

Different concentration of NCs were prepared, 0.05 mg/ml, 0.1mg/ml, and 0.2mg/ml from 0.4mg/ml stock using sterile distilled water in the laminar air flow chamber to prevent the contamination of the solution. The solutions were prepared in the 50 ml centrifuge tube and sealed with the parafilm. and sonicated for (??) minutes. 10ml of each concentration were transferred to separate test tubes or glass vials and actively growing fungal mycelial plugs were incubated with NC. The sclerotium was also cut into two equal pieces after sterilization and inoculated into the NC solution. The plugs and sclerotia were also inoculated in the sterile water as a control. Maximum of 12 plugs and 12 sclerotia pieces in 10ml of NC is used for complete exposure to the NCs in separate tubes for plugs and sclerotia. NCs with the fungal plugs and sclerotia were inoculated in the bright sunlight around 500-700 Wm-2 light intensity. Unactivated controls were also maintained in the laboratory condition. The fungal plugs and sclerotia were inoculated in the different concentrations of NCs and maintained in the laboratory (devoid of sunlight exposure-dark). After 2 hours of incubation in the sunlight, the plugs were placed such that the mycelia touches the PDA media and the cut end of the sclerotia were also placed in the PDA media. Then they are incubated at 22oC. The growth of fungal mycelia from the plugs and sclerotia were observed after 24, 48 and 72 hours.

Trial 1

Screening the antifungal activity follows Method-1
S.no Plates No. of sclerotia or plugs Observation at 24 hours Observation at 48 hours Observation at 72 hours
1. C-1 4 + + +
2. C-2 4 + + +
3. C-3 4 + + +
4. CS-1 4 + + +
5. CS-2 4 + + +
6. CS-3 4 + + +
7. UN 4 + + +
8. UNS 4 + + +
9. T-1 4 - - -
10. T-2 4 - - -
11. T-3 4 - - -
12. TS-1 4 - - +
13. TS-2 4 - - +
14. TS-3 4 - - +
15. subcultured plate 1 + + +

 Stock- 1mg/ml ; Working standard- 0.2 mg/ml ; light intensity- 450 Wm-2

C- Sterile water control for fungal mycellial plugs; CS- Sterile water control for sclerotia ; UN- unactivated NC control at 0.2 mg/ml concentration for fungal plugs ; UNS- unactivated NC control at 0.2 mg/ml concentration for Sclerotia; T- 0.2mg/ml of NC concentration treatment forfungal plugs ; TS-0.2mg/ml of NC concentration treatment for sclerotia.

+ve- Normal growth of fungal mycelia

-ve- growth was inhibited

    Fig. 2.a
      Fig. 2.b
        Fig. 2.c

          Fig. 2.d
            Fig. 2.e
              Fig. 2.f

              Representative pictures of plates from experimental testing the efficacy of NC to use it as fungicide. Fig. 2.a- Control plate(C ), Fig. 2.b- Control plate sclerotia(CS), Fig. 2.c-Treatment (T), Fig. 2.d-Treatment sclerotia(TS), Fig. 2.e -Unactivated NC (UN), Fig. 2.f-Normal Subcultured      

              Trial-2

               Screening the antifungal activity-Trial 2 follows Method-1
              S.no Plates Conc. of NC mg/ml No. of sclerotia or plugs Observation at 24 hours Observation at 48 hours Observation at 72 hours
              1. C Sterile water 12 +++ +++ +++
              2. CS Sterile water 12 + ++ +++
              3. T-1 0.05 12 - - -
              4. T-2 0.1 12 - - -
              5. T-3 0.2 12 - - -
              6. TS-1 0.05 12 - - +
              7. TS-2 0.1 12 - + ++
              8. TS-3 0.2 12 - - +++
              9. UN-1 0.05 12 +++ +++ +++
              10. UN-2 0.1 12 +++ +++ +++
              11. UN-3 0.2 12 +++ +++ +++
              12. UNS-1 0.05 12 + ++ +++
              13. UNS-2 0.1 12 + ++ +++
              14. UNS-3 0.2 12 + ++ +++
              15. subcultured plate - 1 +++ +++ +++

               Stock- 1mg/ml ; Working standard- 0.05 mg/ml, 0.1 mg/ml, 0.2 mg/ml ; light intensity- 350 Wm-2

              1)  +++ normal growth of fungal mycelia, 2) ++ growth less than normal, 3) + slow growth, 4)  - growth was inhibited    

              C- Sterile water control for fungal mycellial plugs; CS- Sterile water control for sclerotia ; UN- unactivated NC control ; UNS- unactivated NC control for Sclerotia ; T-activated NC concentration treatment forfungal plugs ; TS- activated NC concentration treatment for sclerotia.

               Trial-3

              Screening the antifungal activity-Trial 3 follows Method-1
              S.no Plates Conc. of NC mg/ml No. of sclerotia or plugs Observation at 24 hours Observation at 48 hours Observation at 72 hours
              1. C Sterile water 12 +++ +++ +++
              2. CS Sterile water 12 + ++ +++
              3. T-1 0.05 12 +++ +++ +++
              4. T-2 0.1 12 +++ +++ +++
              5. T-3 0.2 12 +++ +++ +++
              6. TS-1 0.05 12 + ++ +++
              7. TS-2 0.1 12 + ++ +++
              8. TS-3 0.2 12 + ++ +++
              9. UN-1 0.05 12 +++ +++ +++
              10. UN-2 0.1 12 +++ +++ +++
              11. UN-3 0.2 12 +++ +++ +++
              12. UNS-1 0.05 12 + ++ +++
              13. UNS-2 0.1 12 + ++ +++
              14. UNS-3 0.2 12 + ++ +++
              15. subcultured plate - 1 +++ +++ +++

              Stock- 1mg/ml; Working standard- 0.05 mg/ml, 0.1 mg/ml, 0.2 mg/ml; light intensity-150 Wm-2

              C- Sterile water control for fungal mycellial plugs; CS- Sterile water control for sclerotia ; UN- unactivated NC control ; UNS- unactivated NC control for Sclerotia ; T-activated NC concentration treatment for fungal plugs ; TS- activated NC concentration treatment for sclerotia.

              Note

              Trial- 3 has been repeated 3 times and same results were observed. The light intensity in the trial-4, 5 and 6 were 280, 250, 340 Wm-2 respectively. No inhibition in the fungal growth was observed. This has been concluded to have problems with

              a)      The poor dissolution because of high stock concentration of 1mg/ml.

              b)      The improper activation of NC due to low light intensity.

              Therefore, to tackle the possibility of poor solubility lower concentration of stocks were prepared and attempt to activate the NC at high light intensity was done.

               Trial-7

              Screening the antifungal activity-Trial 7 follows Method-1
              S.no Plates Conc. of NC mg/ml No. of sclerotia or plugs Observation at 24 hours Observation at 48 hours Observation at 72 hours
              1. C Sterile water 15 - - -
              2. CS Sterile water 15 - - +
              3. T-1 0.05 15 - - -
              4. T-2 0.1 15 - - -
              5. T-3 0.2 15 - - -
              6. TS-1 0.05 15 - - -
              7. TS-2 0.1 15 - + ++
              8. TS-3 0.2 15 - + ++
              9. UN-1 0.05 15 +++ +++ +++
              10. UN-2 0.1 15 +++ +++ +++
              11. UN-3 0.2 15 +++ +++ +++
              12. UNS-1 0.05 15 + ++ +++
              13. UNS-2 0.1 15 + ++ +++
              14. UNS-3 0.2 15 + ++ +++
              15. subcultured plate - 1 +++ +++ +++

               Stock- 0.2 mg/ml, Working standard- 0.05 mg/ml, 0.1 mg/ml, 0.2 mg/ml; light intensity-240 Wm-2

              C- Sterile water control for fungal mycellial plugs; CS- Sterile water control for sclerotia ; UN- unactivated NC control ; UNS- unactivated NC control for Sclerotia ; T-activated NC concentration treatment for fungal plugs ; TS- activated NC concentration treatment for sclerotia.

              Note

              Trial-8 was also performed with similar experimental conditions and the light intensity was 350 Wm-2 and same results were observed, there was no growth in the sterile control plates too. One possible reason could be due to heating of the solution in which fungal plugs and sclerotia has been inoculated at the time of NC activation. Higher temperature might have killed the fungus, therefore the alternate methods have been tried to study the inhibition of the growth and to verify the antifungal activity of the NC. This problem has not been encountered in the initial experiment.

              Alternate methods

              To overcome the above said problems other 2 alternate methods were performed to study the antifungal acitivity of NC.

              Method-2

              In this method, the activation of NC in sunlight and later the fungal plugs are inoculated in the activated NC. Initial stages of stock and working standard preparation for dissolution follows same as method-1 . Once the working standard of different concentration are prepared then they are exposed to sunlight for activation without the fungal plugs and sclerotia. The activation of NC is indicated by the degradation of methylene blue dye added with NC. Once activated NC are brought to the laminar hood and the fungal plugs and sclerotia are inoculated for 3-4 hours and overnight incubation was also done. Then the treated plugs and sclerotia were placed on the media and incubated at 22oC for observing the inhibition of growth. Sterile water controls and unactivated NCs were also maintained.

              Method-3

              As reported earlier, addition of NC to the growth media has been attempted to know the effective method for studying the antifungal activity. Required working standards were prepared from the stock and dissolution was achieved as such using water bath sonicator illustrated in the method-1. Then the required ml of NC solution was exposed to the sunlight for activation with methylene blue dye degradation as an indicator. Once after activation they were spread on the media using glass rod. Then the plugs and sclerotia were placed on the activated NC containing PDA media. Then the plates were incubated at 22oC for observing the pattern of growth. Sterile water controls and NC unactivated that is the NC not exposed to sunlight were also spread on the media and plugs and sclerotia were inoculated on the spread media and incubated at 22oC. Completely dissolved NCs of different concentration were mixed with the PDA media and poured on the petri plates. 5ml of NCs of different concentration was mixed with 15ml PDA media. Then poured and allowed for complete solidification. Then the plugs and sclerotia were placed on the media containing NCs and incubated in sunlight for 2 hours. Then the plates were incubated at 22oC for further analysis of inhibition or growth of the mycelia.

              These different methods were done to know the effective way to achieve complete inhibition of the growth. Observation of different experimental condition were studied and the results were discussed in the trial-9.

               Trial-9

              Screening the antifungal acitivity trial-9 follows method 2 and 3.
              S. No. Conc. mg/ml Conditions No. of plugs Mycellial radii(cm) at 24 hrs. Observation
              1. - Control- Sterile water 2 hrs. in sunlight and plugs incubated for 3 hrs. in laboratory condition 12 1-2.5 Normal growth
              2. 0.1 Treatment- NC activated in sunlight for 2 hrs. and plugs incubated in activated NC for 3 hrs. in laboratory condition 12 0.2- 0.8 Browning of plugs and slow growth was observed.
              3. 0.1 Treatment- NC kept in dark and plugs incubated in the NC for 3 hrs. in laboratory condition 12 0.8- 1.5 Normal growth
              4. 0.1 10ml NC mixed with the media, plugs plated and incubated in Sunlight for 2 hrs. 12 - No growth
              5. 0.1 10ml of NC+ Fungal plugs incubated in 2 hrs. of sunlight 12 - No growth
              6. - 10ml of sterile water + Fungal plugs incubated in 2 hrs. of sunlight 12 - No growth
              7. 0.1 10ml of unactivated NC+ Fungal plugs incubated in 2 hrs. in the laboratory condition 12 1-2.5 Normal growth
              8. 0.1 NC 2 hrs. in sunlight, 0.1ml spread on the media and plugs subcultured 12 1-2.5 Normal growth
              9. - Plates 2 hrs. in sunlight and subcultured(9 plugs) - - No growth
              10. - Normal subcultured 2 1-2.5 Normal growth

               Stock- 0.2 mg/ml, Working standard- 0.1 mg/ml ; light intensity-360 Wm-2

              Note

              This experiment has been performed with different experimental setups with more fungal plugs to identify the better method to proceed the study more significantly, S. no. 2 shows inhibition in the growth of the fungus in which the NC is activated in the sunlight and plugs have been treated in the laboratory condition in the activated NC for 3 hours. Therefore further experiments have been conducted with the above said conditions which following the method-2.

              Trial-10

              Screening the antifungal activity follows method-2
              S.no Plates Conc. of NC mg/ml No. of sclerotia or plugs Observation at 24 hours Observation at 48 hours Observation at 72 hours
              1. C Sterile water 12 +++ +++ +++
              2. CS Sterile water 12 + ++ +++
              3. T-1 0.05 12 +++ +++ +++
              4. T-2 0.1 12 +++ +++ +++
              5. T-3 0.2 12 +++ +++ +++
              6. T-4 0.4 12 +++ +++ +++
              7. TS-1 0.05 12 + ++ +++
              8. TS-2 0.1 12 + ++ +++
              9. TS-3 0.2 12 + ++ +++
              10. TS-4 0.4 12 + ++ +++
              11. UN-1 0.05 12 +++ +++ +++
              12. UN-2 0.1 12 +++ +++ +++
              13. UN-3 0.2 12 +++ +++ +++
              14. UN-4 0.4 12 +++ +++ +++
              15. UNS-1 0.05 12 + ++ +++
              16. UNS-2 0.1 12 + ++ +++
              17. UNS-3 0.2 12 + ++ +++
              18. UNS-4 0.4 12 + ++ +++
              19. subcultured plate - 1 +++ +++ +++

               Stock- 0.4 mg/ml, Working standard- 0.05 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.4 mg/ml; light intensity-380 Wm-2

               Note

              Trial 10 has been repeated 3 times but there was no inhibition observed in the growth of the fungus. It has been suggested that it must be because of improper sunlight required for activation of NC. The indicator methylene blue used for identifying the activation has shown the similar level of degradation when NC is added and when without NC when exposed to sunlight. Methylene blue shows no significant enhanced degradation on addition of NC, therefore it has been illustrated that probably the winter has come and the light intensity may not be sufficient to activate the NC.

              CONCLUSION AND RECOMMENDATIONS

              In general nanoparticles are reported to have high level of antibacterial and antifungal activity, elaborated studies on these particles would allow them to be efficiently used in agriculture to control disease causing pathogens. However the effective concentration and conditions should be studied to implicate them in the field. This study has been conducted to test one such nanocomposite against Sclerotinia sclerotiorum. This nanocomposite requires high sunlight to get activated. Because of poor sunlight experienced during the study (winter period -Delhi), it has been suggested to recheck when there is bright sunlight during summer season. Additionally, When the NCs are exposed to sunlight, the solution gets heated up and as a result the pathogen is getting killed at higher temperature. Therefore the experimental setup should include a step provided that the sunlight activates the NC without affecting the pathogen inoculated. Since the study has been conducted in the winter season in Delhi where the sunlight is very low in addition to smog activation becomes the major disadvantage of using the NC. Therefore the experiments can be optimized with the artificial light source or conducted in appropriate season when the required sunlight is available. Under the modified conditions, NC can be efficiently checked against several pathogens of interest.

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              3)     Derbyshire MC et al., (2019) A whole genome scan of SNP data suggests a lack of abundant hard selective sweeps in the genome of the broad host range plant pathogenic fungus.

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              ​ACKNOWLEDGEMENTS

              With immense pleasure, I would like to convey my sincere gratitude to the guide Dr. Jagreet kaur, Assistant professor, Department of Genetics, University of Delhi, South Campus, New Delhi for providing me with the opportunity to work in her laboratory.

              I also extend my heartful gratitude to the lab mates and PhD scholars Ms. Rashmi Verma, Ms. S. Hamsa, Ms. Pratibha Pant who guided and helped me to work in the JK lab efficaciously.

              I take this opportunity to thank my home institute mentor, Dr. Gnanam, R. Head of the department, Plant Biotechnology, Tamil Nadu Agricultural University who has been guiding me with utmost care.

              My sincere thanks to my coordinator Dr. Sudha, M. and Tamil Nadu Agricultural University, Coimbatore for giving me the chance to undergo internship for Biotechnological work experience.

               I also thank Indian National Academy of Sciences for offering me with the Summer Research Fellowship Program 2019 and their efforts to fulfill the facilities for the trainees to work efficiently.

              Last but not the least, my whole hearted thanks to my friends Swaathy, Pavithra, Jayachendrayan, Ajay, Sathyaseelan, Jegadeesh for being there whenever I needed their help.

              Source

              • : A Textbook on Fundamentals and Applications of Nanotechnology by K. S. Subramanian-2018
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