The Antifungal Activity Of Dispersed Water

Published Date: 02 Nov 2017

ABSTRACT

The antifungal activity of dispersed water based solution of copper oxide (CuO), zinc oxide (ZnO), titanium dioxide (TiO2), silver (Ag) nanoparticle and antiviral nanoparticle (AVNP) prepared by sonication method was applied for in-vitro study on Leptosphaeria maculans and Leptosphaeria biglobosa on culture disc with PDA as media. It was also applied for in-vivo study on the oil seed rape plants i.e. Catana and Topas. From the above applied nanoparticles the studies revealed that different concentrations brought about substantial inhibition in the germination of spores of Leptosphaeria maculans and Leptosphaeria biglobosa. However, In In-vivo study it was revealed that the CuO, ZnO and TiO2 nanoparticle particles were the best constitute material due to their less damage rate i.e. 0% all of them respectively. On the other side Ag and AVNP showed high damage rate i.e. 66.66% and 100% respectively. Thereby Ag and AVNP nanoparticles can`t be beneficial to the oil seed rape plants.

Introduction:

Nanoparticle:

Over a past few years the nanoparticles have developed a huge interest in applications of science. The reason of enormous interest is because of its size, surface morphology and properties. It is smaller than one micrometre i.e. 1e-9m. All NP’s have different physical and chemical properties[1, 2] then other nanoparticles [3], even though they have different properties then a normal size particles of its own kind. The materials physical and chemical property alters as they reach to nano scale in size.

Nanoparticles are used in many fields to increase the efficiency of many products. It has been discovered that a nanoparticles are more effective to kill different kind of bacteria's and fungi which can be harmful to the environment, such as plants, crops[4]. To fight back bacteria`s these tiny particles can be a hopeful material. Different research shows that properties can be found in metallic and metal oxide nanoparticles and they have been examined extensively as a possible antibacterial agent. The surface interaction of nanoparticles with bacteria’s provides antibacterial activity. The wide range of possible antibacterial activities carries out by the large surface morphology of the nanoparticles which increases their contact with the bacteria’s. There are few nanoparticles which are already toxic and effective enough to kill bacteria. This report will show the importance of nanoparticles to kill bacteria's on plants without harming plant itself. Further nanoparticles can be applied to kill bacterial fungus over the plants. Figure 1 shows the applicability of the particles and how the inhibition process works.

Figure . Inhibition zone over the test material after applying nanoparticles.

Figure 1 illustrates the mechanism of inhibition activity a) Inhibitory band the area where no growth of bacteria is possible; b) Test Material and c) Area where growth of bacteria is observed. Chen et al., investigated that the metal oxides antimicrobial activity depends on many environmental and treatment factors such as humidity, temperature, UV irradiation time, surface treatment, and nanoparticles surface morphology [5].

Later a good example of fibre coating for protection from virus and bacterial infusion can be presented from figure 2. The figure represents the coated tiny nanoparticle and micro particles on the surface of the fibre.

Figure . The coating of giber with micro and nanoparticles for antimicrobial.

Literature Survey:

Approaching nanoparticle as an antimicrobial agent is increasing on large scale. With the time the different viruses, bacteria’s and small organism that are harmful to the nature are increasing due to the use of high toxic technological advancements. Fighting back mechanism is being innovated to kill these bacteria. For this different scientists from various departments are working to kill these bacterial forms by means of new pesticides or some new materials. In the same manner material scientists has devised that the nanoparticle is beneficial due to their surface morphology, toxic properties and particle size. Out of these nanoparticles the best nanoparticle that is being used widely is Silver nanoparticles (SN). Later scientists have also found that the TiO2 is also good to use as a disinfectant to water and helps in water purifications. In the similar manner other metal oxide nanoparticles are green synthesised by ordinarily mixing of nanoparticles to moderately non-lethal chemicals to integrate nanomaterials and incorporate the utilization of non-lethal solvents for example water. In the same attribute the Arachis hypogaea leaves forces biomolecules for example proteins [6], which could be utilized as decreasing operator to respond with chromium particles and as frameworks to helps in the development of Cr2O3 NPs in result. These chromium particles help to retaliate the E.Coli in the PDA mixture and acts as a antimicrobial agent [7].

Tissue culture techniques are conventionally used in the academic court of conduct and in plant pathology [8]. Currently the SN and TiO2 nanoparticles are most widely used nanoparticles for consumer product and industrial applications to fight back the bacterial fungi.

2.1 Silver nanoparticles:

The use of silver was first recognized as an antibacterial agent in 19th Century for medicinal use. Silver compound has been utilized to deal with scorch, Cut and infections [9]. Different silver salts have been used as antibacterial agents [10, 11]. According to the studies antibacterial activities have been found in silver nanoparticles. To retaliate the plant pathogens, bacteria`s and fungi silver nanoparticles has immerged as high usage nanomaterial in applications such as retaliating of plant phytopathogens [23,24]. Ag Nps on large overview having a capability of antimicrobial activity can diminish different plant diseases initiated by spores generating fungal pathogens [25]. In an overview the Ag nanoparticles has shown high potential nanoparticle for antifungal activity like in overcoming fungal plant pathogens. However good thing about silver is that it has less effect on human and animal toxicity. Different strategies of applying these nanoparticles on a broad range of biological pathways of microbes helps in enhancing the resistance capabilities of plants, which has gained an importance in terms of issues like the chemical management of plant fungal diseases. Silver nanoparticles efficiency depends on the applicability time period, but preventative measures are necessary before the spores start to penetrate and colonize it within the plant tissues. Researches has also revealed that on extended applicability of silver nanoparticle for can control B. sorokiniana and M. grisea plant pathogens ref (Antifungal Activity of Silver Ions and Nanoparticles on Phytopathogenic Fungi Young-Ki Jo).Furthermore the researches have shown that the silver nanoparticles releasing silver ions have high impact over the chloride ions as well as the have high stability as a carrier to fight fungi pathogens. Environmental tracking of toxicity and harmfulness of silver nanoparticle is necessary for future recognition of the nanoparticle as a fungicide for crop protection. The silver nanoparticle was used to enhance the antifungal effect of the fluconazole agent over the testfungi. Maximum inhibition was presented by Fluconazole in a comibined form with SN against C. albicans as well as on P. glomerata and Trichoderma but over P. herbarum and F. semitectum the combined mixture of SN with Fluconazole did not show any inhibitive activity.

The use of SN nanoparticles is on high rise due to the antimicrobial property of silver has been investigated and applied more broadly than any other inorganic antibacterial agent. The non-toxic nature of SN shows high applicability in eliminating microorganisms, fungus, bacteria and viruses. The SN also shows antimicrobial activity towards biological processes in microorganisms i.e. cell membrane structure and functional alterations. The lipids, proteins and lipopolysaccharides (LPS) intact with cell wall of the Gram-negative provide active protection against biocides whereas there is no LPS in Gram-positive [7]. Protein inhibitions can be found with SN that are associated with ATP productions, although some of SN`s antimicrobial mechanisms are not completely understood. However, SN`s in low concentration have no toxic effects on humans. Thereby pharmaceutical and biological industries are on high rise of using silver as an antibiotic or antimicrobial agent [8].

2.2 CuO Nanoparticles:

CuO nanoparticles due to their toxic effect [12]are highly antibacterial in nature and many studies show that they are capable of fighting [13] and killing the bacterial viruses on plants. Krithiga investigated that by culturing the bacterial colony in YPD broth culture plates later the CuO nanoparticles were applied to inhibit the production of the colonies [13]. They further on tested the antibacterial activity of the CuO nanoparticles against skin infected pathogens like E.coli and S.aureus by Resazurin dye reduction method and disc diffusion method. The antifungal activity of the CuO nanoparticles are in huge focus.

The effect of antifungal was successfully tested on different fungus and bacteria’s by Ramyadevi as shown in figure --[14]. The CuO nanoparticles were applied for the antibacterial and antifugal testing over various fungus and bacteria. The antibacterial effect was recorded to be higher than the antifungal effect i.e. M.Luteus 16nm, S. aureus 21nm, E.Coli 26nm, K.Pneumoiae 15nm and P. aeruginosa 5nm have greater inhibition zone than the antifugal effect on the A.Niger 16nm, A.Falvus 13nm and C.albicans 23nms.

Figure . CuO antibacterial and antifungal effect.

Nicola Cioffi, Luisa Torsi et al. investigated in their study of antibacterial and antifungal effect of CuO nanoparticles that when CuO is mixed with Polymeric composites the rate of release of CuO are proportional to the rate of inhibition and the rate of release of ions with different polymer composites are presented in figure 2. This means that the Cu when composite with PVMK releases more ions in culture broth than any other composite Cu. But the release of Bulk Cu is obvious to be higher than the composite ones [15].

Figure . Release rate of Cu-composite in Culture broth.

2.3 TiO2 Nanoparticles:

TiO2 nanoparticles having high refractive index and photocatalytic properties, particularly in the anatase form induces big impact on the fungus and bacteria killing. This activity of TiO2 nanoparticles makes a thin coatings of a film material demonstrating self-cleaning and disinfecting properties under exposure to ultraviolet(UV) radiation. There by they are widely used in consumer products like sunscreen, cosmetics and etc. TiO2 is also use as a water treatment agent since it is non-toxic by ingestion and low-cost. TiO2 material properties make it a candidate for applications such as medical devices, food preparation surfaces, air conditioning filters, and etcetera. Both Gram-negative and Gram-positive bacteria can be killed by TiO2, but due to the formation of spore in Gram-positive bacteria they are less sensitive to TiO2 nanoparticles. Recently these nanoparticles have shown a high inhibition towards killing of viruses including poliovirus, hepatitis B virus, Herpes simplex virus, and bacteriophage [5]. In plant pathology, there has been low research work of using nanomaterial in plant tissue culture for remove of bacteria. Until now it`s hard to investigate any work that used TiO2 in plant tissue culture media.

Later TiO2 films were applied on wood under UVA (365 nm) to investigate irradiation experimentally. The illumination of light was stopped and the spore growths were observed under the influence of intermittent UVA irradiation. Observations evaluated the inhibition and inactivation effects of TiO2 by comparative regrowth. Two main studies were analysed 1) Inhibition of spores of mold fungi A. niger. the UVA light photocatalytic disinfection processes are effective for mold infestation on moisture-damaged wood boards. They can be further inhibited during cultivation under UVA irradiation. However in door lights are not highly capable enough to deactivate fungi on TiO2; 2) after stopping UVA irradiation on TiO2-treated wet wood board spores were found to be still reproducible. Nevertheless, mold growth can be postponed by irradiation on wet wood material [5].

2.4 Antiviral nanoparticles:

Antiviral nanoparticles also known as AVNP. AVNP was first patented by QinetiQ Material England the contributors were Dr. Guogang Ren from Queen`s Mary University of London and his partner Dr. Paul Reip. They awarded a research prize of 2million GBP to enhance their research and production of nanoparticles by South East England Development Agency (SEEDA). They claimed that these particles are really good in fighting back the SARS flu and Avian flu virus on contact with the virus. There by these particles have not been reported in antifungal activity there by the testing of these particles was necessary to overlook the greater perspective of these particles.

2.5 ZnO nanoparticles:

ZnO same like TiO2 nanoparticles is really effective to light sources and shows high antibacterial and antifungal effect under light. The ZnO films antifungal activity was investigate by R. Renuka and Ramamurthi earlier in year 2000 [16]. The Antifungal effect of the ZnO films was recorded to be really effective for inhibiting the fungus and bacteria as noted in Table—about fungus.

Fungus

Antifungal effect

Inihibition zones

Anodized specimen (m)

Oxidized Specimen (m)

Aspergillus terreus

0.002

0.004

Aspergillus nidulans

0.0008

0.0015

Penicillium frequentans

0.002

0.004

Penicillium rubrum

0.0015

0.005

Penicillium perpurogenum

0.002

0.005

Paecilomyces varioti

0.0008

0.0015

Candida albicans

0.0009

0.002

Aspergillus flavus

0.002

0.005

Aspergillus Niger

0.0015

0.005

Aspergillus Terreus

0.002

0.004

This gives an over view that if the films of ZnO are so effective than the ZnO nanoparticles due to their nanoparticles properties will have higher possibilities to inhibit the fungus and bacteria. The confined small diameter size of ZnO helps to maintain the transparency in water and gives high antifungal and antibacterial properties to fight back and kill bacteria and fungi. Due to the small sizes of super fine nanoparticles of ZnO that are produced helps the particles to hardly observe the light and giving sunscreen properties.

Further Sumitomo Osaka Cement Co., Ltd produced really fined nanopowder of ZnO and tested its antifungal and antibacterial properties which are presented in figure 4. The use of ZnO nanopowder is also similar to CuO nanoparticle in killing of bacteria since the bacteria used to impede were similar as presented in figure 2. This shows that the ZnO nanoparticles are even highly capable of killing the bacteria and fungi. For bacterial killing really small amount of concentration was used to kill bacterial roughly around 100-500 ppm.

Figure . ZnO nanoparticles antibacterial and antifungal effect.

The figure 4 shows the killing of bacteria and fungi using ZnO nanoparticles. The different sign conventions used in the graph donate the inhibition and impeding activity of nanoparticle towards the fungi and bacteria. The highest inhibition can be seen in the case of fungi.

Now applicability of the nanoparticles is really wide and for this research the nanoparticles were applied on LM and LB to overcome phoma stem canker.

2.6 Phoma stem canker:

In oil seeed rape cultivation worldwide a common problem that is prevailing is the increase of the phoma stem canker due to the two very common funus in oil seed rape cultivation i.e. Leptosphaeria maculans and Leptosphaeria Biglobosa. This pathogen in mycological studies is described as pseudothecal loculo-ascomycete containing pycnidial phase termed as phoma lingam [17]. The name of the disease has been called on the basis of fungus asexual stage i.e. Phoma lingum [18, 19]. This disease has gain extreme importance due to its effect in the oil seed rape plant around Australia, Canada, and Europe [19]. But in other countries such as China and India, it`s rarely found.

L. Maculan is one of the kind of hemibiotrophic pathogen. It is fungi that requires living host cells but is able to survive on host cell once it`s passed away. There are two types of Leptosphaeria species derived from their ancestor. 1) Leptosphaeria Maculan (LM) and 2) Leptosphaeria Biglobosa (LB). LM is more virulent and causes grey-green lesions on the leaves and infect the host causing large damage to basal part of stem. Whereas the LB is avirulent and induces small and dark leaf spots and causes superficial lesions on the upper part of stem but they don’t cause extreme damage to yield output [19]. Figure 6 show two different types of fungi that are common to grow on oil seed rape plants i.e LM and LB. In vitro, it is observed that the LB grows quickly than LM in culture media. LB produces yellow pigments (water soluble) that gives a contrast in between LM and LB. The LM and LB can also be distinguished on in vivo test by the germination speed and phytotoxins such as serodesmins of LM.

Figure . Two different types of Leptosphaeria fungi. LM is on the lower part of stem and LB is on the upper part of stem.

In-Vitro:

The reason to use in-vitro is to experimentally test that are conducted for fiction studies before implementing it to the real object i.e. towards nature (Human or animal or plants). If In-vitro is not successful so real application tests are avoided, it might be hazardous to the nature. There by it is a good practice in scientific world of plant pathology, human anatomy, zoology and botany to apply in-vitro study before proceeding towards the real experiment without acquiring the knowledge base or consequences of the material or technique being imposed to real object.

In-Vivo:

The Vivo-Tests are conducted on real applications after the accomplishment of the in-vitro test. These tests are conducted on ethical basis. These tests can be hazardous but after high end in-vitro test it is implemented to real applications.

Materials and methodology:

Dispersion of Nanofluids:

Nanofluids was prepared before the testing was carried for In-Vitro and in-vivo experiments. The preparation of nanofluids was carried using ultrasonic device. Three different concentrations of nanoparticles i.e. 100ppm, 500ppm, and 1000ppm were dispersed in Deionized water.

Ultrasonic device:

The ultrasonic device was used to disperse nanoparticles in water; without this device it would been hard to disperse the nanoparticles homogeneously.

Further this and the work that will be followed after this has been shown in figure –

Figure . Dispersion mechanism of nanoparticles and applicability on fungus.

3.1 In vitro materials:

The materials which were used for in-vitro experiment were nanoparticles, antibiotics, Potato Dextrose Agar, fungi (LB and LM) and water as basefluid.

EQUIPMENT USED

Characterisation of Nanoparticles:

The nanoparticles were characterized for the analysis of the nanoparticle size and shape.

Transmission Electron Mission:

The transmission electron microscopy images were used to analyse the size and the shape of the nanoparticles.

3.1.1 Nanoparticles:

Nanoparticles that were approached for the experiment in through this study are mentioned below:

Table

Nanoparticle TEM image (All right reserved by Dr. Guogang Ren papers)

Figure CuO nanoparticles TEM Image [20] Figure Silver nanoparticles TEM Image [21]

Figure ZnO nanoparticles TEM Image [22] Figure TiO2 nanoparticles TEM image[23]

Antiviral nanoparticles AVNP (composite of different nanoparticles)

3.1.2 Fungus:

Fungus that was implemented used for in-vitro experiment was L.Maculans and L.Biglobosa. The fungi can be seen in figure 6.

3.1.3 Culture media:

Potato Dextrose Agar (PDA) was used as culture to cultivate the fungi and to impede the growth of the fungi and to apply nanoparticles for inhibition of fungi.

3.1.4 Antibiotic

The antibiotics that were used to implant in the media culture were Penicilin and Streptomycin.

3.1.5 Method in Vitro

In-Vitro experiment preparation was carried using dispersion of nanoparticles in basefluid water by help of sonication. There were 3 different nanoparticles that were dispersed with 3 different concentrations i.e. 100ppm, 500ppm and 1000ppm. Different nanoparticles that were used are mention in table1. After the dispersion the nanofluid was applied for in-vitro test. In in-vitro test the PDA was prepared by autoclaving method at temperature 150-180°C.

PDA was transferred to 9cm petridish by using pipette boy. Later two different sets of petridishs were made. Each set containing 13 petridishes. Initially in the beaker holding PDA antibiotic was dispersed by pipette. All petridishes will be negative controlled i.e. media+ antibiotics. First set was a control one with PDA and 100μL antibiotic (Penicillin and streptomycin) that was transferred from beaker into a petridish.

To calculate the amount of petri dishes needed for treatment it was obtained from:

Where ω is the amount of petri dishes needed for treatment experiment. There by after calculations it was found that approximate 13 petri dishes will be needed.

15 ml of PDA with antibiotic was pipette out by pipette boy at this stage carefully transfer the media into petri dish and avoid bubbling of solution, make sure the surface of media being distributed on petri dish is uniform and make sure the media is transferred as quick as possible so that the media does not solidify and give rooms to inaccuracies. Then repeat the pipette transfer in 13 petri dishes for 13 replicates of experiment for control one and other sets with nanoparticle.

Before the sample is poured mark at the bottom of petri dishes about the concentration of nanoparticle, name of nanoparticles and name of fungi. The name convention will be applied on it to make it easy for ourselves to recognise the dish in future.

Figure . In vitro experiment flow chart.

Second set was PDA+antibiotic and further nanofluid with different concentration was added into other petri dish. For different concentrations for e.g. 100ppm in 15ml PDA 20μL nanoparticles was added as shown in figure 11. While adding different concentrated nanoparticles it necessary to change pipette boy since the contamination of the old sample can affect the transfer in new petri dish, also make sure while adding avoid contacting the pipette boy tip to any surface otherwise it will be contaminate.

After putting PDA in the dishes we have to keep these dishes in refrigerator and let PDA dry. When it is dry apply fungi on it. Procedure to apply fungi on it is as follow. The fungus was taken from the tray, by using a puncture whose size was 0.4x0.4cm.Then it was transferred to petridishes in which it was inoculated, at the centre of the dish, on the top of the PDA’s surface. The puncture tip was heated so that the fungus which is being picked up from the fungus plate other contaminations can be killed before applying it to the sample of our interest. Other reason is to softly immerse the puncture in the PDA media. After heating the puncture don’t take the fungus directly or else due to extreme temperature it might kill the fungus itself so it`s better to give 4-5 minutes after heating and then apply the fungus to the media. These prepared samples were then contained in an contained and shielded with a black cover so that no light can affect the samples or else the fungus will not germinate, since the dark environment helps in germination process of fungus. After every 3 days the size was measure and this was continued for 21 days. These reading were taken to analyse the effect of nanoparticle antifungal effect on the fungi growth.

Calculating these reading were done using equation 2 as shown below:

3.2 In vivo materials:

Materials that were used for in vivo experiment were Oilseed rape plants (Catana and Topas), nanoparticles and basefluid. The materials that we used for in vivo is plain plastic tray, rectangle rug form, plastic tray with 24 pockets, seeds, two different types of composed, water, plant moisturizer .

In the in-vivo material the nanoparticles that were used were a) CuO nanoparticles, b) Silver nanoparticles, c) TiO2 nanoparitlces, d) ZnO nanoparticles, and f) Antiviral nanoparticles.

Different nanoparticles were mostly in the range of 50-200nm. These particles are best for most of the antibacterial and antifungal effect suggested from different studies [24-29].

Method:

In vivo two seed will be used i.e. Catana and Topas (Oilseed rape). For doing experiments 3 different trays were used, 3 different trays were used so that 3 sets of replicates can be makeover. In each tray there are 24 pockets containing 6 rows and 4 columns. From the very left in 5 columns there will be catana plants and on the very right column there will topas. Catana was put in 3 seed per pockets and topas was inserted in 2 seed per pockets.

From the composed soil, the first things which is required is to remove any unwanted dust or stones or else they will not help in germination of plant properly. Then mix the two different soil composed by help of hands. In the plain tray the rectangular rug will be placed then 24 plastic trays will be placed over the surface of rug. Later the composed which was mixed will be put into the pockets of the tray. After this press the composed soil with hands firmly so that no air/bubbles or pores are open or else germination process might take more time. If the composed is compressed too much then more soil composed shall be added the pocket until the pockets are filled completely. Then after this make a hole in the centre of each pockets for inoculation of seeds but make sure that hole not too much deep inside the soil or else the plant will take longer time to germinate. Now after all this the 3 seeds will be inserted in the centre of the hole 5 sets of Catana in left columns and in very right column 2 Topas seed will be inserted each hole very right column. After inserting the seed them smooth the surface of the composed soil by hands and then provide plant moisturizer over the tray. Then after this transfer water in the tray that is underneath the composed soil tray, by doing this rug will suck the water and later on helps for germination of plants. Later throw some water on the composed soli tray surface. After this take the trays in the glass house and place them under sunlight. Later on look after the plants by providing water on weekly basis and check if there are any damaged leave so cut them off.

Now applying nanoparticles:

After one month the plants were ready for in-vivo experiment. First stick was inserted in the soil and the stem of plants were attached to the stick so that they germinate in nicely straight forward method so that later on its easy to apply nanoparticles. Before applying the nanoparitcles the chart will be made for storing the reading regarding the nanopartciles.

Characterization of Samples:

After the inoculation of plants the nanoparticles were applied over the surface of plants. Later they were characterized for effect that was produced by nanoparticles on the surface of the plants.

Microscope:

The characterization of samples for in-vivo was performed using Microscope. Microscope was used to analyse the surface and the locations of the burns created due to the applied nanoparticles. Further the un-burn surfaces of the similar leaves were compared with the burnt ones.

Results:

The potential results of the research are given in this section.

4.1Nanofluid preparation:

The preparation of nanofluids was carried by sonication. The sonication was carried at 350 watts for 10 minutes each sample.

4.2 In-Vitro results:

As the solution was mixed with nanofluid i.e. roughly 200 μL, it was then vortexed but as its already known that the solution solidifies as it comes to room temperature it was hard to disperse it homogeneously well enough to get nanoparticles dispersed properly in solution. This gives rise to higher inaccuracies of results that were achieved. This showed that to mix or disperse the solutions it’s necessary to homogeneously dispersed them before applying the fungus.

Remedy:

To remove the inaccuracies, it will be better in future to carefully, homogeneously and uniformly disperse the nanofluid in the solution containing PDA and antibiotic to be dispersed well.

4.1.1 TiO2 nanoparticles effect on fungi:

Figure . TiO2 nanoparticles applied on fungi; a) TiO2 nanoparticle on L.M; b) TiO2 nanparitcles on L.B.

TiO2 nanoparticles were effective on both the fungi i.e. LM and LB. From the figure 12 it can be seen that the as the concentration was increased to 1000 ppm the nanoparticles showed high rate of inhibition. Thereby from both the graphs i.e. Figure 12 a and b it can be concluded that the TiO2 inhibition is way better than control in-vitro experiment. But in case of 100 ppm used for LM the inhibition rate was similar as control as seen from figure 12a.

4.1.2 ZnO Nanoparticles effects on fungi:

Figure .ZnO nanoparticles applied on fungi; a) ZnO nanoparticle on L.M; b) ZnO nanparitcles on L.B.

ZnO nanoparticles were not effective on both the fungi i.e. LM and LB. From the figure 13 it can be seen that the as the concentration was increased to 1000 ppm the nanoparticles showed no inhibition. Thereby from both the graphs i.e. Figure 13 a and b it can be concluded that the ZnO is not capable of inhibition in-vitro experiment.

4.1.3 CuO Nanoparticles effects on fungi:

Figure . CuO nanoparticles applied on fungi; a) CuO nanoparticle on L.M; b) CuO nanparitcles on L.B.

CuO nanoparticles were effective on both the fungi i.e. LM and LB. From the figure 14 it can be seen that at lower concentration the effect of inhibition was less, but as the concentration was increased to 1000 ppm the nanoparticles showed high rate of inhibition. Thereby from both the graphs i.e. Figure 14 a and b it can be concluded that the 1000ppm CuO inhibition is way better than control in-vitro experiment. But in case of 100 ppm used for LM the inhibition rate was not good as control as seen from figure 14a.

Figure . Maximum inhibition by different nanoparticles applied on fungi; a) L.M; b) L.B.

The maximum inhibition applied on fungi by nanoparticles after 21days can be seen from the figure 14 and 15. The maximum impeding of the growth by different nanoparticles has been shown in figure 14 and 15, from those figure it can be seen that for the first case i.e. in LM CuO shows high rate of impeding than that of other nanoparticles and for LB the best impeding of fungi growth is shown by TiO2. In both the case ZnO is not as good as TiO and CuO.

Figure . Moderate inhibition by different nanoparticles applied on fungi; a) L.M; b) L.B.

The moderate inhibition applied on fungi by nanoparticles after 21days can be seen from the figure 16. The moderate impeding of the growth by different nanoparticles has been shown in figure 16, from those figure it can be seen that for the first case i.e. in LM TiO2 shows moderate rate of impeding than that of other nanoparticles and for LB the moderate impeding of fungi growth is shown by ZnO.

Figure . Minimum inhibition by different nanoparticles applied on fungi; a) L.M; b) L.B.

The minimum inhibition applied on fungi by nanoparticles after 21days can be seen from the figure 17. The minimum impeding of the growth by different nanoparticles has been shown in figure 17, from those figure it can be seen that for the first case i.e. in LM ZnO shows low rate of impeding than that of other nanoparticles and for LB the low impeding of fungi growth is shown by CuO. In both the case TiO2 has good inhibition rate than CuO and ZnO.

In-Vivo results and figures:

The test of invivo was carried to analyse the toxicity of the nanoparticles on the plants. In the in-vivo test the nanoparticles represented different effects on Catana and Topas plant. The effect that were analysed was the change of the toxicity and how the plant structure changes due to applying of nanoparticles. Secondly the in-vivo test were performed to analyse the damage rate on the plants. The equation that was used in this work was derived on the basis of the equation by Kabir Lamsal [30] i.e. related to the percentage inhibition rate.

%

Where R is the rate of growth of Fungi in control plate and r is the radial growth of fungi in nanoparticle palte.

These damage rate are shown in the section --.

In-Vivo Discussion of inaccuracies of results:

Below are the figure and table showing the nanoparticle effect on in-vivo experiments that was conducted on Oil seed rape plant i.e. Catana and Topas.

In-Vivo Test results of oil seed rape plants damage rate and leaf conditions are mentioned and were analysed for toxicity effect if nanoparticles over plants.

Figure . Damage rate of Catana leaves using different nanoparticles Damage rate(Catana Leaf 1&2).

The damage rate was tested and analysed to investigate the effect of the toxicity of nanoparticles on the Catan leaves. Different concentration nanofluid was prepared for testing.

Figure . Leaf condition after applying nanoparticle (Leaf Condition (Catana leaf 1&2)).

Leaf conditions were tested by visual observations as shown in figure 21, from figure 21 it can be concluded that he best nanoparticles that represented the leaf health stable after the application of nanoparticles were CuO and ZnO on most of the different concentrations.

Figure . AVNP rate of damage on Catana and Topas.

AVNP was tested on two different type of oil seed rape i.e. Topas and Catana. The damage rate of AVNP on Topas was much better as compared to Catana specially the 1000ppm concentration was the best one since that was around 1 unit of damage.

Figure . Damage rate of different nanoparticles over plants.( Damage rate units = unit)

Damage rate was estimated using the visual observation analysis. After doing this it was analyse that the CuO, ZnO and TiO2 were the best nanoparticles since they did not damage the plants. On the other hand AVNP was not that good and the Ag was the worst of all.

Figure . % damage rate of nanoparticle on plants.

The% damage rate in figure24 shows that the CuO,TiO2 and ZnO were best in sences of toxicity effect and also there conditions were way better than Ag and AVNP. The % damage was calculated by equation --.

Discussion:

Nanofluid discussion:

Most of the nanoparticles showed nice dispersion in water but the silver nanoparticles did not show high dispersibility and this can be possibly due to the hydrophobic nature of the Ag nanoparticles as shown in figure --. The best dispersibility was shown by TiO2 and ZnO nanoparticle whereas the CuO was even smoothly dispersed. AVNP was also homogeneously dispersed. All the dispersion uniform and smooth dispersion was characterized by visual inspections.

Figure . Ag nanoparticles dispersion in water and their hydrophobic effect.

Ag nanoparticles this phenomena of undispersibility improvement can be seen in the study stated by Isabella romer [31].In her they prepare the silver nanoparticles with different media and the best nanoparticle size which they got was with citrate ions in that solutions. This means citrate attachment can help in the improvement of the nanoparticles dispersibility in water. If the nanoparticles are not well dispersed in water the concentration will have huge impact since the ideal condition of concentration was not achieved that why when Ag nanoparticles were applied on the plant showed unrealistic effect on plants. This conclude that it`s necessary that dispersion is properly achieved before approaching any application that is related to dispersibility of nanoparticle in fluid and later use. This is why lots of studies is being put together to make homogeneous dispersion of all the nanofluids in aqueous solution due its wide range of applications.

Before applying the nanoparticles to plants, first wipe the plants with tissue so that the wax is removed from the surface of the plant, if this wax is not removed the nanofluid solution will not settle on the top of the plant surface. Since the surface of the plant with wax is more hydrophobic so it will repel the nanofluid as shown in figure 13.

Figure . Removal of wax from the top surface of plant. a) The wax not removed surface. b) The wax removed from the top surface.

After removing the wax from the top surface then we will put 6 droplets of nanofluid on the surface of plant each droplet will be of 15 μL. Over the two leaves of similar plant 6 droplets will be dropped so that 2 replicates can be recorded. In 3 trays randomly the concentration of nanoparticles will be distributed. Random distribution will help in removing the inaccuracies of readings.

In-Vitro Discussion:

The nanoparticles test on in-vitro were quite significant and represented good inhibition growth towards the fugi. The maximum inhibition applied on fungi by nanoparticles after 21days can be seen from the figure 14 and 15. The maximum impeding of the growth by different nanoparticles has been shown in figure 14 and 15, from those figure it can be seen that for the first case i.e. in LM CuO shows high rate of impeding than that of other nanoparticles. The nanoparticles showed high rate of impeding and this can be back from the study of patented work by Isolde [32, 33] that copper oxide nanoparticle can be a benefit to fight the L. Maculans, in one of their patent they state about the phytotoxic effect of different compounds that can be used to fight different fungicide out of which the LM is one probable fungicide that can be killed using the nanoparticles. For LB the best impeding of fungi growth is shown by TiO2. In both the case ZnO is not as good as TiO and CuO.

To forestall any photocatalytic movement of TiO2 NP in medium, societies were brooded in an altogether dark regulated condition. From the effects, it is discovered that TiO2 NPs have a huge impact on callus measure and embryogenesis of calli while for callus measure between the redundancies of every medication state in in Vitro study of TiO2 nanoparticles that Influences on Barley (Hordeum vulgare L.) tissue Culture [34]. In our case it was seen similar that the TiO2 had a big influence on inhibiting the fungus growth rate this can be due to the photocatalytic effect of TiO2 nanoparticles.

The moderate inhibition applied on fungi by nanoparticles after 21days can be seen from the figure 16. The moderate impeding of the growth by different nanoparticles has been shown in figure 16, from those figure it can be seen that for the first case i.e. in LM TiO2 shows moderate rate of impeding than that of other nanoparticles and for LB the moderate impeding of fungi growth is shown by ZnO.

The minimum inhibition applied on fungi by nanoparticles after 21days can be seen from the figure 17. The minimum impeding of the growth by different nanoparticles has been shown in figure 17, from those figure it can be seen that for the first case i.e. in LM ZnO shows low rate of impeding than that of other nanoparticles and for LB the low impeding of fungi growth is shown by CuO. In both the case TiO2 has good inhibition rate than CuO and ZnO.

ZnO was not seem to work properly in this study of in-vitro but as for the antibacterial effect or antifungal effect is considered as a nanocomposite material the ZnO shows high possibility of applicability as investigated by Mahnaz Mandeh [34], in their work they state that Polyurethane based nanocomposites with 0.4 and 0.7 wt.% of a ZnO nanoparticles were ready for test of polyurethane varnishes on standard wood ground flooring based tests. The samples had nanocomposite coatings comprise of polymer framework in which micrometer silica and nanosized zinc oxide particles are scattered. It was indicated that the original polyurethane varnishes don't show antibacterial activity, however the antimicrobial impact of the ZnO nanoparticles was affirmed. In this way, development of the states of Pseudomonas aeruginosa and Saccharomyces cerevisiae was altogether hindered in presence of the ZnO nanoparticles. What's more, is needed 0.4 and 0.7 wt.% of the ZnO nanoparticles restrain development of Staphylococcus aureus by more than 85% and 95%.

Sources of Error:

No bubble formation:

While transferring the PDA into the Petri dishes makes sure the solution is transferred homogeneously and the bubbles formation is avoided since this effect the growth of fungi in culture media.

Inclination of petri dish:

After and during the transferring of the media to pertidish make sure the surface on which the petri dish is sitting is smooth and flat or else this will affect the growth of fungi on the culture media.

Fungi placement:

When you are taking the fungus to the media by help of scalpel make sure you don’t touch the media except from the centre point. Since if the media is touched from anywhere else it will cause the fungi to grow from that particular point. Or else we won’t be able to find the correct growth rate.

Fungi Puncture contamination:

If the puncture that was used previously for the first sample is used over the other sample without heating it can cause the previous fungi to germinate on the new media culture plates. Thereby its necessary to heat the puncture to remove contamination.

In-Vivo Discussion:

The effect of nanoparticle on in-vivo helps to kill fungi bacteria and in this study of In-Vivo the Catana and Topas plant were used. The application of nanoparticels over the plant had a huge impact. This was characterized by help of visual observations. Visually it was observed that different nanoparticles acts in different way to plant depending on their properties they pose. The best effective nanoparticle for in-vivo study was the CuO nanoparticle and TiO2.

In In-vivo experiment the nanoparticle showed high response after applying them in nanofluid form. The scale of the damage rate is signified by the 0-6 and even the other leave condition scale is from 0-6. For damage rate the significance of the damage is best at 0 and mostly this is used to signify plant as in good condition after applying the nanoparticles. After applying it was seen that nanoparticle has a big impact on leave condition and leave growth and damage rate. The best nanofluid that represented good result against toxicity by visual observation seem to be of TiO2 and ZnO nanoparticles due to their photocatalytic activity at the same time leave was even good there were no burnt present, the colour change of nanoparticle was visible that was blue this can be also due to photocatalytic effect of TiO2 and ZnO nanoparticles which help it to emit different light on excitation with light molecules. The concentration that showed amazing result were by 1000ppm TiO2 and 100ppm, 500ppm ZnO nanoparticles. Other concentrations were also not that bad but the overall conditions of the leave were not seemed to be highly proactive.

The nanoparticle that showed the worst case scenario was Ag and AVNP. The Ag nanoparticle leave condition was better quite similar like TiO2 and ZnO. The leave condition can be better due to its antibacterial effect but later on it made burns on the plant. Leave condition in AVNP was not that good as Ag and TiO2 nanoparticles. But the burns generated by AVNP were less severe than Ag nanoparticles.

Source of error for in –vivo

Concentration

While making the nanofluid the concentration that is being dispersed in fluid need to be homogeneously dispersed so that chance of error due to concentration are less. If the nanoparticle somehow like Ag nanoparticles does not disperse properly in the water it`s better to use some kind of surfactant to disperse the particle homogeneously. The concentration has a huge effect on the burning of leaves if the concentration is too high it can burn or damage the leave as it happened in case of Ag.

Droplet allocation

The allocation of droplet should be well oriented so that the droplet does not go to any other leave rather than the one on which its being dropped or else nanoparticles will mix and it will be hard to investigate and analyse them later on in future.

Plant health and safety

For plant health and safety make sure adequate amount of water is being supplied time to time so that plants don’t die out.

Waxing properly

While removing the wax from the leaves of the plant gently remove the wax by a tissue paper so that the plant does not torn out.

In-Vivo fungi plant apprearance

After doing all the above experiment some plants were left so further testing was done to analyse study of fungi on plants. It was approached by means of similar procedure to the earlier in-vivo plant except in this we applied fungi onver those plants. The main reason to inoculate the fungi was to apply nanoparticle over it and check the toxicity and the inhibitory rate of the nanoparticles but due to lack of time the application of nanoparticles over fungi could not be met and also the grown fungi was not that proper. Any how the plant on which fungi was applied was not properly grown but it was visually characterized for knowing what can be the possible reasons that let fungi not to grown properly. The visual characterization of results is:

Possible reason Fungi not appeared:

The fungi`s not appearing on plants can be caused due to few reasons

After inoculation water must be sprayed on inoculated leaves by water sprayer for few days which help fungi to grow.

Inoculated Plants must be covered in a dark because fungi germinate in dark.

Plants that were being used were not too fresh.

Plants need to be punctured on the areas where fungi had to apply.

Figure . CuO nanoparticles 500ppm applied on catana plant. a) Catana plant with the area where no nanoparticles were applied. b) Catana plant where nanoparticles were applied.

CuO C1 100ppm

ZnO C3 1000ppm

ZnO nanoparticle in fig -- were applied on the surface of plant and it was seen that the plant were really healthy only effect that was visualized was that there was a colour change due to photocatalytic effect.

Ag nanoparticles c1

. Ag nanoparticle 1000ppm.

In figure -- the image on top is the plant where the nanoparticle was not applied and other 6 figures below are the plant where nanoparticles were applied. It can be seen from the figure that the nanoparticles had severe effect on the plant condition and the plant had burns due to the applied nanoparticles. This can be possibly due to higher concentration.

Avnp catana c3

Avnp catana c1

Avnp catana c2

Cocnlusions:

In this study investigation was carried on the nanoparticle toxicological effect on the plants and in-vitro study of the nanoparticle on fungi. The nanoparticles have large applications in different fields of science in the same way scientist and researchers have extrapolated the study regarding the nanoparticles use as antimicrobial, antibacterial and antifungal. In the same way in this study we carried the work regarding the nanoparticle toxicology. The toxicological effect of nanoparticle can be benefit or either disastrous to plant growth and might be positive or negative to fungi inhibitions.

In this study it was found that the nanoparticle poses different properties when they come in contact with the in-vitro fungi. Some nanoparticles can inhibit fungi at high level of inhibition. In our study it was found that TiO2 and CuO represented the high rate of inhibition activity. For in-vivo study the CuO, ZnO and TiO2 showed the low rate of damage whereas the AVNP and Ag nanoparticle proceeded in damaging the plant on large scale. The leave conditions were best while using CuO, ZnO and TiO2 whereas for AVNP and Ag nanoparticle the conditions were not that well.

Future Work:

After doing this research many aspects of other research could not be overcome due to the short time period. Thereby for our future work it is suggested that the analysing of the properties such as toxicity of nanoparticle will be found. On the plant when fungus grows over the plant the nanoparticle can be applied to analyse the toxic and antifungal effect of nanoparticle on the fungus. Later other plants and in-vitro fungi will be tested for the antifungal activity of nanoparticles. The inhibition properties that were shown by particular nanoparticles in our study will be used for further research so that best concentration can be targeted to achieve the best results. Further on the modified nanoparticles will be tested on the fungal activity of plants. After the study of the nanoaprticles application over plants it was found that the CuO nanoparticles were best in sense that the particles did not harm the plant and showed the antibacterial effect. There by it can be suggested that the use of the Cu-O nanoparticles in future will be beneficial.