FIELD OF THE INVENTION
[0001] The enclosed describes an antimicrobial composition comprising inorganic
silver salt nanoparticles. More particularly, the present invention relates to a
composition of Trichoderma fused silver-based nanoparticles effective against
different human and agricultural pathogens and the production method thereof.
BACKGROUND OF THE INVENTION
[0002] Trichoderma have been known since at least the 1920s for their ability to
act as biocontrol agents against phytopathogenic fungi. The classical
mechanisms of control have included antibiosis, mycoparasitism, and
competition for nutrients. Trichoderma strains inhibit or kill plant-pathogenic
fungi through production of antifungal antibiotics and/or hydrolytic enzymes
(Shameli et al., 2012; Sarkar et al., 2007; Sato et al., 2004; Solomon et al.,
2007; Shahverdi et al., 2007). Trichoderma is a very effective biological mean
for plant disease management especially the soil born. It is a free-living fungus
which is common in soil and root ecosystems. It is highly interactive in root,
soil and foliar environments. It reduces growth, survival or infections caused
by pathogens by different mechanisms like competition, antibiosis,
mycoparasitism, hyphal interactions, and enzyme secretion. Trichoderma is
known as an important and significant bio-control agent. The classical
mechanisms of control have included antibiosis, myco-parasitism, and
competition for nutrients. The significant combination of Trichoderma with
synthetic salts/metallic ions can be utilized in treatment of different infections
within the plants and animals both.
[0003] Nanoparticles are viewed as the fundamental building blocks of
nanotechnology and are the starting points for preparing many nanostructured
materials and devices. Nanoparticles themselves have useful applications in
areas such as medicine and molecular biology research. In
particular, silver nanoparticles may be used as antimicrobial agents against

bacteria, viruses, and fungi, including drug-resistant strains of these
microorganisms. Typically, bacteria have diameters in the micron range, while
viruses have diameters less than a micron in size. The fact that
the silver nanoparticles are so small allows them to interact readily with such
microorganisms. These can be utilized as alternatives to different antibiotics
etc.
[0004] In the present invention relates to overcome the obstacles of the prior art
and emphasizes that, Trichoderma secretes some potent secondary metabolites
responsible for antimicrobial activity against opportunistic and nosocomial
infections causing pathogens. Both Trichoderma (biological) and silver
(Inorganic metallic ions) are known to have significant antimicrobial potency.
The combination of secondary metabolites from Trichoderma and Ag ions in
terms of fused nanoparticles can results in preparation of potent antimicrobial
and seed germination agents.
[0005] The information disclosed in this background of the disclosure section is
only for enhancement of understanding of the general background of the
invention and should not be taken as an acknowledgement or any form of
suggestion that this information forms the prior art already known to a person
skilled in the art.

OBJECTS OF THE INVENTION
[0006] The principal object of the present invention is to overcome the
disadvantages of the prior art and provide a composition of Trichoderma fused
silver-based.
[0007] Another object of the present invention is to provide a composition of
Trichoderma fused silver-based nanoparticles effective against different human
and agricultural pathogens and the production method thereof.
[0008] Still another object of the present invention is to provide a composition
with novel molecules and secondary metabolites, having effective antipathogenic activities against wide variety of plant and animal pathogens.
[0009] Yet another object of the present invention is to provide nanoparticles after
fusion with some metallic salts (green synthesis) such as silver which will have
profound effect on pathogens.
[0010] Another object of the present invention is to characterize the nanoparticles
via UV- absorption spectra and TEM.
[0011] Still another object of the present invention is to provide low cost and
inexpensive raw materials.
[0012] These and other objects and advantages of the present subject matter will
be apparent to a person skilled in the art after consideration of the following
detailed description taken into consideration with accompanying drawings in
which preferred embodiments of the present subject matter are illustrated.

SUMMARY OF THE INVENTION
[0013] In an important embodiment, the present invention relates to provide a
composition of Trichoderma fused silver-based nanoparticles effective against
different human and agricultural pathogens and the production method thereof.
[0014] In another embodiment the functionalizing substance from the fungus is
Kojic acid and it acts as a capping and reducing agent.
[0015] In a preferred embodiment the method of synthesizing Trichoderma fused
silver nanoparticles comprises of : a. isolation of Trichoderma species from
soil samples; b. providing a bio-filtrate of Trichoderma harzianum fungal
cells; c. exposing the Trichoderma harzianum biofltrate to a solution
containing 1mM silver nitrate under dark conditions and shaking for 70-80

hours, in which the fungal cells produce at least one enzyme or metabolite that
reduces silver ions to silver nanoparticles
[0016] In another embodiment the the solution is kept in dark condition at
29±10C under continuous shaking at 200 rpm for 72 h.
[0017] In yet another embodiment ethyl acetate is used as a solvent for the
extraction and purification of secondary metabolites.
[0018] In still another embodiment the antimicrobial compounds in Trichoderma
culture filtrate are identified by GC-MS.
[0019] In another preferred embodiment the biosynthesized silver nanoparticles
(AgNPs) are characterized by UV–Vis spectroscopy and Transmission electron
microscopy (TEM).
[0020] The foregoing summary is illustrative only and is not intended to be in any
way limiting. In addition to the illustrative aspects, embodiments, and features
described above, further aspects, embodiments, and features will become
apparent by reference to the drawings and the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS
[0021] It is to be noted, however, that the appended drawings illustrate only typical embodiments of the present subject matter and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments. The detailed description is described with reference to the accompanying figures. Some embodiments of system or methods in accordance with embodiments of the present subject matter are now described, by way of example, and with reference to the accompanying figures, in which:
[0022] Fig. 1 illustrates the (A) Trichoderma supernatant; (B) Change in colour of silver nitrate (from white to green) resulting in formation of Trichoderma fused silver nanoparticles, in accordance with an embodiment of the present invention;
[0023] Fig. 2 illustrates the Preparation of Trichoderma fused silver nanoparticles (TR-Ag Nps), in accordance with an embodiment of the present invention;
[0024] Fig. 3 illustrates the Observation of Trichoderma fused silver nanoparticles (TR-Ag Nps) as determined by SEM, in accordance with an embodiment of the present invention;
[0025] Fig. 4 illustrates the Antimicrobial activity of solvent extracts of Trichoderma, in accordance with an embodiment of the present invention;
[0026] Fig. 5 illustrates the Antimicrobial activity determination by well diffusion method of solvent extracts of Trichoderma, in accordance with an embodiment of the present invention;
[0027] Fig. 6 illustrates the Antimicrobial activity of solvent extracts of Trichoderma fused silver nanoparticles (TR-Ag Np), in accordance with an embodiment of the present invention;
[0028] Fig. 7 illustrates the Antimicrobial activity determination by well diffusion method of Trichoderma fused silver nanoparticles (TR-Ag Np), in accordance with an embodiment of the present invention;
[0029] Fig. 8 illustrates the GC-MS spectra of metabolites extracted in Trichoderma harzianum, in accordance with an embodiment of the present invention;
[0030] The figure depicts embodiments of the present subject matter for the purposes of illustration only. A person skilled in the art will easily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the disclosure described herein.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0031] While the embodiments of the disclosure are subject to various modifications and alternative forms, specific embodiment thereof have been shown by way of example in the figures and will be described below. It should be understood, however, that it is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternative falling within the scope of the disclosure.
[0032] The terms “comprises”, “comprising”, or any other variations thereof used in the disclosure, are intended to cover a non-exclusive inclusion, such that a device, system, assembly that comprises a list of components does not include only those components but may include other components not expressly listed or inherent to such system, or assembly, or device. In other words, one or more elements in a system or device proceeded by “comprises… a” does not, without more constraints, preclude the existence of other elements or additional elements in the system or device.
[0033] The present subject matter relates to provide a composition of Trichoderma fused silver-based nanoparticles effective against different human and agricultural pathogens and the production method thereof.
[0034] Reference may be made to Figure 1 illustrating the the (A) Trichoderma supernatant; (B) Change in colour of silver nitrate (from white to green) resulting in formation of Trichoderma fused silver nanoparticles, in accordance with an embodiment of the present invention;
[0035] Reference may be made to Figure 2 illustrating the Preparation of Trichoderma fused silver nanoparticles (TR-Ag Nps), in accordance with an embodiment of the present invention;
[0036] Reference may be made to Figure 3 illustrating the Observation of Trichoderma fused silver nanoparticles (TR-Ag Nps) as determined by SEM, in accordance with an embodiment of the present invention;
[0037] Reference may be made to Figure 4 illustrating Antimicrobial activity of solvent extracts of Trichoderma, in accordance with an embodiment of the present invention;
[0038] Reference may be made to Figure 5 illustrating the Antimicrobial activity determination by well diffusion method of solvent extracts of Trichoderma, in accordance with an embodiment of the present invention;
[0039] Reference may be made to Figure 6 illustrating the Antimicrobial activity of solvent extracts of Trichoderma fused silver nanoparticles (TR-Ag Np), in accordance with an embodiment of the present invention;
[0040] Reference may be made to Figure 7 illustrating the Antimicrobial activity determination by well diffusion method of Trichoderma fused silver nanoparticles (TR-Ag Np), in accordance with an embodiment of the present invention;
[0041] Reference may be made to Figure 8 illustrating the GC-MS spectra of metabolites extracted in Trichoderma harzianum, in accordance with an embodiment of the present invention;
[0042] In accordance with an embodiment of the present invention relates to Trichoderma, which is a free-living fungi common in soil and root ecosystems. They are opportunistic, avirulent plant symbionts and produce a variety of compounds that induce plant defense mechanisms. Interestingly, Hypocrea/Trichoderma spp. is even able to induce systemic resistance, which is characterized by the occurrence of disease control in the plant at a site distant from the location of Hypocrea/Trichoderma. They stimulate the production of low-molecular weight compounds that have antimicrobial activity like e.g. phytoalexins which are normally produced by plants in response to an attack by pathogens.
[0043] Methodology:
[0044] Example 1: Isolation of Trichoderma species from soil samples: To isolate Trichoderma from soil, a Trichoderma selective medium was prepared (Mohammad Akrami et al., 2011). The basal medium consisted of 0.2 g MgSO4 (7H2O), 0.9 g K2HPO4, 0.15 g KCl, 1.0 g NH4NO3, 3.0 g D-glucose anhydrous, 0.15 g rose bengal and 20g agar. These constituents were added to 950 ml of distilled water and autoclaved at 121°C for 30 minutes. The biocidal ingredient, 0.25g tetracycline was mixed in 50 ml of sterilized distilled water and added to the autoclaved basal medium where it cooled to 40 to 50°C. 10 grams of soil were suspended in 50 ml of sterile distilled water and agitated for 30 min at 200 rpm in a rotary shaker. Serial dilutions were made and 0.1 ml of each was spread on the Trichoderma selective medium plates with a glass rod. Three plates of each sample were prepared and incubated for 5 days at 30°C. Trichoderma isolates were collected and transferred onto potato dextrose agar (PDA) plates for maintaining pure culture (Guerillot et al., 1993) for 5 days at 28°C. Three hundred milli litres flasks were incubated for 14 days at 28°C on a laboratory incubator. The fungal filtrate was collected for further use.
[0045] Example 2: Preparation of Trichoderma filtrates and extraction of the filtrate: The filtrate of each fungus was extracted several times with ethyl acetate (v/v) in a separating funnel. The extracts from both mycelia and filtrate were evaporated under vaccum at 500C till dryness. The obtained solid material was dissolved in ethyl acetate to form the crude extract and tested for antimicrobial activity. For day optimization the fungus was grown in the malt extract medium at pH 6.2. Inoculated flasks were incubated at 270C on an incubator shaker for 8 days. The filtrate biomass production was determined each day for antibacterial activity.
[0046] Example 3: Preparation of silver nanoparticles: For the preparation of silver nanoparticles two stabilizing agents, sodium dodecyl sulphate (SDS) and sodium citrate were used. For the synthesis of silver nanoparticles, silver nitrate solution (from 1.0 mM) and 8% (w/w) sodium dodecyl sulphate (SDS) were used as a metal salt precursor and a stabilizing agent, respectively. Hydrazine hydrate solution with a concentrate (1. 0 mM) was used as reducing agents. The occurrence of colour was indicated by the formation of silver nanoparticles. The silver nanoparticles were further purified by centrifugation. To remove excess silver ions, the silver colloids were washed at least three times with deionized water under nitrogen stream. A dried powder of the nanosize silver was obtained by freeze-drying (Harman, 2004a; 2006b; Hider and Zhou, 2005; Howell, 1998; Initaj and Tae-Soo, 2007).
[0047] Example 4: Preparation of Trichoderma fused Silver nanoparticles: The silver nitrate (1 mM) solution was prepared in 50 ml deionised water. Fungal bio filtrate (excluding almost with biological mass) was brought in contact with the silver nitrate solution in a 200 ml Erlenmeyer flask. The solution was then kept in dark condition at 29±1 0C under continuous shaking at 200 rpm for 72 h. After 72 h of reaction time, the colour change was observed (Irtwange, 2006; Jarchem Industries, 2000; Kharchenko, 1999; Lee et al., 1950).
[0048] Example 5: Characterization of prepared nanoparticles via UV- absorption spectra and Transmission electron microscopy (TEM): The formation of AgNPs and TR-AgNPs by the bioreduction of Ag+ to Ag0 was easily monitored using UV–Vis spectroscopy. The scanning was performed in the range of 200–700 nm. The morphology and size was determined by TEM.
[0049] Example 6: Determination of antifungal activity of Trichoderma-Ag* fused nanoparticles against the fungal phyto-pathogens: Plant pathogenic fungal cultures viz. Fusarium oxysporum, Rhizoctonia solani, Fusarium monoliforme and Sclerotenia sclerotiorum were grown separately in 20 ml potato/dextrose/broth (PDB) in 250 ml flasks. The culture flasks were incubated on an orbital shaker at 150 rpm at 30 0C. Further the cultures were poured along with PDA in sterilized petriplates. Sterilized paper discs soaked with fused nanoparticles in different concentrations mentioned [viz. 5 (a), 10 (b), 15 (c), 20 (d), 25 (e) ppm] were placed on the culture plates at the peripheral region of the culture plate. The plates were further kept for 48-72 h for observation of growth inhibition if any. The inhibition of the radial growth was observed and recorded (Miller, 1959; Monte, 2001; Morton et al., 1950; Moubasher et al., 1977a; 1979b; Nallathambi et al., 2009; Shameli et al., 2012; Sarkar et al., 2007; Sato et al., 2004; Solomon et al., 2007; Shahverdi et al., 2007).
[0050] Example 7: Separation and identification of antimicrobial compounds in Trichoderma extract by GC-MS analysis : The GC-MS spectra was obtained by using Perkin Elmer GC Claurus 500 system and Gas Chromatograph interfaced to a Mass Spectrometer (GC/MS) equipped with Elite-1 fused silica capillary column (30 m × 0.25 mm) composed of 100 % dimethyl poly siloxane) from Perkin Pvt. Ltd., Germany. The carrier gas was helium. 70°C hold for 5 minutes and hold for 5 minutes. The control of the GC-MS system and the data peak processing were controlled by means of Shimadzu‘s GC-MS solution software, version 2.21. The crude metabolites extracted wasinjected within the column. Compound identification was verified based on the relative retention time and mass fragmentation pattern spectra with those of standards. The samples were prepared in methanol before analyzed by GC/MS.
[0051] Results:In the present investigation, Trichoderma fused silver nanoparticles (TR-Ag Nps) were produced [Figure 1 and 2]. The particle size was determined by SEM and their absorption spectrum was determined at 200-700 nm. It was observed that the TR-Ag Nps produced were of very fine shape and size having optimal 20 nm size. These nano particles were having slightly rough spherical structures which were observed in free and interconnected form (Figure 3). The UV absorption spectra of the fused nanoparticles recorded the maximum wavelength at 415 nm. The antifungal activity of the fused nanoparticles was evaluated against the plant pathogenic fungal cultures viz. Fusarium oxysporum, Rhizoctonia solani, Fusarium monoliforme and Sclerotenia sclerotiorum at different doses varying from 5 -25 ppm. It was observed that the fused nanoparticles had strong antifungal activity against the concerned pathogens. It was also observed that, by varying the concentration of doses of the fused nanoparticles, the radial axis of the fungal was getting decreased in more effective manner. The results are shown in Table 1 and figure 4& 5
[0052]
[0053] The antimicrobial activity of Trichoderma solvent extract and Trichoderma-Ag fused nanoparticles was evaluated against the bacterial and fungal pathogens viz. Pseudomonas aeruginosa, Bacillus subtilis, Micrococcus luteus, Leuconostoc mesentroides, Aspergillus niger and Aspergillus ochraceous by well diffusion method. The results were found to be surprising as both Trichoderma solvent extract and TR-Ag fused nanoparticles were having significant antimicrobial activity against the pathogens studied. Trichoderma solvent extract and Trichoderma fused silver nanoparticles (TR-Ag Np) showed any antimicrobial activity. Also no antifungal activity of each of these was found against Aspergillus niger and Aspergillus ochraceous at small doses but at 1000 µl were found to be effective against Aspergillus niger. The results are shown in Table 2 and Figure 6&7. The GC/MS analysis of ethyl acetate extract revealed the presence of 12 compounds. These compounds were determined as 4 volatile alcohols (1-4) and fatty acid esters (5-12). The GCMS spectra of Trichoderma metabolites showed the separation of fatty acids viz. Kojic acid, citric acid and acetic acid as the important components as determined by different peaks. The results showed the highest percentage of Kojic acid (65%), acetic acid (15%) and followed by citric acid (5%). The GC-MS spectrum is shown in Figure 8

[0054] Although embodiments for the present subject matter have been described in language specific to structural features, it is to be understood that the present subject matter is not necessarily limited to the specific features described. Rather, the specific features and methods are disclosed as embodiments for the present subject matter. Numerous modifications and adaptations of the system/component of the present invention will be apparent to those skilled in the art, and thus it is intended by the appended claims to cover all such modifications and adaptations which fall within the scope of the present subject matter.

We Claim:

1. An antimicrobial composition effective against different human and agricultural pathogens, comprising atleast one inorganic salt and a functionalizing substance from the fungus.
2. The antimicrobial composition as claimed in claim 1, wherein the inorganic salt is silver salt.
3. The antimicrobial composition as claimed in claim 1, wherein the fungus is Trichoderma.
4. The antimicrobial composition as claimed in claim 1, wherein the functionalizing substance from the fungus is Kojic acid.
5. A method of synthesizing Trichoderma fused silver nanoparticles, the method comprising:
a. isolation of Trichoderma species from soil samples;
b. providing a bio-filtrate of Trichoderma harzianum fungal cells;
c. exposing the Trichoderma harzianum biofltrate to a solution containing 1mM silver nitrate under dark conditions and shaking for 70-80 hours, in which the fungal cells produce at least one enzyme or metabolite that reduces silver ions to silver nanoparticles.
6. The antimicrobial composition as claimed in claim 5, wherein the solution is kept in dark condition at 29±10C under continuous shaking at 200 rpm for 72 h.
7. The antimicrobial composition as claimed in claim 1 or 5, wherein ethyl acetate is used as a solvent for the extraction and purification of secondary metabolites.
8. The antimicrobial composition as claimed in claim 1 or 5, wherein the antimicrobial compounds in Trichoderma culture filtrate are identified by GC-MS.
9. The antimicrobial composition as claimed in claim 1 or 5, wherein Trichoderma harzianum secretes secondary metabolites acting as a capping and reducing agent.
10. The antimicrobial composition as claimed in claim 1 or 5, wherein the biosynthesized silver nanoparticles (AgNPs) are characterized by UV–Vis spectroscopy and Transmission electron microscopy (TEM).