Description:Herbal nano-formulation for sustainable management of stalk and ear rot diseases in maize
FIELD OF INVENTION:
The present disclosure relates to the field of Agriculture, Nanotechnology, Herbal formulations and Plant Pathology. More particularly, it relates to an herbal nano formulation which can be utilized for sustainable management of stalk and ear rot diseases of maize.
BACKGROUND:
There are various technologies to deal with the attack of fungal phytopathogens but most of these are made up through synthetic chemicals which ultimately leave toxic residues in the environment. Various traditional practices have also been adopted by local farmers for the management of fungal pathogens which focus on chemical treatments, cultural practices, biological control agents, resistant crop varieties and traditional plant-based extract.
Chemical treatments are one of the most widely used techniques in agriculture by farmers for frequent action against the attack of fungal phytopathogens. Various systemic fungicides like Tebuconazole are used to minimize the attack of devastating pathogen Fusarium. The limitation of using this fungicide is the development of resistance in Fusarium strains and also they have toxic residual effects in the ecosystem. Chemicals like thiabendazole and fludioxonil are used as seed applicators to provide a shield against the infection caused by Fusarium. Various soil fumigants like methyl bromide are used to reduce the attack of pathogens but these fumigants deteriorate the environment.
Cultural practices are also one of the traditional methods to reduce the attack of pathogens. Farmers used various techniques like crop rotation, field sanitation, irrigation management and soil solarization. Munkvold, 2003 suggested that rotating the non-host crops helps to interrupt the pathogen life cycle and reduces their population in agricultural fields. In field sanitation practices, the destruction and removal of infected plants from the fields is essential because sometimes pathogen also survives on the residues of the plant. In soil solarization, the field soil is covered with transparent polyethene sheets to trap the sunlight which can reduce the pathogen diversity. In the technique of irrigation management, farmers abstain from over-irrigation which can reduce the arrival of pathogens through water.
Biological Control is also one of the existing techniques used to control the harmful effects of phytopathogens. It includes utilization of various natural antagonistic fungi like Trichoderma spp. and Gliocladium spp. to suppress the attack of Fusarium.
Resistant Crop Varieties is one of the most sustainable existing strategies against the pathogen Fusarium. Various molecular and traditional breeding techniques were utilized for the development of genetically resistant varieties of crops which are susceptible to pathogens. Hybrid varieties of maize are also developed with partial resistance to pathogens against Fusarium ear rot.
Traditional Plant-based extracts with vast antifungal potential are used to reduce the attack of Fusarium. Various plant extracts like neem, garlic, and turmeric are used to inhibit mycelial growth, disruption of the fungal cell wall and suppression of wilt disease in several crops respectively.
There are various problems and limitations associated with the existing technologies for the management of fungal phytopathogens. The excessive use of chemical treatments can lead to the development of resistant varieties and the accumulation of these toxic chemicals in crops which leads to detrimental health hazards. The cultural practices methods of reducing the attack of pathogens requires large manpower and it is not applicable for large fields and in severely infected crops. The biological control method of minimizing the pathogen attack requires very precise conditions and their effectiveness is not shown up to mark in large fields. The usage of resistant and hybrid varieties against the pathogen are strain-specific, a new strain of pathogen can easily deteriorate the entire crop fields.
There have been some attempts in the prior art to resolve the above-mentioned issues. Some attempts also have been made to synthesize silver nanoparticles using plant extracts.
US9637807B1: The invention discloses a method of metal nanoparticle synthesis by using an extract of terfeziaceae and the synthesized nanoparticles were used as an anti-microbial and an anti-parasitic agent.
Khan Merajuddin et.al, studied the antimicrobial and anticancer potential of seeds of C. colocynthis by using three different solvent extracts.
Ogundare Segun A. et.al, synthesized the silver nanoparticles by using the nanocrystalline cellulose (NCC) isolated from the melon seed shells (MSS) of Citrullus colocynthis. The synthesized silver nanoparticles were used for the reduction of nitrobenzene to aniline using NaBH4.
Therefore, there is a need of an environmentally friendly solution to reduce the ill effects caused by the attack of various phytopathogenic fungi.
Therefore, there exists a need to overcome the existing prior arts.
OBJECTS OF THE INVENTION:
The principal object of the present invention here is to develop the herbal nano formulation for sustainable management of stalk and ear rot diseases in maize.
Another object of the present invention is to develop the herbal nano formulation, which will be an alternative for the usage of synthetic fungicides.
Yet another object of the present invention is to develop the herbal nano formulation which is biodegradable, safe for the environment and have vast antifungal potential.
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:
The present invention discloses an herbal nano formulation for sustainable management of stalk and ear rot diseases of maize.
In an aspect of the invention, the herbal nano formulation comprising; an aqueous extract of Citrullus colocynthis (L.) seeds and a silver nitrate solution; wherein, the nano formulation shows antifungal activity against Fusarium verticillioides is disclosed.
In another aspect of the invention, a process for preparation of herbal nano formulation is disclosed wherein the process comprises steps of; collecting healthy and mature fruits of Citrullus colocynthis; drying of the fruits followed by separation of seeds from the fruits of C. colocynthis; and pulverizing the seeds to obtain a seed powder; preparing an aqueous seed extract from the seed powder; adding a silver nitrate solution in the aqueous seed extract and incubating the obtained mixture for 24hrs at room temperature to obtain the herbal nano formulation; conforming the colour change in the herbal nano formulation after completion of incubation period.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: An overview of the preparation of herbal nano-formulation
Figure 2: A general representation to produce AgNPs from plant material.
Figure 3: UV-Visible spectra of 1mM AgNPs + different concentrations of
C. colocynthisis seeds extract: (A) 2.5 ml of Seed extract in 1mM AgNO3 (B) 5 ml of Seed extract in 1mM AgNO3 (C) 7.5 ml of Seed extract in 1mM AgNO3 (D) 10 ml of Seed extract in 1mM AgNO3 (E) Control (C. colocynthis Seed extract).
Figure 4: XRD pattern of biosynthesized AgNPs.
Figure 5: FT-IR spectra of biosynthesized AgNPs.
Figure 6: SEM micrograph of C. colocynthis mediated AgNPs.
Figure 7: (A) Area EDS spectrum of AgNPs (B) Weight (%) of elements
Figure 8: (A) HR-TEM micrograph of biosynthesized AgNPs (B) SAED
pattern of the AgNPs.
Figure 9: Antifungal activity of different concentrations of biosynthesized
C. colocynthis mediated AgNps against Fusarium verticilloides.
Figure 10: Pot experiment to analyze the effect of herbal nano formulation on maize for sustainable management of stalk and ear rot diseases of maize crop.
DETAILED DESCRIPTION
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.
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.
The present invention discloses an herbal nano formulation for sustainable management of stalk and ear rot diseases in maize.
In an aspect of the invention, the herbal nano formulation comprising; an aqueous extract of Citrullus colocynthis (L.) seeds and a silver nitrate solution; wherein, the nano formulation shows antimicrobial activity against Fusarium verticillioides is disclosed.
In another aspect of the invention, a process for the preparation of herbal nano formulation is disclosed wherein the process comprises steps of; collecting healthy and mature fruits of Citrullus colocynthis; drying of the fruits and followed by separation of seeds from the fruits of C. colocynthis; and pulverizing the seeds to obtain a seed powder; preparing an aqueous seed extract from the seed powder; adding a silver nitrate solution in the aqueous seed extract and incubating the obtained mixture for 24hrs at room temperature to obtain the herbal nano formulation; conforming the colour change in the herbal nano formulation after completion of incubation.
The present invention wherein, the process of preparation of herbal nano formulation starts with collection and separation of seeds from the fruits of C. colocynthis (Figure 1 and 2). The fully mature and diseased-free fruits were selected. The fruits were then dried for the separation of seeds. Seeds were then pulverized to obtain the seed powder. The aqueous seed extract were then prepared by mixing 10 g of seed powder with 90 ml of double distilled water by using a magnetic stirrer. Then for silver nano particle preparation, 1 mM concentration of silver nitrate solution was prepared and then 10 ml of aqueous seed extract of C. colocynthis is added in 25 ml of 1 mM silver nitrate solution. The prepared solution was incubated at room temperature for 24 hours. After 24 hours, the colour of the resulting solution changes to deep brown due to the reduction of silver ions.
The present invention wherein, the herbal nano-formulation for the management of Fusarium against stalk and ear rot diseases in maize offers an inclusive approach that deals with the major challenging limitations in the existing technologies like environmental and health concerns, resistance, effectiveness and high-cost inputs. The potential of herbal nano-formulation is to increase effectiveness, stability, and target delivery which makes it a promising invention for the management of sustainable agriculture practices. The Herbal nano-formulation enhances the penetration of silver nanoparticles (AgNPs) with the help of various bioactive plant compounds into pathogen cells and maize tissues which allow the treatment to reach the target specific site. It provides controlled and prolonged release of active ingredients against pathogens. Silver nanoparticles offer a synergistic approach which shows antifungal properties. This approach makes the formulation more potent than traditional methods which increases its effectiveness against a wider range of pathogens. The plant-based nature of herbal nano-formulations makes them sustainable and does not raise any environmental and health concerns. The herbal nano-formulation contributes to healthier crops of maize by effectively controlling stalk and ear rot diseases with improved quality and yield.
In conclusion, the present invention demonstrates that C. colocynthis seed extract is a green, eco-friendly tool for synthesizing AgNPs with efficient antifungal activity, particularly against plant pathogenic fungi F. verticillioides. Thus, offer a valuable understanding that could be implemented for several applications in the sustainable management of plant protection against F. verticillioides.
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.
Example 1: Preparation of herbal nano formulation:
The healthy fruits of C. colocynthis were obtained from the Bahadurpur site of Dayalbagh Educational Institute, Dayalbagh, Agra. The collected plant material was cleaned and washed under the running tap water followed by double-distilled water to get rid-off from all the dust and adhere impurities. Thoroughly washed and cleaned plant materials were dried in the shade for about 5-7 days and were pulverized to fine powdered form with the aid of a mixer grinder. The prepared powder from the seeds was kept in an airtight container at 4°C in a refrigerator for further experimentation. The aqueous seed extract of C. colocynthis was prepared separately in a sterilized 250 ml conical flask by dissolving 10 g of each prepared plant powder in 90 ml of double distilled water and heating the mixture at 60º C for 30 min at a magnetic stirrer. After stirring, the prepared extracts were cooled down and then filtered twice through Whatman filter paper no. 1 to obtain the extract. The obtained extract was further centrifuged at 10,000 rpm for 10 minutes to eliminate biomass and then it was kept in the refrigerator at 4°C in a sterilized capped glass tube. The analytical grade aqueous solution of silver nitrate (AgNO3, 99 %) of 1 mM concentration was prepared by taking 0.004 g of AgNO3 and adding 25 ml of double-distilled water in a conical flask. This solution was further used for the biogenically synthesis of AgNPs. Now, 10 ml of prepared seed extract of C. colocynthis was mixed separately with 1 mM concentration of AgNO3 for the reduction of Ag+ ions into Ag. The prepared concentration was then incubated at room temperature for 24 hours for visual colour change. After 24 hours, the visual colour change of the solution was observed and recorded.
Example 2: Characterization of herbal nano formulation:
The pH of the AgNO3 solution was checked before and after the incorporation of plant extract, it was found somewhat alkaline and nearly unaffected. The biogenically synthesized herbal nano-formulation from the seed extract of C. colocynthis was further characterized with advanced techniques which include UV-Vis, XRD, FT-IR, SEM-EDS and TEM. The UV-visible spectral analysis of C. colocynthis seed extract-mediated AgNPs (the herbal nano formulation) showed the absorption peak at 444 nm which is attributed to the SPR displayed by AgNPs (Figure 3). The XRD spectral investigation exposed the crystalline nature corresponding to the face-centered cubic (FCC) lattice of silver (Figure 4). The FT-IR study showed the existence of various functional groups like alcohols, phenols, amines, etc. which plays a crucial role as reducing and stabilizing agents for the synthesis of AgNPs (Figure 5). The SEM equipped with EDS displayed small and spherical shape nanoparticles. (Figure 6). The EDS analysis showed the presence of a strong metallic signal at 3 KeV for silver (Figure 7). Furthermore, the HR-TEM results displayed spherical to oval shape AgNPs, with an average size of 20 nm. The SAED pattern displayed the concentric fringes which determine the crystalline nature of biosynthesized AgNPs (Figure 8).
Example 3: Testing of antifungal potential of herbal nano formulation:
The antifungal potential of biosynthesized AgNPs was tested against phytopathogenic fungi viz. Fusarium verticillioides was evaluated by in vitro study in the Microbiology laboratory of the Department of Botany, DEI, Agra. The result showed that the 100-ppm concentration of C. colocynthis mediated AgNPs displays significantly higher effectiveness in suppressing the mycelial growth of F. verticillioides. The in vitro experimental study demonstrated that the fungal mycelial radial growth was inhibited by raising the concentration of C. colocynthis seed extract-mediated AgNPs (Figure 9). After analyzing the in vitro antifungal potential, it was concluded that,100-ppm concentration of AgNPs was found to be most effective in suppressing the mycelial growth of F. verticillioides. Thus, a pot trial study for the efficacy of a liquid-based formulation of 100 ppm concentration of AgNPs was observed under protected conditions in maize crops (Figure 10). In the selected crop, statistical differences were observed among five treatments (i.e., T1, T2, T3, T4 and T5) based on morphological studies like plant height, number of leaves and physiological parameters like fluorescence (Fs) and normalized difference vegetation index (NDVI) (Table 1). Out of all the treatments, the (T3) in which biogenically synthesized herbal nano-formulation was applied at regular interval of time showed the greatest positive effect. The study demonstrated the positive effects of the herbal nano formulation on maize plants, responded with accelerated growth, higher greenness index, and a greater number of leaves with no signs of necrosis, chlorosis, leaf drying, or other symptoms of decreased plant quality in comparison with other treatments. The growth parameters were observed to be maximum in (T3) which was followed by positive control (T5) in which treatment of commercial fungicides Mancozeb 75% was applied. In the treatment (T4), the infection of fungal mycelia was amended along with the treatment of biosynthesized herbal nano-formulation was also provided to check the potential of the nano formulation against targeted pathogen in protected conditions. In the treatments T2 and T4, fungal mycelia of F. verticillioides. were infested in the soil before the sowing of maize seeds. The treatment (T2) was considered negative control in which the treatment of herbal nano formulation was not provided. Thus, in vivo investigation revealed that 100 ppm concentration of the herbal nano formulation in maize plants (T3) was found to be effective in comparison with other treatments. The effect of biosynthesized herbal nano formulation were found in the following order: T3 ? T5 ? T4 ? T1?T2.
Table. 1. In vivo study to analyze the effect of 100 ppm concentration of herbal nano formulation (AgNPs) on maize plants

, Claims:We claim;
1. An herbal nano formulation comprising; an aqueous extract of Citrullus colocynthis (L.) seeds and a silver nitrate solution; wherein, the nano formulation shows antifungal activity against Fusarium verticillioides.
2. A process for preparation of herbal nano formulation comprising steps of;
a) collecting diseased free and mature fruits of Citrullus colocynthis;
b) drying of the fruits and followed by separation of seeds from the fruits of C. colocynthis;
c) drying and pulverizing the seeds to obtain a seed powder;
d) preparing an aqueous seed extract from the seed powder;
e) adding a silver nitrate solution in the aqueous seed extract and incubating the obtained mixture for 24hrs at room temperature to obtain the herbal nano formulation;
f) conforming the colour change in the herbal nano formulation after completion of incubation.
3. The process for preparation of herbal nano formulation wherein, the silver nitrate solution is in 1mM concentration.
4. The biogenically synthesized AgNPs from the seed extract of C. colocynthis exhibits a predominantly spherical shape with an average size of 20 nm.