Description:Title of the Invention:
Gano-immune stimulator and methods of preparation of the same from Ganoderma lucidum mushroom for the control of fusarium wilt disease of chickpea caused by Fusarium oxysporum f.sp.ciceris

Applicant (s):
1. Dr. Chandan Singh (Ph.D)
Address: Dept of Botany, Dr. Harisingh Gour Vishwavidyalaya, Sagar
2. Ms.Smita Mishra (Ph.D Student)
Address: Dept of Botany, Dr. Harisingh Gour Vishwavidyalaya, Sagar
3. Dr. Deepak Vyas (Professor)
Address: Dept of Botany, Dr. Harisingh Gour Vishwavidyalaya, Sagar

Preamble to the description
The following specifications describe the use of Ganoderma lucidum extract as a bio-fungicide to control the fusarium wilt of chickpeas, the preparation of formulations and the method of applications.
Field of Invention
This invention is related to Ganoderma lucidum-based bioformulation as an agrochemical to be used to control the Fusarium wilt of chickpea, particularly caused by a fungal pathogen (Fusarium sp).
Background
Ganoderma lucidum (G. lucidum) is a member of basidiomycetes; Chinese Taoist monks traditionally use this mushroom to have a healthy life, and most importantly, it has also been used by the Japanese and is commonly called the "mushroom of immortality". G. lucidum has diverse bioactive compounds that boost human immunity and various ailments; apart from this, it has antimicrobial properties. This mushroom is a well-known background of human immune system modulator. Recently, it has been explored for its inducible effect on plant immunity boosters. Considering the bioactive compound of the mushroom, we used the extract to control the fusarium wilt of chickpea. Fusarium wilt of chickpeas is caused by the notorious fungi Fusarium sp., which is a common pathogen of wilt in chickpeas, and in severe cases, it causes the loss of 50-100% of chickpeas worldwide. Though different methods have been used to control this disease of the chickpeas, none of the methods has been found completely curable of this disease; however, using the chemical method of control, this disease is controlled on a large scale, but fusarium pathogen develops resistance against these chemicals and also develops resistance against chickpea resistant variety in one or two years and of course using synthetic chemical imparts negative impact on the environment, therefore inducing chickpea systemic resistant using bio-stimulators to induce resistance against the fusarium is a sustainable way to manage fusarium wilt along with existing methods of disease management. Therefore, we have explored G.lucidum extract for this purpose. We prepared an emulsion-based product, which we tested under pot conditions as well as on field conditions, and we obtained surprising positive results. This invention provides new insight into applying mushroom-based bioformulation to manage the fusarium wilt of chickpeas.
Japan, Korea, China, North America, and Malaysia largely commercialize the production of G. lucidum and have generated employment; in India, the production of G. lucidum and many other products has started. However, there is no available data on the production status.
Key phytochemicals of the G. lucidum are polysaccharides ( beta-1-3, beta-1-6 homo D-glucan), Terpenoids (ganoderic acid A-Z, lucidenic acids), amino acids (D-aspartic acid, glycine, phenylalanine, Tyrosine) and other compounds like oleic acid, coumarins, volatile oils, alkaloids, laccase, Phelonic compounds, flavonoids etc.
Managing the fusarium wilt of chickpeas is challenging, and no single control measure is entirely effective. Therefore, an integrated disease management strategy could provide environmentally and economically sustainable means to control the disease.
There are a number of resistance varieties of chickpea developed against the Fusarium wilt of chickpeas to combat the pathogens, but still, a serious concern is a changing climate, where the resistance variety fails to cope with the passage of time. The durability of chickpea resistance is compromised, and hence, the yield and the disease incidence index are raised, which may be due to the adaptations of the pathogens.

Therefore, agricultural technology is to be increased to enhance host resistance durability, which likely involves molecular modification of the host plants and the development of novel biological products, including microorganisms and metabolites.
Secondary metabolites as elicitors in plant's systemic resistance could be safe alternatives to fungicides, bacteriocides and insecticides; hence, adopting secondary metabolites as elicitors could also help reduce the toxic effect of chemical products that may pose threats to the environment and human health.
Elicitors are substances that turn on a plant's chemical defence system. Mushrooms contain numerous bioactive compounds with various health effects and elicitors for plants, stimulating plant growth and development and activating those vital pathways responsible for resistance in plants.
Singh & Vyas, (2023) used the crude extract of G.lucidum to induce systematic resistance in chickpeas against fusarium wilt.
Similarly, Zhang et al., (2019) have studies on the G. lucidum polysaccharide (GLP) to induce resistance in the cotton plant against the fusarium wilt; they sprayed and irrigated the GLP bioformulation, and after treatment of cotton with GLP, they found the increased level of the peroxidase(POD), superoxide dismutase and polyphenol oxidase in leaves. Also, in QRT-PCR results, the gene expression related to the jasmonic acid pathway in cotton increased significantly.
No such applied research has been reported on the proposed invention as per our search.
Non-Patent Literature Study
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Arvayo-Ortiz, R. M., Esqueda, M., Acedo-Felix, E., Sanchez, A., & Gutierrez, A. (2011). Morphological Variability and Races of Fusarium oxysporum f.sp. ciceris Associated with Chickpea (Cicer arietinum) Crops. American Journal of Agricultural and Biological Sciences, 6(1), 114–121. https://thescipub.com/pdf/10.3844/ajabssp.2011.114.121
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Kim, D. S., & Hwang, B. K. (2014). An important role of the pepper phenylalanine ammonia-lyase gene (PAL1) in salicylic acid-dependent signalling of the defence response to microbial pathogens. Journal of Experimental Botany, 65(9), 2295–2306. https://doi.org/10.1093/jxb/eru109
Mayer, A. M., Harel, E., & Shaul, R. B. (1965). Assay of catechol oxidase, a critical comparison of methods. Phytochemistry, 5, 783–789.
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Naz, R., Nosheen, A., Yasmin, H., Bano, A., & Keyani, R. (2018). Botanical-chemical formulations enhanced yield and protection against Bipolaris sorokiniana in wheat by inducing the expression of pathogenesis-related proteins. PLoS ONE, 13(4), 1–22. https://doi.org/10.1371/journal.pone.0196194
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Singh, C., & Vyas, D. (2023). Use of Ganoderma lucidum extract to elevate the resistance in chickpea against the Fusarium oxysporum f. sp. ciceris. Archives of Phytopathology and Plant Protection, 56(8), 605–624. https://doi.org/10.1080/03235408.2023.2207955
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Objects of the invention
I. The object of the invention is to provide G. lucidum-based bioformulation for agronomical use.
II. Emulsion preparation suitable for preparations of formulation from G.lucidum extract
III. To check the induced change of enzyme related to resistance in chickpeas against the Fusarium wilt under the treatment of emulsifiable concentrate
IV. To provide alternative agrochemicals obtained from biological sources like mushrooms, which will boost new ventures for the agrochemical industries.
Summary of the inventions
According to the embodiments illustrated herein, there is provided the process of Ganoderma lucidum extracts in Chloroform and G.lucidium based bioformulation in water in oil (W/O) emulsion to form emulsifiable concentrate (EC) and then formulation concentrate of Ganoderma extracts and screening its potential against Fusarium oxysporum f. sp. ciceris (FOC) the causal organism of Fusarium wilt of chickpea, a devastating pathogen of chickpea worldwide.
A new method was developed to quickly assess the effects of extracts as crop (herbs and shrubby) stimulators, like inducing a change in enzymes involved in the phenylpropanoid pathway involved in the production of different substances related to the crop defence system. The current invention is based on the stimulator system, which induces changes in the enzymes involved in the phenylpropanoid pathway.
Crude extract was extracted using Soxhlet unit, and antifungal activity was checked against FOC after getting the potential result by comparative study; the extract was subject to bioformulation using W/O emulsion. Formulated bioformulation was then examined in the lab, and pot conditions were used to reveal the potential of the extract.
The FOC challenge chickpeas treated with different concentrations of the bioformulation showed enhanced resiliency in the chickpea against FOC. A susceptible variety of chickpea (JG-62) was obtained from the ICRISAT, Hyderabad, India.
Enzymes like PO, PPO, and PAL, along with the phenolic content of chickpeas, were analyzed under different bioformulation concentrations. Increased level of phenolic content in chickpeas indicates the inducible effect of chickpeas; moreover, the level of PAL, PO and PPO under bioformulation challenged with FOC clearly indicated the role of formulation in enhancing resistance in chickpeas.
GC-MS analysis revealed that fatty acids (FAs) were the dominant compound in the extract, and individual studies of the FAs showed that FAs are the active players in inducing resistance in the chickpea.
The present invention provides a new approach in the preparation of new bio-agrochemicals, which will promote organic agrochemical practices and environmental safety.
The aforementioned features and advantages of the present disclosure may be appreciated by reviewing the detailed description, figures, and tables.
Advantages and novelty of the invention
1. The present invention has added new insight into applying the mushroom bioactive compound to increase induced resistance in the chickpea against the Fusarium wilt of chickpea.
2. This invention will help explore and design more formulations for the diverse diseases of crops other than chickpeas.
3. The present invention established the fact that the bioactive compound of G.lucidum helps reduce chemical pesticides and boosts agricultural health.
4. The novelty of the work is that mushrooms are used not only for exploitation for food and medicine but also to get bioactive compounds, which are used as biocontrol substances as well as environmentally friendly.
Brief description of the Drawings
Figure 1: Microscopic visual of Emulsion (A2) under 10x and FC (formulation concentrate) under 10x; scale-100mm.
Figure 2: Leaf scorching test as a measure of phytotoxicity in a pot experiment.
Figure 3: Showing FC-GCE effects on seed infection and seed germination of chickpeas challenged with FOC.
Figure 4: GC-MS Profile of crude chloroform extract of Ganoderma lucidum.
Figure 5: Showing the effect of Fusarium wilt of chickpea in field conditions.
Figure 6: Working methodology of the whole process.
Figure 7: Testube method to assess the effect of formulation on chickpea.
Figure 8: Pot experiment to check the inducible effect of formulation under FOC-challenged conditions.
Figure 9: Emulsion preparation (W/O).
Figure 10: Emulsifiable concentrate of Ganoderma extract.
Brief description of the table
Table 1: Combinational ratio of water in oil and Tween-80 (surfactant) to get the best and most stable emulsion.
Table 2: Emulsion stability test of W/O with Tween 80.
Table 3: Stability test of FC-GCE (Formulation concentrate-Ganoderma Chloroform Extracts).
Table- 4: Total Phenolic content of chickpea under treatment of FC-GCE.
Table 5: Effect of FC-GCE on PO activity in Chickpea leaf challenged with FOC.
Table 6: Effect of FC-GCE on Polyphenol oxidase (PPO) activity in chickpea leaf challenged with FOC.
Table 7: Effect FC-GCE on PAL activity in Chickpea leaf challenged with FOC
Table 8: Compound fractions detected by GC-MS in the Chloroform extract of G. lucidum crude.

Detailed description of the invention
Extraction of Ganoderma lucidum
G. lucidum fruiting bodies were washed and dried in a hot air oven at 500C. The dried mushroom was ground using an electrical grinder. 50 gm powder of mushroom was taken for the crude extraction using the Soxhlet unit in 250ml of Chloroform. The extraction was continued till the colour of the extraction in the siphon become faded. Extraction was done at the boiling point of the solvent. The crude extract was heated in a rotary evaporator to concentrate the extract to solid form and was kept at 40C in the refrigerator for further study and analysis.
Preparation of emulsion and its stability test
Initially, an emulsification process was carried out using water, castor oil, and Tween 80. The water-in-oil (W/O) proportion was made using Table -1 combinations to get the best combination. A suitable emulsion is characterized by a milky appearance, no separation zone, and stabilized water-oil and Tween-80.

After the stability test, the best stable combination was selected. The test was done by observing the formation of destabilization, which was recorded every 10 minutes. The formulation with no separation was further characterized by optical microscopy. The observation was conducted for 3 hours with measurement intervals every 10 minutes. The selection of the optimal surfactant composition was based on the most stable emulsion test result (Table 2).
Preparation of Emulsifiable Concentrate (EC) and its stability test
To create safe and useful products from the large range of agrochemical formulations that are currently accessible, a variety of various formulation additives are needed. Surface active agents are the most significant formulation additives (Arvayo-Ortiz et al., 2011). The surfactants frequently determine the maximum concentration of the formulation that can be achieved, the size of the particles or droplets, its long-term stability, and occasionally even its biological activity (Feng et al., 2018). The major goals of the formulation are to maximize the inherent activity of the active component and to give the user a convenient, safe product that won't degrade over time (Naz et al., 2018). Here we have prepared a water-in oil formulation with the surfactant Tween 80 for developing the formulation concentrate using the Ganoderma chloroform extract. To obtain the stability and water in oil combination along with Tween 80, a series of combinations were made, and an emulsion stability test was conducted.
50mg of GCE (Ganoderma Chloroform extracts) was dissolved in DMSO solvent to semi-liquid and was added to the stable emulsion to form an emulsifiable concentrate (EC). To develop the EC, 10 ml of the stable emulsion was taken, and the semi-liquid dissolved extract was added and shaken mechanically to achieve uniform formulation and stabilization (concentration extract of 5mg/ml), and the stability of the EC was checked visually as well as optically (Figure-1). EC formulation was added with water at a ratio of 1:10 for the emulsion stability test (Table 3). The stability test was carried out in the same way as the emulsion stability test.
The formula combination of W/O A2 was selected for the preparation of formulation concentrate; the stability test showed that the A2 have higher stability than all other combinations after visual and microscopic study (Table-3 and Figure-1). The appearance was milky, and no separations of oil were observed in A2 till 2hrs; no sedimentations were observed in A2. Therefore the A2 was selected as the emulsion for the preparation of formulation, which was denoted as FC-GCE (Formulation Concentrate- Ganoderma Chloroform Extract). Developing stable emulsion is a crucial part of the preparation of emulsion-based agrochemicals and in this invention.
Different formulation concentrations used in this invention
• 10% FC-GCE
• 20% FC-GCE
• 30% FC-GCE
• 40% FC-GCE,
• 50% FC-GCE were made.
The phytotoxicity of the different concentrations of EC was done using a leaf-scorching test:
The formulation (FC-GCE) at different concentrations, namely 10% FC-GCE, 20% FC-GCE, 30% FC-GCE, 40 % FC-GCE and 50% FC-GCE were sprayed on potted seedlings of chickpea to observe leaf scorching as a measure of phytotoxicity the result shown in figure-2 shows no leaf scorching was observed on chickpea leaf sprayed with different concentrations of the FC-GCE. This indicates that the formulations were nontoxic to chickpea.
The induced change in total phenolic content of the treated chickpea was determined using different treatments of the FC-GCE challenged with the FOC using the standard protocols.
FC-GCE 50% 84.5
FC-GCE effect on the seed germination and seed infection % under FOC
The seeds of chickpeas were pre-infected with FOC by being soaked in spore suspensions of the FOC overnight. Pre-infected seeds were soaked in different concentrations of the Emulsifiable Concentrate for 18 hrs and then shaded dried. Ten seeds were uniformly placed on a standard germination paper roll-towel medium and incubated at 25 ± 2°C and 90 ± 2 per cent relative humidity. After ten days, the per cent germination was recorded.
The induction of defence enzymes of chickpeas under different treatments of FC-GCE
To evaluate the change in the defence enzyme of chickpea under different treatments of formulation, a mature chickpea was uprooted, and the roots were washed with distilled water; 5ml of formulation of each concentration was poured into 25 ml flask, and in each flask washed uprooted chickpea was dipped and allowed to stand for 10 min under laboratory condition. At the same time, a suspension of Fusarium oxysporum f. sp. ciceris (FOC) was prepared with water at the concentration of 3 x106spores/ml and poured 15 ml in a culture tube and was arranged on a test-tube stand and labelled accordingly. After 10 min of chickpea treatment with all the formulation concentrations, the chickpea was inserted in the FOC suspension test tube and allowed to stand overnight. The next day, the treated chickpeas were collected and stored at 40C in a refrigerator for enzyme analysis.

Defence enzyme peroxidase (PO)
PO, one of the enzymes involved in the phenylpropanoid pathway, increased with the treatment of different concentrations of the FC-GCE in the chickpea, indicating the positive and inducible role of the G.lucidum extracts. The enzyme assay was carried out according to Hammerschmidt et al.(1982)
Defence enzyme Polyphenol oxidase (PPO)
Polyphenol oxidase assay was carried out according to Mayer et al. (1965), and the result shows that the activity of the PPO in chickpeas increases under different FC-GCE concentrations compared to water-treated control.
Defence enzyme Phenylalanine Ammonia Lyases (PAL)
Phenylalanine ammonia-lyase assay was carried out according to Dickerson et al. (1984).
GC-MS analysis of G.lucidum chloroform extracts
The sample was analyzed at the CytoGeneResearch and Development Lucknow (UP) India (https://www.cytogene.in/). The GC-MS analysis was carried out in Agilent 7890B GC connected to 5977A MSD, Column HP_5MS 5% Phenyl Methyl Silox-60ºC-325ºC, 30m x 250 µm x 0.25µm, injector temperature 250ºC, detector temperature 280ºC, sample dissolved in Chloroform and injected (2µl), splitless mode and carrier gas was helium at 1 mL/min. The total running time of the GC was 40 min. NIST MS2011 library was then searched to compare the structures of the compounds with that of the NIST database (National Institute of Standard and Technology). Compounds were then identified based on the retention times and mass spectra with already known compounds in the NIST library (C:\Database\NIST14.L) and the major compound detected (table-8).
Increased in phenolic content and the enzyme activity involved in the phenylpropanoid pathway were enhanced in the FC-GCE-treated chickpea under a challenged environment. After GC-MS analysis, the chance of chemical compounds responsible for this change came to light; nearly 40 fractions of the different compounds were derived based on the peak area and the retention time (Figure-4). We noted that fatty acids (FAs) have major dominancy in the fraction (Table-8). The fractions were classified into major classes and were checked individually in the different databases for their role in the antifungal activity and induced systemic resistance in the plant. It has been established that FA plays a significant role in plant defence (Kachroo & Kachroo, 2009). FAs and their breakdown products induce various modes of plant defence (Naguib, 2019; Shah, 2005). FAs containing 16- and 18-carbon FAs participate in defence to modulate systemic immunity in the plants (Kachroo & Kachroo, 2009; Shah, 2005); in Ganoderma extracts, n-hexadecanoic acid (14 % of total peak area), 16-carbon FA is dominant among all other fatty acids present in the extract. Moreover, Oleic acid (10.67 % of total peak area) and 18-carbon FAs indicate direct links with the enzyme activity involved in phenylpropanoid pathways of the treated chickpea.
Apart from the involvement in plant defence, FAs play a significant role in the heat stress physiology of plants; for instance, Linolenic acid helps in modifications of protein during heat stress in the plant (Yamauchi et al., 2008), polyunsaturated fatty acids (PUFAs) found to have active roles in the regulations of chloroplastic membranes' lipid fluidity and determine the plant's ability to acclimatize in temperature stress (Yaeno et al., 2004).
In the series of different defence signalling pathways, the emergence of the phenylpropanoid pathway in the plant is an important pathway that enables plant defence against biotic and abiotic constraints. The phenylpropanoid pathway produces compounds that serve as precursors to a wide range of phenolic compounds, plant hormones, phytoalexins, and lignins; phenylpropanoid compounds are derived from cinnamic acid, which is formed from phenylalanine; PAL catalyzes the non-oxidative deamination of phenylalanine to trans-cinnamate (Al Jitan et al., 2018; Kim & Hwang, 2014; Van Loon et al., 2006).
The activity of the PO in the treated chickpea indicated that the extracts induced the PO activity. Peroxidase belongs to a large multigene family and participates in a broad range of physiological processes, such as (a) lignin and suberin formation, (b) cross-linking of cell wall components, (c) synthesis of phytoalexins, or (d) participating in the metabolism of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Both ROS and RNS switch on the hypersensitive response (HR), a form of programmed host cell death at the infection site associated with limited pathogen development (Almagro et al., 2009)
Increased PPO in the treatments of chickpea indicated that the FC-GCE formulation has inductive effects on PPO genes that are induced as part of the general defence response in chickpea (Fuerst et al., 2014). Plants have evolved multiple defence signalling pathways to defend themselves from abiotic and biotic constraints.
PAL plays an important role in plant defence as it is involved in the biosynthesis of Salicylic acid (SA) and is an essential signal compound in the plant (Kim & Hwang, 2014). The significantly different activity of PAL in Chickpea treated with FC-GCE challenged with the FOC showed that the extracts of the Ganoderma have an inducible effect on the enzymes associated with the phenylpropanoid pathway.

Claims:We Claim:

1. The use of Ganoderma lucidum extracts to control Fusarium wilt of chickpeas and the process of applications.

2. The materials and the combination of emulsion that suitable for the preparation of emulsifiable concentrate using G.lucidum.

3. The test tube method of analyzing the stimulator effect of any formulations in crops under lab conditions.