Bioinsecticide composition and its method of preparation thereof

Abstract

The present invention relates to a bioinsecticide (19) composition comprising a diluted Cordyceps fumosorosea culture (12), a kaolin clay (13), a sodium citrate (14), a citric acid (15), and a carboxymethyl cellulose (16). The present invention further relates to a method (100) for preparing a bioinsecticide powder (20), comprising steps of growing (101) a Cordyceps fumosorosea (10) to obtain a Cordyceps fumosorosea stock culture (11), diluting (102) the Cordyceps fumosorosea stock culture (11) to obtain a diluted Cordyceps fumosorosea culture (12), blending (103) the diluted Cordyceps fumosorosea culture (12) with a kaolin clay (13) to obtain a wet powder texture (17), drying (104) the wet powder texture (17) to obtain a dry powder texture (18), mixing (105) the dry powder texture (18) with a sodium citrate (14), a citric acid (15), and a carboxymethyl cellulose (16) to obtain a bioinsecticide (19) composition, and drying (106) the bioinsecticide (19) composition to obtain a bioinsecticide (20) powder. Accordingly, the present invention also relates to a method of using a bioinsecticide (19) composition.

Description

[0001] BIOINSECTICIDE COMPOSITION AND ITS METHOD OF PREPARATION THEREOF

[0002] FIELD OF INVENTION

[0003] The present invention relates to a bioinsecticide derived from an entomopathogenic fungus called Cordyceps fumosorosea (formerly Isaria fumosorosea) against target insects from Lepidopteran, Hemipteran, or Spodoptera orders, predominantly Metisa plana, known as bagworms. More particularly, the present invention relates to a bioinsecticide formulation using Cordyceps fumosorosea as an active ingredient in wettable powder composition, and its method of preparation thereof.

[0004] BACKGROUND OF THE INVENTION

[0005] The impact of pests on crops constitutes one of the many insect-related issues faced by Malaysia's agriculture sector. Malaysia is the world’s largest producer and exporter of palm oil (Elaeis guineensis) with 52.0% of global production and 61 .1% of global exports. The rapid growth of oil plantations in Malaysia has matched with the rise of various pests that threaten oil palm production which cause significant losses in the annual oil palm crop production.

[0006] In Malaysia, bagworms, nettle caterpillars, hairy caterpillars, and other leaf-eating caterpillars are among the major pests of oil palm. There are about 1000 known species and 300 genera in the bagworm family (Lepidoptera: Psychidae), which can be found all over the world. The common species of bagworms of oil palm in Malaysia can be identified as Pteroma pendula, Metisa plana, and Mahasena corbetti.

[0007] Synthetic pesticides are often employed to defend crops from pests and diseases. However, non-target toxicity, biodegradability, and residual effects have grown to be significant concerns, necessitating the adoption of more cost-effective and environmentally friendly pest control techniques.

[0008] Biological pesticides constructed on pathogenic microorganisms specific to a target pest offer an ecologically sound and effective solution to pest problems. They are less harmful for both the environment and public health. The most widely utilized biopesticides are pathogenic living organisms that target the target pest. To aid in the registration of biopesticides, the EPA formed the Biopesticides and Pollution Prevention Division within the Office of Pesticide Programs in 1994. As part of IPM programs, this division advocates for the use of safer pesticides, such as biopesticides.

[0009] Among bioinsecticides derived from an entomopathogenic fungus, Cordyceps fumosorosea has the potential to be an environmentally benign biocontrol agent because of its potential to control a variety of insects. The fungi could be produced economically by submerged fermentation and fungal propagules comprised of conidia, mycelia, and blastospores and formulated similarly to conventional insecticides.

[0010] Cordyceps fumosorosea offers several advantages as a bioinsecticide. It is environmentally friendly, targeting specific insect pests while being safe for humans, animals, and other beneficial insects. This allows the ecosystem to be maintained specificity helps maintain ecological balance and biodiversity. Cordyceps fumosorosea does not contribute to pesticide resistance, ensuring long-term effectiveness, unlike chemical insecticides. Cordyceps fumosorosea- based bioinsecticide is non-toxic which makes it an ideal component for integrated pest management (IPM) strategies, thus reducing harmful chemical reliance and emergence of insecticide resistance organisms.

[0011] Additionally, Cordyceps fumosorosea can be used in organic farming, aligning with sustainable agricultural practices. Its ability to control a wide range of pests, including aphids, whiteflies, and caterpillars, makes it versatile and valuable for various crops. The fungus also degrades naturally, leaving no harmful residues, thus preserving soil health and water quality. In addition to that, Cordyceps fumosorosea- based bioinsecticide provides an effective, sustainable, and safe alternative for pest control in agriculture.

[0012] European patent EP04945922A1 discloses an invention that teaches a process, formulations, and method for producing a biopesticide which comprises a prilled formulation comprising a carrier and pathogenic fungal mycelium particularly Paecilomyces fumosoroseus ATCC 20874. The invention disclosed the application of pathogenic fungal mycelia from the genus Cordyceps and that the invention targets a wide range of pests, including insects from the Lepidoptera order, which includes the Metisa plana or bagworm. Furthermore, the invention uses a submerged fermentation technique for fungi propagules and conidia to create a biopesticide formulation where the invention also discloses that Kaolin clay is one of the suitable carriers for the formulation. Furthermore, the invention disclosed the fermentation condition where the condition is set at a preferred temperature range of 28-30°C, in pH value is controlled within the range of about pH 4.0 to 6.0, preferably about 5.0 with agitation set at a minimum agitation rate of about 400 to 600 rpm. The invention also disclosed that the application dose for the bioinsecticide composition is recommended to be about 107 conidia spores / ml. However, the invention did not specifically disclose the Cordyceps fumosorosea fungus species. The invention did not disclose the structure of the biopesticide as a wettable powder. The invention formulation does not mention the ranges of the kaolin clay and the incorporation of carboxymethyl cellulose, sodium citrate, and citric acid as surfactant or any organic acid in the formulation. Furthermore, the invention also did not disclose the culture is placed at room temperature in a dark area for 4 days to optimize the culture growth. Nonetheless, the invention did not disclose the characteristics of the bioinsecticide which include the wettability, suspensibility, foaming and sedimentation, and dispersibility properties of the bioinsecticide.

[0013] China patent CN104642392A with priority to CN201510015670A, discloses relates to the technical field of biological control of insect pests and particularly discloses Isaria fumosorosea conidia wettable powder. The invention further disclosed that kaolin clay acts as a carrier, while carboxymethyl cellulose is used as a stabiliser. The bioinsecticidal concentration of the invention is disclosed to be within the ranges from 1.0x107spores / mL to 2.0x107spores / mL, and the invention discloses the wettability of the bioinsecticide wettable powder to be less than 3 min and the suspensibility is 81.25% with the efficacy of 14 days. However, the invention did not disclose having submerged propagules and conidia combination, the specific amount of kaolin clay, carboxymethyl cellulose, and citric acid used and also the incorporation of sodium citrate, citric acid, and organic acid in the invention composition. Furthermore, the invention did not disclose the culture condition used specifically the culture condition pH value and that the culture was placed at room temperature in a dark area for 4 days to optimize the culture growth. The invention also did not mention filtration and centrifugation as a method for harvesting propagules and conidia in the culture. Furthermore, the invention did not mention the recovery techniques that involve drying using an oven at 25-29°C of the propagules and conidia in the culture. The invention also did not mention the foaming, sedimentation, and dispersibility properties of the composition.

[0014] Another China patent CN111961598A with priority to CN202010971228A discloses an invention that discloses the utilisation of Isaria javanica bioinsecticide in a wettable powder form and also teaches the development of a fungal preparation using Isaria javanica ljH6102 and the importance of conidia for an ideal fungal preparation. The invention stated that the culture condition is done in a temperature range between 25-28°C with agitation at 150 rpm for 1-2 days. The recovery of bioinsecticide supernatant mentioned in the invention is done through filtration and centrifugation, where the supernatant of the bioinsecticide composition is then applied by spraying. The bioinsecticidal concentration of invention formulation in the wettable powder is 5.50x109conidia / g with the wettability of the bioinsecticide wettable powder being less than 3 min and the suspensibility is 79.32%. The efficacy of the invention is disclosed in 4 days. Nonetheless, the invention did not mention having a submerged propagules and conidia combination and any incorporation of kaolin clay, carboxymethyl cellulose, sodium citrate, citric acid, and organic acid in the invention composition. The invention also did not mention the culture condition pH value and that the culture is placed at room temperature in a dark area for 4 days to optimize the culture growth. Furthermore, the invention did not mention drying using an oven at 25-29°C, foaming, sedimentation, and dispersibility properties of the bioinsecticide.

[0015] Meanwhile, China patent CN116836815A and United States patent US 8574566B2, discloses the utilization of Cordyceps fumosorosea and Isaria fumosorosea as one of the compositions in their respective invention. CN116836815A and US 8574566B2 utilised conidia in the composition, and the culture condition mentioned in CN116836815A is set to a specific condition whereby the culture was done for 7 days on Potato Dextrose Agar solid medium, with a temperature of 27±1 °C, humidity of 70%±5% RH, and a photoperiod of 14L-10D. Meanwhile, US 8574566B2 discloses that the fungi are grown at 23°C and the growth medium contains 2% glucose, 2% maltose, and 2% peptone for bacteriology, with a pH of 5.5, in an orbital shaker at 140 cycles per minute for 120 hours (equivalent to 5 days). US 8574566B2 also discloses the uses of filtration and centrifugation to isolate blastospore biomass from growth media, with centrifugation at 2500 rotations per minute for 20 minutes. The insecticidal concentration observed in CN116836815A is 1x108spores / mL showing the highest mortality rate, whereas in US 8574566B2 is 5x10 blastospores / ml. Both CN116836815A and US 8574566B2 discloses the efficacy rate can be observed after 3 days and that both inventions can caused infection to the targeted insects. Nonetheless, both CN116836815A and US 8574566B2 did not mention the composition to be in a wettable powder form. CN116836815A did not mention targeting lepidopteran insects, and US 8574566B2 did not specifically mention targeting Metisa plana or bagworms. Both inventions did not specifically mention the use of submerged propagules and conidia of the fungi and the composition of both CN116836815A and US 8574566B2 did not mention the specific amount of kaolin clay and carboxymethyl cellulose used along with the incorporation of sodium citrate, citric acid, and organic acid in their respective compositions. Furthermore, both CN116836815A nor US 8574566B2 did not disclose the drying using an oven at 25-29°C and the characterisation of the bioinsecticide in terms of wettability, suspensibility, foam and sedimentation ability, and dispersibility properties. The development of bioinsecticides from Cordyceps fumosorosea is crucial because it provides an environmentally friendly alternative to chemical pesticides, reducing harm to non-target organisms and preventing pesticide-resistant insects. Furthermore, Cordyceps fumosorosea fungus acts to target specific pests, thus, preserving beneficial insects and maintaining ecological balance. Additionally, it supports sustainable agriculture, integrating well with organic farming practices and promoting soil and water health. Cordyceps fumosorosea bioinsecticide is effective against a wide range of pests from the genera Aphis, Bemisia, Plutella, Spodoptera, Frankliniella, Helicoverpa, Liriomyza, Tetranychus, Diaphorina, and Trialeurodes, also including those that are resistant to chemicals. In addition to that, Cordyceps fumosorosea can enhance crop yields and quality alongside offering a safer, sustainable, and efficient solution for pest management, crucial for long-term agricultural productivity and environmental conservation.

[0016] SUMMARY OF THE PRESENT INVENTION

[0017] It is an objective of the present invention to provide a teaching of bioinsecticide composition where the bioinsecticide is composed of Cordyceps fumosorosea, an entomopathogenic fungus with the ability to infect and control the pest insect population, where the pest insect is from the genus Lepidoptera predominantly bagworms species.

[0018] Accordingly, the present invention relates to the method of preparation of Cordyceps fumosorosea bioinsecticide in which Cordyceps fumosorosea is the main active ingredient and the bioinsecticide is in a wettable powder form with good efficiency and the capability to reduce and control pest insects with low toxicity to human. The bioinsecticide is in a wettable powder form, achieved through the air drying process. The bioinsecticide produced can be applied as an alternative to chemical-based pesticides in the agriculture industry.

[0019] Accordingly, the objective of the present invention can be achieved where the bioinsecticide active ingredient is an entomopathogenic fungus from the family Cordycipitaceae, preferably from the genus Cordyceps, more preferably Cordyceps fumosorosea. The Cordyceps fumosorosea bioinsecticide is in the form of liquid, powder, gel-like materials, encapsulated forms, granules or pellets, and sprayable formulations, or a combination thereof, preferably in a wettable powder form.

[0020] Accordingly, Cordyceps fumosorosea fungus is isolated from but not limited to soil, forests, agricultural fields, grasslands, areas with decaying organic matter, lab-grown stock culture, mycotized dead infected pests, or a combination thereof.

[0021] Accordingly, the Cordyceps fumosorosea culture media composition comprises potato dextrose broth combined with potato dextrose agar, glycerol and distilled water. Subsequently, the media mixture is autoclaved at 121 °C for 15 minutes.

[0022] The embodiment of the present invention where the bioinsecticide composition comprises a diluted Cordyceps fumosorosea culture, a kaolin clay, a sodium citrate, a citric acid, and a carboxymethyl cellulose.

[0023] Additionally, the embodiment of the present invention discloses the bioinsecticide composition further comprises 50-1000 mL of diluted Cordyceps fumosorosea culture, 30-90 g kaolin clay, 30-90 g sodium citrate, 20-60 g citric acid, and 5-30 g carboxymethyl cellulose.

[0024] Additionally, the embodiment of the present invention is where the bioinsecticide composition contains submerged propagules and conidia for effective bioinsecticidal action.

[0025] In an aspect of the present invention, where the kaolin clay acts as a carrier and the amount of kaolin clay is in the range of 10-70% weight of the bioinsecticide composition.

[0026] Another aspect of the present invention is where the sodium citrate, citric acid, and carboxymethyl cellulose mixture act as surfactants which range between 10-40% by weight of the bioinsecticide composition. In another aspect, the present invention discloses the steps of the bioinsecticide powder preparation method where Cordyceps fumosorosea is grown in lab conditions, where 1-3 cm, preferably 1 cm of Cordyceps fumosorosea disk culture is isolated and inserted into the potato dextrose broth media mixture to obtain a Cordyceps fumosorosea stock culture. This step is followed by diluting the Cordyceps fumosorosea stock culture to obtain a diluted Cordyceps fumosorosea culture, then the diluted Cordyceps fumosorosea culture is blended with kaolin clay to obtain a wet powder texture which is then dried to obtain a dry powder texture. The sodium citrate, the citric acid, and the carboxymethyl cellulose are mixed with the dry powder texture to obtain a bioinsecticide mixture and finally dried to obtain a bioinsecticide powder.

[0027] Another aspect of the present invention discloses the diluted Cordyceps fumosorosea culture contains submerged propagules and conidia in a concentration of 106-101° CFU / mL, preferably 108CFU / mL.

[0028] Another embodiment of the present invention is where the bioinsecticide powder wettability is in the range of 10-120 s, preferably 90 s.

[0029] The embodiment of the present invention where the suspensibility of the bioinsecticide powder is in the range of 60-90%, preferably 79-80%, more preferably 79.5%.

[0030] Another aspect of the embodiment of the present invention is where the bioinsecticide powder foam and sedimentation ability score is between 90-100%, preferably 100%.

[0031] In another embodiment of the present invention, the bioinsecticide powder dispersibility is in the range of 70-100%, preferably 88%.

[0032] The embodiment of the present invention where at least one part of the bioinsecticide composition is mixed with at least one part of water, then the composition is stirred to obtain a homogenous mixture and finally is packed into a container for application on an affected plant area.

[0033] An aspect of the embodiment of the present invention where the efficacy of the bioinsecticide composition is determined 1-9 days after application.

[0034] Accordingly, the Cordyceps fumosorosea infection on the target insect pest can be observed within 1-4 days.

[0035] Accordingly, the target insect is from Lepidopteran, Hemipteran, or Spodoptera order or bagworm species, Metisa plana, Pteroma pendula, and Mahasena corbetti.

[0036] The present invention consists of features and a combination of parts hereinafter fully described and illustrated in the accompanying drawings, it is understood that various changes in the details may be made without departing from the scope of the invention or sacrificing any of the advantages of the present invention.

[0037] BRIEF DESCRIPTION OF THE DRAWINGS

[0038] To further clarify various aspects of some embodiments of the present invention, a more particular description of the invention will be rendered by references to specific embodiments thereof, which are illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail through the accompanying drawings in which:

[0039] Figure 1 is an illustration showing a production method for preparing a Cordyceps fumosorosea bioinsecticide powder. Figure 2 shows the external symptoms of bagworm larvae exposed to Cordyceps fumosorosea bioinsecticide after: A; Control (0 day), B; after 1 day, C; 3 days, D; 5 days, E; 7 days, F; 9 days, and G; 12 days.

[0040] Figure 3 shows the hyphae developed with low conidia density on the Metisa plana surface within 24 hours, where conidia is annotated as “Co” and hyphae is annotated as “Hy”.

[0041] Figure 4 shows the hyphae and conidia growth more extensively on the Metisa plana surface within 48 hours, where conidia is annotated as “Co” and hyphae is annotated as “Hy”.

[0042] Figure 5 shows the conidia detached from conidiophores and proliferated on the Metisa plana surface within 72 hours, where conidia is annotated as “Co” and hyphae is annotated as “Hy”.

[0043] Figure 6 shows a high density of matured conidia attached to the Metisa plana surface within 96 hours, where conidia is annotated as “Co” and hyphae is annotated as “Hy”.

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that current invention may be practised without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.

[0046] Pathologically, Cordyceps fumosorosea is an entomopathogenic fungus that infects and kills insects through a specialized pathogenic process. The infection begins when fungal spores come into contact with an insect's cuticle. The spores germinate and produce hyphae that penetrate the exoskeleton using mechanical pressure and enzymatic degradation of the cuticle. Once inside the host, the fungus proliferates by growing mycelium throughout the insect's body, consuming its internal tissues.

[0047] This internal invasion disrupts the insect's physiological functions, often producing secondary metabolites that are toxic to the host. These toxins further weaken the insect, leading to its death. The symptoms in the infected insect include lethargy, loss of coordination, and eventual paralysis.

[0048] After the insect dies, the fungus continues to grow, eventually breaking through the host's body and producing new spores on the cadaver. These spores are then released into the environment to infect other susceptible insects, perpetuating the pathogenic cycle. This effective mode of action makes Cordyceps fumosorosea a valuable bioinsecticide for pest control.

[0049] The embodiment of the present invention relates to the bioinsecticide composition and the preparation method of bioinsecticide powder from Cordyceps fumosorosea that can be used for the agriculture industry to combat insect pest such as but not limited to the genera Aphis, Bemisia, Plutella, Spodoptera, Frankliniella, Helicoverpa, Liriomyza, Tetranychus, Diaphorina, and Trialeurodes, also including those that are resistant to chemicals. The present invention can be used as an alternative to chemical-based pesticides which in long-term used can lead to soil and water pollution.

[0050] In particular, the present invention notably described the present invention explores the production of a bioinsecticide with high efficacy in controlling bagworm species and less harmful to the affected plant and soil. Producing a high-efficiency bioinsecticide is crucial for protecting the environment, ensuring human health and food safety, and providing economic benefits. The present invention also provides a holistic approach to pest management that helps secure food production systems while preserving ecological balance and public health. Additionally, the Cordyceps fumosorosea bioinsecticide provides effective, targeted pest control, along with promoting sustainable farming practices where Cordyceps fumosorosea bioinsecticide has a lower risk of causing the emergence of pest resistance insects compared to chemical insecticide. Furthermore utilising the Cordyceps fumosorosea bioinsecticide integrates well with the integrated pest management programs leading it to be a holistic pest management approach.

[0051] The Cordyceps fumosorosea bioinsecticide is also able to enhance plant health and yield. The eco-friendly nature and compatibility with the integrated pest management programs further support a safer and more productive agricultural system. Moreover, Cordyceps fumosorosea bioinsecticide goes along with the public demands for organic and sustainable products, which offer economic advantages to farmers.

[0052] The present invention relates to a biodegradable mycoinsecticide composition for controlling bagworm infestation in a plant. Preferably, the composition comprises a fungal insecticide having submerged propagules and conidia derived from an entomopathogenic fungus strain of Cordyceps fumosorosea, a carboxymethyl surfactant mixture and carrier. Advantageously, the mycoinsecticide composition is effective in controlling bagworm and lepidopteran order infestation in a plant, particularly oil palm and agricultural crops. The mycoinsecticide composition also presents the benefit of selectively infecting the target pest, particularly bagworm and lepidopteran insects, without harming beneficial insect pollinators.

[0053] Specifically, the present invention discloses a bioinsecticide (19) composition comprising a diluted Cordyceps fumosorosea culture (12), a kaolin clay (13), a sodium citrate (14), a citric acid (15), and a carboxymethyl cellulose (16), where the bioinsecticide (19) composition further comprises 50-1000 mL, preferably 250 mL of the diluted Cordyceps fumosorosea culture (12), 30-90 g, preferably 61.08 g of the kaolin clay, 30-90 g, preferably 72.38 g of the sodium citrate, 20-60 g, preferably 45.94 g the citric acid, and 5-30 g, preferably 20.88 g of the carboxymethyl cellulose.

[0054] Accordingly, the embodiment of the present invention discloses the fungal bioinsecticide has an insecticidally effective dosage rate of submerged propagules and conidia in a range of 10-40% (w / w) of the mycoinsecticide composition which consists of 1 O6-1 O1oCFU / mL fungal concentration. The fungal insecticide described herein comprises submerged propagules and conidia of the strain Cordyceps fumosorosea. More preferably, the submerged propagules derived from the strain of Cordyceps fumosorosea comprise blastospore, sclerotia, mycelia fragments and conidia.

[0055] Alternatively, the embodiment of the present invention Cordyceps fumosorosea bioinsecticide composition can be produced in other various forms but not limited to wettable powder, dried powder, liquid formulations, gel-like materials, encapsulated forms, granules or pellets, and sprayable formulations. With this, each form can offer specific advantages tailored to different applications. The versatility in production forms allows Cordyceps fumosorosea bioinsecticide composition application to be tailored according to the most suitable method for specific crops, pests, and environmental conditions.

[0056] The description of the embodiment of the Cordyceps fumosorosea bioinsecticide composition form can be further explained below: i. Wettable powdered: where the powder can be mixed with water to form a sprayable suspension. This bioinsecticide form is easy to store and transport, has a long shelf life, and is cost-effective. ii. Suspension concentrates: which is a form of liquid formulation that provides a stable suspension of fine particles of the active ingredient in a liquid phase and offers a ready-to-use bioinsecticide composition after dilution with water, and ensures an even application that is suitable for foliar spraying. iii. Emulsifiable concentrates: a form of liquid concentrates whereby the liquid formations form an emulsion when mixed with water. This form has a good penetration on the plant surface and is easy to apply with standard spraying or sprinkling equipment. iv. Granules: in which the bioinsecticide composition is in solid granulated form that can be applied directly to the soil. This provides a long-term soil bioinsecticide application to effectively ditter soil pests. v. Dust: a finely grounded dried bioinsecticide composition powder for a dry application that is suitable for crops that are sensitive to water. vi. Bait formulations: where the bioinsecticide composition is incorporated in a food source for insect pests such as nectar, sugary solution, or rotten fruits. This form targets more specific pests, reducing non-target impacts and therefore effective for certain pests. vii. Effervescent tablet: where the tablet is dissolved when in contact with water to form a solution which is convenient for small-scale farmers or gardeners, and easy to measure and mix. viii. Slow-release pellets: in which the bioinsecticide composition is pressed into a pellet form and the pellets slowly release the active ingredient over time to allow long-term pest control and reduce the bioinsecticide application frequency.

[0057] The embodiment of the present invention provides the preparation method to produce a Cordyceps fumosorosea bioinsecticide powder (21 ) comprising the steps of growing (101 ) a Cordyceps fumosorosea (10) to obtain a Cordyceps fumosorosea stock culture (11 ), followed by diluting (102) the Cordyceps fumosorosea stock culture (11 ) to obtain a diluted Cordyceps fumosorosea culture (12), then blending (103) the diluted Cordyceps fumosorosea culture (12) with a kaolin clay (13) to obtain a wet powder texture (18), drying (104) the wet powder texture (18) to obtain a dry powder texture (19), followed by mixing (105) the dry powder texture (19) with a sodium citrate (14), a citric acid (15), and a carboxymethyl cellulose (16) to obtain a bioinsecticide mixture (20), and finally drying (106) the bioinsecticide mixture (20) to obtain a bioinsecticide (21) powder.

[0058] Accordingly, the fungal insecticide is obtained via isolation of the mycelia of Cordyceps fumosorosea emerging from mycotised dead pests. The entomopathogenic fungi are produced from the mycelia in a liquid culture media or a solid culture media fermentation process known in the technique. Accordingly, the present invention of the bioinsecticide powder preparation method discloses that the Cordyceps fumosorosea stock culture is grown in a sterilised potato dextrose broth media mixture where the broth media is autoclaved at 121 °C for 15-20 minutes to ensure that the broth is free from contamination. The culture is grown with agitation for 3 days at a temperature of 25-30°C, and the pH of the potato dextrose broth media mixture is between pH 4-7. The broth culture is regularly monitored for the proliferation of Cordyceps fumosorosea within the broth, forming visible mycelial growth. The culture is then moved to a dark area for 4 days at room temperature to optimize the conidia and mycelia growth. The culture is also regularly sampled to monitor the fungal growth, biomass concentration, and metabolite production through biomass dry weight determination or optical density measurements.

[0059] The bioinsecticide composition is formed from 10-40% (w / w) of surfactants in a preferred embodiment. Accordingly, the bioinsecticide composition is comprised of one or more surfactants. The surfactants include but are not limited to water soluble food grade surfactants derived from the carboxyl group or a combination thereof. Preferably, the bioinsecticide composition comprises a mixture of carboxymethyl surfactants comprising carboxymethyl cellulose, organic acid, sodium citrate and citric acid. The mixture of carboxymethyl surfactants in the described ranges is suitable to enhance the bioinsecticide composition dispersibility and suspensibility in diluted conditions. Therefore, following modification of the carrier, the bioinsecticide composition will be able to be administered efficiently, through a delivery device, without causing adherence of the submerged propagules and conidia of Cordyceps fumosorosea to the inner walls of the delivery device or obstructing the delivery components of the delivery device. It is to be understood that the mixture of the surfactants in the bioinsecticide composition may have minimal, if not at all detrimental effects on the viability and stability of the entomopathogenic fungi insecticide.

[0060] The present invention also teaches that the bioinsecticide composition is formed from 10-70% (w / w) of a carrier. Accordingly, the bioinsecticide composition is comprised of at least one solid carrier, preferably kaolin clay. The kaolin clay makes up the total amount of bioinsecticide composition. Advantageously, the kaolin clay allows the entomopathogenic fungal insecticide to remain efficacious, thus allowing the bioinsecticide to be delivered to the body of the pest, particularly the delivery of submerged propagules and conidia of Cordyceps fumosorosea onto the bag casing of bagworms, with an effective degree of lethality, enabling the bioinsecticide composition to be re-dispersed and dispensed into a delivery container and preventing sedimentation of the submerged propagules and conidia of Cordyceps fumosorosea. Furthermore, the incorporation of kaolin clay enables the bioinsecticide composition to be formulated as a wettable powder and enhances its effectiveness and usability. Kaolin clay also ensures the even distribution of fungal spores and stabilises the formulation against environmental stresses. Kaolin clay also enhances adhesion to plant surfaces, prolonging residual activity and reducing wash-off during rain or irrigation. Additionally, it can act as a physical barrier to pests, providing dual protection.

[0061] Moving forward, as shown in Figure 1 , the embodiment of the present invention discloses a preparation method (100) for the production of Cordyceps fumosorosea fungal bioinsecticide powder (20), which comprises the following steps:

[0062] 101 , growing a Cordyceps fumosorosea (10) to obtain a Cordyceps fumosorosea stock culture (11 ) whereby 0.1-1 .0 cm, preferably 0.5-1 .0 cm, more preferably 1 cm of Cordyceps fumosorosea is inoculated into a sterile potato dextrose agar broth media. The inoculated Cordyceps fumosorosea is then incubated at a temperature of 25-30°C, preferably 25-28°C, and the culture pH is between 4-7, preferably between pH 5.5-6.5, with constant agitation or aeration at least at 120-150 rpm to enhance growth. The Cordyceps fumosorosea mycelial growth will proliferate within the broth.

[0063] 102, diluting the Cordyceps fumosorosea stock culture (11 ) to obtain a diluted Cordyceps fumosorosea culture (12), where the dilution is done in a serial dilution technique in which 1 ml of Cordyceps fumosorosea stock culture (11 ) is added into 9 ml of sterile distilled water for a 101dilution. Mixed the 101diluted culture and transfer 1 ml from the 101dilution to 9 ml of sterile distilled water for a 102dilution. The steps are repeated until 1010dilution. The serially diluted cultures are left for 7 days. 0.1 ml for dilutions from 106, 107, 108, 109, and 101° are taken and plated on potato dextrose agar for colony-forming unit (CFU) calculations. The bioinsectide powder (20) is produced, preferably from the 108dilution. Diluting the Cordyceps fumosorosea stock culture (11 ) ensures optimal spore concentration for effective pest control, improves handling and application, prevents equipment clogging, and enhances cost-effectiveness. It maintains spore viability, reduces phytotoxicity risk, and allows for better mixing with other formulation components, improving overall bioinsecticide powder (20) performance and stability.

[0064] 103, blending the diluted Cordyceps fumosorosea culture (12) with a kaolin clay (13) to obtain a wet powder texture (17) where the diluted Cordyceps fumosorosea culture (12) is slowly blended with the kaolin clay (13) at 200-800 rpm to avoid splashing and ensure that the diluted Cordyceps fumosorosea culture (12) is fully incorporated into the dry ingredients. The blending is done for 2-10 minutes, preferably 5 minutes, to achieve a uniform, wet powder texture (17) without any lumps or uneven distribution.

[0065] 104, drying the wet powder texture (17) to obtain a dry powder texture (18) where the drying process can be done by not limited but includes air drying, oven drying, freeze drying, vacuum drying, spray drying, infrared drying or a combination thereof. The drying of the wet powder texture (17) is done by spreading the powder thinly and evenly on a tray, and dried at a low drying temperature of 25-35°C, with good air circulation, control humidity, and the drying time is between 1-4 hours, preferably 2 hours to obtain the dry powder texture (18). This will ensure that the wet powder texture (17) is effectively dried without damaging the fungal spores.

[0066] 105, mixing the dry powder texture (18) with a sodium citrate (14), a citric acid (15), and a carboxymethyl cellulose (16) to obtain a bioinsecticide (19) composition where the mixing is done at the speed of 1000-1500 rpm, preferably 1200 rpm, to achieve a more thorough and even mixing. Furthermore, this ensures a homogeneous mixture without causing excessive heat that could harm the fungal spores.

[0067] 106, drying the bioinsecticide (19) composition to obtain a bioinsecticide powder (20) where the bioinsecticide (19) composition is dried by air drying, oven drying, freeze drying, vacuum drying, spray drying, infrared drying or a combination thereof at drying temperature of 25-35°C, for 1-4 hours, preferably 4 hours ensures optimal spore viability and stability. The low-temperature range prevents heat damage to the fungal spores while effectively removing moisture, thus preserving the bioinsecticide's efficacy and extending its shelf life.

[0068] In another embodiment of the present invention where the bioinsecticide powder (20) wettability time is in the range of 10-120 seconds, preferably, 90 seconds. The rapid wettability of the bioinsecticide powder (20) ensures rapid and uniform dispersion in water, facilitating efficient, and consistent application. This further improves the ease of use for farmers, ensuring the bioinsecticide is readily available for immediate pest control upon mixing.

[0069] An aspect of the present invention where the suspensibility of the bioinsecticide powder (20) is 60-90%, preferably 79-80%, more preferably 79.5%. This ensures the optimal distribution of active spores in the solution. This high suspensibility prevents settling, promoting uniform application and effective pest control, thereby enhancing the bioinsecticide's overall efficacy and reliability in agricultural use.

[0070] Another aspect of the embodiment of the present invention is that the bioinsecticide powder (20) foam and sedimentation ability score is between 90-100%, preferably 100%. This indicates that the bioinsecticide powder (20) has minimal foaming and forms a stable suspension. This ensures efficient application without clogging equipment and consistent delivery of active ingredients.

[0071] The embodiment of the present invention further discloses the dispersibility of the bioinsecticide powder (20) between 70-100%, preferably 88%. This characteristic allows the bioinsecticide powder (20) to mix well in water, forming a uniform solution and facilitating effective application, ensuring even coverage on crops and maximizing the bioinsecticide's pest control efficacy, thereby enhancing agricultural productivity and ease of use.

[0072] The method of using the bioinsecticide (19) composition provided by the embodiment of the present invention has the following steps comprising mixing the bioinsecticide (19) composition in water, stirring the bioinsecticide (19) composition and the water to obtain a homogenous mixture, and finally packing the homogenous mixture into a container for application on an affected plant. Preferably the working total volume ratio between bioinsecticide (19) composition to water is in the range of 1 :500-1 :1000. The bioinsecticide (19) composition in solution form then can be applied on the affected plant by spraying or putting the bioinsecticide (19) composition in a sprinkling system.

[0073] According to one embodiment, the efficacy of the efficacy of the bioinsecticide (19) composition is determined 3-9 days after application on the affected plant by observing Cordyceps fumosorosea infection on a target insect pest. This is to allow adequate time for Cordyceps fumosorosea spores to infect and manifest visible symptoms in the target insect pests. This is crucial for the fungal spores to germinate, penetrate the insect cuticle, and proliferate within the host, leading to noticeable infection and mortality. Furthermore, this ensures a reliable assessment of the bioinsecticide’s effectiveness. The described period is also important to avoid inaccurate evaluation of the product’s pest control capability because assessing the bioinsecticide (19) composition too early may not show the full impact of the infection, while too late could miss the peak of the bioinsecticide (19) composition infection stages.

[0074] According to one embodiment, the present invention of the bioinsecticide (19) composition can suppress one or more plant pests. Particularly, the bioinsecticide (19) composition can suppress pest infestation targeted at harmful insects from Lepidopteran, Hemipteran, or Spodoptera order of insects preferably including the Psychidae family consisting of bagworm species of Metisa plana, Pteroma pendula and Mahasena corbetti which is more prominent in oil palm plantations as defoliators.

[0075] Overall, the Cordyceps fumosorosea bioinsecticide is environmentally friendly, targets a wide range of pests, and reduces resistance development. It is safe for non-target organisms, leaves no harmful residues, and fits well in integrated pest management. Additionally, it supports plant health, is cost-effective, and meets organic farming standards. The present invention may be embodied in other specific forms without departing from its essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore indicated by the appended claims rather than by the foregoing description. All changes, which come within the meaning and range of equivalency of the claims, are to be embraced within their scope.

[0076] Description of the reference numerals used in the accompanying illustrations according to the present invention:

[0077] Reference Numerals Description

[0078] 10 Cordyceps fumosorosea

[0079] 11 Cordyceps fumosorosea stock culture

[0080] 12 diluted Cordyceps fumosorosea culture

[0081] 13 kaolin clay

[0082] 14 sodium citrate

[0083] 15 citric acid

[0084] 16 carboxymethyl cellulose

[0085] 17 wet powder texture

[0086] 18 dry powder texture

[0087] 19 bioinsecticide

[0088] 20 bioinsecticide powder

[0089] 100 Preparation method for bioinsecticide powder (20)

[0090] 101 G row ( Cordyceps fumosorosea)

[0091] 102 Dilute (Cordyceps fumosorosea stock culture)

[0092] 103 Blend

[0093] 104 Dry (wet powder texture(17))

[0094] 105 Mix

[0095] 106 Dry (bioinsecticide (19))

Claims

CLAIMS1 . A bioinsecticide (19) composition, comprising of: a diluted Cordyceps fumosorosea culture (12); a kaolin clay (13); a sodium citrate (14); a citric acid (15); and a carboxymethyl cellulose (16), characterised in that the bioinsecticide (19) composition further comprises 50- 1000 mL of the diluted Cordyceps fumosorosea culture (12), 30-90 g of the kaolin clay, 30-90 g of the sodium citrate, 20-60 g of the citric acid, 5-30 g of the carboxymethyl cellulose, and contain submerged propagules and conidia for effective bioinsecticidal action of the composition.

2. The bioinsecticide (19) composition according to claim 1 , wherein the kaolin clay (13) acts as a carrier in amounts ranging from 10-70% weight of the bioinsecticide (19) composition.

3. The bioinsecticide (19) composition according to claim 1 , wherein the sodium citrate (14), the citric acid (15) and the carboxymethyl cellulose (16) mixture are surfactants in the bioinsecticide (19) composition.

4. The bioinsecticide (19) composition according to claim 1 , wherein the total amount of the surfactant mixture ranges between 10-40% by weight of the bioinsecticide (19) composition.

5. A method (100) for preparing a bioinsecticide powder (20), comprising steps of: growing (101 ) a Cordyceps fumosorosea (10) to obtain a Cordyceps fumosorosea stock culture (11 ); diluting (102) the Cordyceps fumosorosea stock culture (11 ) to obtain a diluted Cordyceps fumosorosea culture (12);blending (103) the diluted Cordyceps fumosorosea culture (12) with a kaolin clay (13) to obtain a wet powder texture (17); drying (104) the wet powder texture (17) to obtain a dry powder texture (18); mixing (105) the dry powder texture (18) with a sodium citrate (14), a citric acid (15), and a carboxymethyl cellulose (16) to obtain a bioinsecticide (19) composition; and drying (106) the bioinsecticide (19) composition to obtain a bioinsecticide (20) powder, characterised in the step of growing (101 ) the Cordyceps fumosorosea stock culture (11 ) further comprises agitating a mixture of a potato dextrose broth and the Cordyceps fumosorosea (10) for 3 days in pH 4-7 until conidia and mycelia of Cordyceps fumosorosea are formed, followed by culturing the mixture in a dark area for 4 days at room temperature, diluting (102) the Cordyceps fumosorosea stock culture (11 ) using sterile distilled water, wherein the dilution is done in 1010dilution series, the step of blending (103) to obtain a wet powder texture (17) is done at a slow speed until a homogenous texture is formed, the step of drying (104) the wet powder texture (17) is done for 2 hours, and the step of drying (106) the bioinsecticide (19) composition is done for 4 hours.

6. The method (100) for preparing the bioinsecticide powder (20) according to claim 5, wherein the diluted Cordyceps fumosorosea culture (12) contains submerged propagules and conidia in a concentration ranging from 106- 1010CFU / mL.

7. The method (100) for preparing the bioinsecticide powder (20) according to claim 5, wherein the wettability is in the range of 10-120 s.

8. The method (100) for preparing the bioinsecticide powder (20) according to claim 5, wherein the suspensibility is 60-90%.

9. The method (100) for preparing the bioinsecticide powder (20) according to claim 5, wherein the foam and sedimentation score ranges between 90-100%.

10. The method (100) for preparing the bioinsecticide powder (20) according to claim 5, wherein the dispersibility range between 70-100%.

11. A method of using a bioinsecticide (19) composition prepared according to claim 1 , comprising: mixing at least one part of the bioinsecticide (19) composition and at least one part of water; stirring the bioinsecticide (19) composition and the water to obtain a homogenous mixture; packing the homogenous mixture into a container for application on an affected plant, characterised in that a working volume ratio between the bioinsecticide (19) composition and the water is in the range of 1 :500-1 :1000, wherein the means of application is by spraying or sprinkling system on the affected plant.

12. The method of using a bioinsecticide (19) composition according to claim 11 , wherein the efficacy of the bioinsecticide (19) composition is determined 3- 9 days after application on the affected plant by observing Cordyceps fumosorosea infection on a target insect pest.

13. The method of using a bioinsecticide (19) composition according to claim 12, wherein the target insect is from Lepidopteran, Hemipteran, or Spodoptera order of insect including bagworm species, Metisa plana, Pteroma pendula, or Mahasena corbetti.

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