Microbial bioformulations and their preparation method for growth promotion and blister blight disease control in plants

Abstract

The present invention relates to a microbial bioformulation specifically designed for the treatment of blister blight disease in tea plants. The bioformulation comprises a consortium of actinobacterial strains, including AIPL1, AIPL2, AIPL6, AIPL_TR2, AIPL_TR9, and AIPL_TR19. These strains are selected from groups including Streptomyces sp. The formulation is optimized to deliver effective concentrations of these microbes, inhibiting the growth of blister blight pathogens. Additionally, the bioformulation includes a carrier medium, carbon and nitrogen sources to enhance the efficacy and shelf life. The invention also encompasses the process for preparing this bioformulation, which involves culturing the selected actinobacterial strains, harvesting them, and mixing them with the carrier substrate. The resulting product, produced by the defined process, offers an environmentally friendly and effective alternative to chemical treatments for blister blight disease in tea.

Description

[0001] TITLE: Microbial bioformulations and their preparation method for growth promotion and blister blight disease control in plants

[0002] CROSS REFERENCE

[0003] The patent application claims the priority date benefit of Indian Patent Application no. 202531000041 filed on Jan 01, 2025.

[0004] FIELD OF INVENTION

[0005] This invention relates to microbial bioformulations for the management and treatment of blister blight disease in tea plants. The present invention discloses a bioformulation comprising consortia of potent actinobacterial strains that exhibit broad antifungal activity, improve plant growth, and do not adversely affect the indigenous microbial communities of tea plants.

[0006] BACKGROUND OF THE INVENTION

[0007] Tea (Camellia sinensis) is a vital agricultural commodity, particularly in Northeast India, which is known for its extensive tea gardens. However, the productivity of these tea gardens is under constant threat due to blister blight, a fungal disease caused by Exobasidium vexans. This disease is highly destructive, leading to significant crop losses, sometimes up to 40%, thereby impacting both the economy of the region and the livelihoods of tea farmers.

[0008] Traditional control measures for blister blight, such as pruning and plucking, are labour-intensive and only provide limited relief. In many cases, chemical fungicides and pesticides are employed as a more immediate solution. However, the use of these chemicals presents several challenges, including the risk of phytotoxicity, environmental contamination, and health hazards due to the presence of chemical residues in tea leaves. Consumers are increasingly concerned about the safety and quality of their tea, driving a demand for safer, more sustainable methods of disease control.

[0009] Biological control methods have been explored as an alternative to chemical treatments. For instance, certain strains of Trichoderma harzianum. Serratia marcescens. Pseudomonas fluorescens,Orchobactrum anthropi and Bacillus subtilis have shown somepromise in controlling fungal diseases in plants. However, these biological controls often fall short in terms of efficacy, and their commercial availability is limited. There is a critical need for a bioformulation that not only effectively controls blister blight but also integrates seamlessly into existing agricultural practices without the drawbacks associated with chemical treatments.

[0010] Some of the patent disclosures are also available, such as

[0011] Australian Patent Application Publication No. AU 2021303289 Al, this disclosure focuses on the use of actinobacteria for enhancing growth characteristics in leguminous plants by co-inoculating with rhizobial microorganisms. However, it requires more intensive media conditions for the growth of these microbes, which may not be practical for widespread agricultural use in varying environmental conditions.

[0012] PCT Application Publication No. WO 2019046909 Al, this application discusses novel actinobacteria and their use in biological control. The compositions disclosed involve cell-free culture filtrates from Streptomyces cultures, intended for broad agricultural applications. However, this method is limited by the requirement for specific strains and their associated culture filtrates, which may not provide a holistic solution for complex plant-pathogen interactions in diverse crops like tea.

[0013] US Patent Publication No. US 11819027 B2: This patent discloses methods and compositions using beneficial endophytes from the genus Streptomyces to promote plant growth. The focus is on specific Streptomyces sequences, which, while beneficial, are limited to certain plant species and do not address the broader spectrum of diseases affecting crops like tea.

[0014] Given the limitations of existing technologies, there is a significant unmet need for a bioformulation that is not only effective against blister blight but also adaptable to various environmental conditions, easy to produce, and safe for both humans and the environment. The present invention addresses these needs by providing a novel microbial bioformulation composed of actinobacterial strains that exhibit broad-spectrum antifungal activity, adaptability to different agro-climatic conditions, and compatibility with the indigenous microbial communities of tea plants.

[0015] OBJECT OF THE INVENTIONThe primary object of the invention is to provide a novel microbial bioformulation that effectively controls blister blight disease in tea plants, particularly in the tea gardens of India.

[0016] Another object of the invention is to develop a bioformulation that is composed of potent actinobacterial strains, which produce bioactive secondary metabolites with broadspectrum antifungal activity against Exobasidium vexans, the causal agent of blister blight, and other secondary pathogens.

[0017] A further object of the invention is to provide a bioformulation that is easily culturable and adaptable to varying agro-climatic conditions, making it suitable for use in diverse teagrowing environments.

[0018] Yet another object of the invention is to create a bioformulation that promotes plant growth by producing extracellular enzymes such as cellulase, protease, amylase, and pectinase, thereby enhancing nutrient availability and supporting overall plant health.

[0019] An additional object of the invention is to offer a bioformulation that can be applied both as a soil treatment and as a foliar spray, ensuring ease of application and integration into existing agricultural practices.

[0020] It is also an object of the invention to develop a bioformulation that is environmentally friendly, safe for human health, and does not disrupt the indigenous microbial communities of tea plants, providing a sustainable alternative to chemical fungicides and pesticides. Yet another object of the invention is to provide a versatile bioformulation that can be used not only in tea plants but also in other crops, offering broad utility against various fungal diseases and contributing to sustainable agricultural practices.

[0021] SUMMARY OF THE INVENTION

[0022] The present invention provides a solution to the problem of blister blight in tea plants through the development of a microbial bioformulation composed of a consortium of potent actinobacterial strains. These strains are isolated from the rhizospheric soil of tea plants and have demonstrated significant antifungal activity against Exobasidium vexans, the causative agent of blister blight, as well as other secondary pathogens that commonly affect tea plants.In one aspect the present invention involves the isolation and identification of twelve actinobacterial strains from tea rhizosphere soil, with six of these strains being selected and were found to have superior antifungal properties and have ability to produce extracellular enzymes like cellulase, protease, amylase, and pectinase. These enzymes not only contribute to the suppression of fungal pathogens but also promote plant growth by enhancing nutrient availability.

[0023] Unlike existing bioformulations that may require intensive media conditions or are limited to specific microbial strains, the bioformulation described in this invention is composed of actinobacteria that are easily culturable, produce bioactive secondary metabolites, and are highly adaptable to a range of environmental conditions. These characteristics make the bioformulation particularly suited for use in the diverse agro-climatic conditions of Northeast India, where tea cultivation is prevalent.

[0024] In one aspect the disclosure provides bioformulations developed using the selected strains and the culture conditions for these bioformulations have been optimized with utmost care and experimentation.

[0025] In yet another aspect of the present invention the disclosed bioformulation are effective in suppressing blister blight when applied preventively, without disrupting the indigenous microbial communities.

[0026] In some aspect of the present invention the disclosed bioformulations are versatile and can be used against a range of fungal diseases in different crops.

[0027] In one aspect the disclosed bioformulation offers a dual functionality as both disease suppressors and plant growth promoters, making them a valuable tool for sustainable agriculture.

[0028] The invention disclosed herein presents a comprehensive and environmentally friendly solution to managing blister blight in tea plants, with the potential for broader applications in agriculture.

[0029] SOURCE OF BIOLOGICAL MATERIAL

[0030] The biological material used in the present invention composition includes actinobacterial isolates streptomyces sp. Some of the identified streptomyces sp. Includes Streptomyces californicus, Streptomyces fagopyri, Streptomyces kanamyceticus, Streptomycescalifornicus, Streptomyces goshikiensis, Nocardia coffeae, Streptomyces vinaceus, Streptomyces parvulus, Streptomyces parvulus which were isolated from tea rhizosphere soil of Assam, India. Accession number for the isolate species will be provided.

[0031] BRIEF DESCRIPTION OF DRAWINGS

[0032] The invention will be better understood and objects other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such description makes references to the annexed drawings wherein:

[0033] Figure. 1 : Flow chart depicting preparation process of the antibacterial consortia.

[0034] Figure. 2: Depicts the real time images of the leaves of plant before and after treatment. Fig. 2(a) and (b) are the leaf images 3 months and 6 months after treatment with bioformulation- 1 respectively, and Fig. 2(c) and (d) are leaf without any treatment (Control). Fig. 2(e) and (f) are the leaf images 3 months and 6 months after treatment with bioformulation- 1 respectively and Fig. 2(g) and (h) are leaf without any treatment (Control).

[0035] DETAILED DESCRIPTION OF THE INVENTION

[0036] The following detailed description is merely exemplary in nature and is not intended to limit the described embodiments or the application and uses of the described embodiments. This description is not intended to be a detailed catalogue of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the scope of the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations, and variations thereof.

[0037] The terms “for example” and “such as,” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise.As used herein, the term “about” is meant to account for variations due to any experimental errors which may be commonly accepted in the field for a numeric value, for example such a variation can be considered as a ±10% of the said numeric value. All measurements reported herein are understood to be modified by the term “about,” whether or not the term is explicitly used, unless explicitly stated otherwise. Further for the purposes of the present invention, ranges may be expressed as from “about” one particular value to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value to the other particular value. The recitation of numerical ranges by endpoints includes all the numeric values subsumed within that range.

[0038] As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in the present disclosure; other suitable methods and materials known in the art can also be used. The materials, methods and examples are illustrative only and not intended to be limiting by any means. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict, the present specification, including definitions, will control.

[0040] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

[0041] The term “including” is used to mean “including but not limited to”, “including” and “including but not limited to” are used interchangeably.

[0042] The term “treat” or “eradicate” are used interchangeably to mean effect of the bioformulation in treating the disease by controlling or inhibiting the growth of the pathogen causing the disease or condition in plant.The term “GLM broth medium” refers to different formulation containing glycerol, L-asparagine, and methionine.

[0043] As used herein, the phrases “bioformulation” or “microbial formulation” is used interchangeably and refers to the composition according to the present invention comprising of broth medium, carbon source, nitrogen source and the antibacterial isolates, the said composition is effective against the causal pathogen of blister blight disease in plants, and act as plant growth promoters.

[0044] As used herein, the phrase “consortia” refers to a combination of two or more actinobacterial isolates that has potential in managing plant diseases and pests and also promotes plant growth and mitigates abiotic and biotic stresses in crops such as tea.

[0045] The present invention provides a microbial bioformulation comprising a consortium of actinobacterial strains isolated from the rhizospheric soil of tea plants.

[0046] In some embodiments the disclosed bioformulation comprises of consortium of actinobacterial strains isolated from the rhizospheric soil of tea plants, wherein the strains selected for preparing the consortia exhibit broad-spectrum antifungal activity and are capable of producing bioactive secondary metabolites that are effective against the pathogen responsible for blister blight disease in tea plants.

[0047] In another embodiment, the bioformulation according to the present invention comprises six potent actinobacterial strains identified as AIPL1, AIPL2, AIPL6, AIPL TR2, AIPL TR9, and AIPL TR19. These strains have been found to be adaptable to diverse agro-climatic conditions, and possess antifungal efficacy, ability to produce extracellular enzymes (cellulase, protease, amylase, pectinase). The composition by employing the consortia of the strains provides an enhanced efficacy in terms of disease prevention as well as plant growth promotion.

[0048] In a further embodiment, the bioformulation the formulation according to the present invention is prepared in liquid form, with each strain cultured in GLM broth at suitable temperature and RPM for a period of at least 5 days.

[0049] In a related aspect of the above embodiment the strains are cultured in the GLM broth at a temperature ranging from about 25°C to about 35°C, and at an RPM of about 110 RPM to about 250 RPM.In a further related aspect of the above embodiment the strains are cultured in the GLM broth at a temperature ranging from about 25°C to about 35°C, preferably at about 27°C to about 32°C and at an RPM of about 150 RPM to about 200 RPM.

[0050] In one aspect of the above embodiment the strains are cultured in the GLM broth at a temperature of about 28°C and at an RPM of about 180 RPM.

[0051] The culture conditions are optimized to maximize the production of antifungal metabolites and extracellular enzymes, ensuring the formulation's effectiveness in field applications. In one embodiment, the present invention provides different set of bioformulations comprising of potent actinobacterial strains identified and selected from AIPL1, AIPL2, and AIPL6, AIPL TR2, AIPL TR9, and AIPL TR19. These formulations are developed to offer a synergistic effect when applied to tea plants, enhancing disease control and promoting plant growth.

[0052] In one aspect according to the above embodiment the bioformulation comprises of a combination of strains AIPL1 and AIPL2, wherein the formulation combines the antifungal properties of AIPL1 and AIPL2, providing a potent solution for controlling blister blight while promoting plant growth.

[0053] In yet another aspect according to the above embodiment the bioformulation comprises of a combination of strainsAIPLl and AIPL6, wherein the bioformulation leverages the unique extracellular enzyme production capabilities of AIPL1 and AIPL6 to enhance plant growth and prevent disease spread.

[0054] In another aspect according to the above embodiment the bioformulation comprises of a combination of strainsAIPL2 and AIPL6, wherein the formulation combining AIPL2 and AIPL6, focusing on synergistic antifungal activity and plant growth promotion through enzyme production.

[0055] In a further aspect according to the above embodiment the bioformulation comprises of a combination of strains AIPL TR2 and AIPL TR19, wherein the combination of AIPL TR2 and AIPL TR19 offers broad-spectrum antifungal activity and adaptability to varying ago-climatic conditions, making it ideal for diverse tea-growing environments. In one aspect according to the above embodiment the bioformulation comprises of a combination of strains AIPL TR2 and AIPL TR39, wherein the strains are combined inthis formulation to maximize the production of antifungal metabolites, offering a robust solution for disease control.

[0056] In another aspect according to the above embodiment the bioformulation comprises of a combination of strains AIPL TR19 and AZPL TR39, wherein the said combination formulation harnesses the antifungal and growth-promoting properties of AZPL TR19 and AZPL TR39, designed for effective disease management in tea plants.

[0057] In one aspect according to the above embodiment the bioformulation comprises of a combination of strainsAIPLl, AIPL2, and AIPL TR2, the said formulation combines strains to provide a broader range of antifungal activity and enhanced plant growth promotion.

[0058] In yet another aspect according to the above embodiment the bioformulation comprises of a combination of strainsAIPLl, AIPL6, and AIPL TR19, wherein the formulation using strains AIPL1, AIPL6, and AIPL TR19, ensuring robust disease suppression and support for plant health.

[0059] In one aspect according to the above embodiment the bioformulation comprises of a combination of strainsAIPL2, AIPL TR2, and AIPL TR39, combines the strengths of these strains, focusing on broad-spectrum disease control and plant growth enhancement. In yet another aspect according to the above embodiment the bioformulation comprises of a combination of strainsAIPLl and AIPL TR2, the said simpler formulation combining AIPL1 and AIPL TR2, offering a balanced approach to disease control and plant growth promotion.

[0060] The above disclosed combinations of bioformulation provide a wide range of formulations that were evaluated for their efficacy in treating blister blight disease and promoting tea plant health. These combinations offer flexibility for different afro-climatic conditions and disease severity levels.

[0061] In another embodiment, the present invention provides a bioformulation comprising of AIPL1, AIPL2, and AIPL6, wherein the said formulation offers a synergistic effect when applied to tea plants, enhancing disease control and promoting plant growth.In yet another embodiment, the present invention provides a bioformulation comprising of AZPL TR2, AIPL TR9, and ALPL TR19, wherein the said formulation offers a synergistic effect when applied to tea plants, enhancing disease control and promoting plant growth. In a related aspect of the above embodiments the said composition is effective against the pathogen responsible for blister blight disease in tea plants. In another related aspect the said pathogen is Exobasidium vexans.

[0062] In one embodiment, the present invention discloses a process for preparing the microbial bioformulation, which includes isolating actinobacterial strains from tea rhizospheric soil, culturing the isolates in a nutrient-rich broth, and optimizing the culture conditions to enhance the production of antifungal metabolites and extracellular enzymes. The prepared formulation was found to be effective against the pathogens responsible for blister blight disease in tea plant.

[0063] In another embodiment, the process specifically involves culturing the selected actinobacterial strains selected from AIPL1, AIPL2, AIPL6, AIPL TR2, AIPL TR9 and AIPL7 in GLM broth under controlled conditions of temperature and agitation for at least 5 days. This process ensures the maximum viability and activity of the microbial consortia in the final bioformulation.

[0064] In one aspect of the above process embodiment the process involves culturing AIPL1 and AIPL2 strains in GLM broth at suitable temperature with agitation for at least 5 days. The controlled conditions are optimized to enhance the production of antifungal metabolites, ensuring the strains' effectiveness in the final bioformulation.

[0065] In yet another aspect of the above process embodiment the process involves culturing involves culturing AIPL1 and AIPL6 strains in GLM broth under controlled conditions of temperature, agitation for suitable period. The combination of these strains in the culturing process focuses on maximizing extracellular enzyme production, contributing to the bioformulation's dual role in disease suppression and plant growth promotion.

[0066] In a further aspect of the above process embodiment the process involves culturing procedure wherein AIPL2 and AIPL6 strains are cultured together in GLM broth at suitable temperature and agitation for a period of at least 5 days. The controlled environment allows these strains to thrive, ensuring a high concentration of bioactive metabolites in the bioformulation.In yet another aspect of the above process embodiment the process involves culturing the AZPL TR2 and AIPL TR9 strains in GLM broth under controlled conditions. The process focuses on maximizing antifungal metabolite production.

[0067] In a further aspect of the above process embodiment the process involves culturing AIPL TR2 and AIPL7 in GLM broth at 28°C suitable temperature with agitation for 7 days. The process aims to optimize the production of bioactive compounds that contribute to the bioformulation's antifungal efficacy.

[0068] In another aspect of the above process embodiment the process involves culturing AIPL TR19 and AIPL TR39 strains in GLM broth under controlled conditions. This embodiment focuses on ensuring the robust growth of these strains and their contribution to the final bioformulation's antifungal properties.

[0069] In some aspects of the above process embodiment the process involves culturing AIPL1, AIPL2, and AIPL TR2 strains together in GLM broth at suitable temperature with agitation for at least 7 days. The combination of these strains enhances the bioformulation's spectrum of antifungal activity and its plant growth-promoting properties. In one aspect of the above process embodiment the process involves culturing AIPL1, AIPL6, and AIPL TR19 in GLM broth under controlled conditions of temperature and agitation for suitable period. The process is designed to maximize the production of both antifungal metabolites and extracellular enzymes, making the bioformulation effective for both disease control and plant growth promotion.

[0070] In a further aspect of the above process embodiment the process involves culturing AIPL2, AIPL TR2, and AIPL TR39 strains together in GLM broth at suitable temperature with agitation for at least 5 days. The focus is on producing a bioformulation with a broad spectrum of antifungal activity.

[0071] In another aspect of the above process embodiment the process involves culturing AIPL1 and AIPL TR2 strains in GLM broth at suitable temperature with agitation for sufficient duration. The process is optimized to ensure the maximum viability and activity of these strains, contributing to an effective bioformulation for disease control and plant growth promotion.In related aspect of the above disclosures in the process the strains are cultured in the GLM broth at a temperature ranging from about 25°C to about 35°C, and at an RPM of about 110 RPM to about 250 RPM, preferably at a temperature ranging from about 27°C to about 32°C, and at an RPM of about 150 RPM to about 200 RPM, more preferably at a temperature of about 28°C and at an RPM of about 180 RPM. Such condition of temperature and agitation for suitable period of 5-10 days is also referred to a controlled condition for the culture of strains according to the present invention.

[0072] These process embodiments provide various combinations of the actinobacterial strains, each cultured under controlled conditions to produce an effective bioformulation. The optimized process conditions allow flexibility in selecting the most suitable strains and processes based on specific field conditions and disease challenges.

[0073] In another embodiment, the process specifically involves culturing the selected actinobacterial strains selected from AIPL1, AIPL2, AIPL6, AIPL TR2, AIPL TR9 and AIPL TR19 in GLM broth under controlled conditions of temperature (28°C) and agitation (180 rpm) for 7 days. This process ensures the maximum viability and activity of the microbial consortia in the final bioformulation.

[0074] In a further embodiment, the process includes the application of the bioformulation to tea plants. The formulation is applied as a foliar spray or soil treatment at specific intervals, with the dosage and application frequency optimized based on field trials to achieve the best results in disease suppression and plant growth promotion.

[0075] In another embodiment, the present invention provides a process for testing the efficacy of the bioformulation in field conditions. This process involves treating specific blocks of tea plants with the bioformulation, monitoring disease incidence, and comparing the results with untreated control blocks to assess the bioformulation's effectiveness.

[0076] In one embodiment, the invention relates to a method of treating blister blight disease in tea plants, which involves applying the microbial bioformulation comprising the actinobacterial strains to the tea plants. The bioformulation acts as a biocontrol agent, suppressing the growth of Exobasidium vexans and reducing the incidence of blister blight. In another embodiment, the invention provides a method for preventing blister blight disease in tea plants. The bioformulation is applied preventively before the onset of thedisease, creating a protective barrier that inhibits the pathogen's ability to infect the tea plants.

[0077] In a further embodiment, the method specifically involves spraying the bioformulation on tea plants, with applications made every 15 days over a period of three months. The effectiveness of this treatment is evaluated by monitoring the number of blister blight-infected leaves in treated versus untreated control blocks.

[0078] In one related aspect the dosage of water to bioformulation to be applied is selected from:

[0079]

[0080] In one preferred embodiment the method according to the present invention involves spraying the bioformulation on tea plants in a dosage of 5 liters of bioformulation mixed with 5 liters of water, with applications made every 15 days over a period of three months. The effectiveness of this treatment is evaluated by monitoring the number of blister blight-infected leaves in treated versus untreated control blocks.In another embodiment, the method according to the present disclosure not only focuses on disease treatment but also promotes plant growth. The bioformulation, when applied to tea plants, enhances plant vigor and yield by providing beneficial extracellular enzymes and maintaining a healthy microbial balance in the soil.

[0081] Exemplary broth medium according to present invention is selected from:

[0082] GLM broth medium comprise of ingredients selected from glycerol and peptone which acts as carbon and nitrogen sources respectively that causes optimum growth of actinobacterial isolates which in turn promotes antimicrobial secondary metabolites production. However, if the microbial strains are put directly in water, the strains won’t be able to grow optimally and thus microbial metabolites won’t be produced.

[0083] In one preferred aspect the GLM broth medium comprises of malt extract, yeast extract, peptone, glycerol, with a slightly alkaline pH in water.

[0084] In one related aspect the GLM broth medium comprises of Malt extract: 3 g; yeast extract: 3 g; peptone: 5 g; glycerol: 10 ml; PH: 7.4; water: 1000 L.

[0085] A suitable carbon source according to the present invention is selected from a group comprising of glycerol, and like molecules or combination thereof.

[0086] A suitable nitrogen source according to the present invention is selected from a group comprising of peptone and like molecules or combination thereof.

[0087] The antibacterial isolates according to the present invention is selected from Streptomyces sp. selected from strains Streptomyces californicus, Streptomyces fagopyri, Streptomyces kanamyceticus, Streptomyces californicus, Streptomyces goshikiensis, Nocardia coffeae, Streptomyces vinaceus, Streptomyces parvulus, Streptomyces parvulus.

[0088] In the present invention the strains are identified and coded as: AIPL1: Streptomyces californicus, AIPL2: Streptomyces fagopyri, AIPL3: Streptomyces kanamyceticus, AIPL4: Streptomyces californicus, AIPL6: Streptomyces goshikiensis, AIPL7: Nocardia coffeae, AIPL TR2: Streptomyces vinaceus, AIPL TR9: Streptomyces parvulus, AIPL TR19: Streptomyces parvulus.

[0089] In one aspect the present invention involves the isolation and identification of twelve actinobacterial strains from tea rhizosphere soil, with six of these strains selected (AIPL1,AIPL2, AIPL6, AIPL TR2, AIPL TR9, and AIPL TR19) were found to have superior antifungal properties and their ability to produce extracellular enzymes like cellulase, protease, amylase, and pectinase. These enzymes not only contribute to the suppression of fungal pathogens but also promote plant growth by enhancing nutrient availability.

[0090] Unlike existing bioformulations that may require intensive media conditions or are limited to specific microbial strains, the bioformulation described in this invention is composed of actinobacteria that are easily culturable, produce bioactive secondary metabolites, and are highly adaptable to a range of environmental conditions. These characteristics make the bioformulation particularly suited for use in the diverse agro-climatic conditions of Northeast India, where tea cultivation is prevalent. In one related aspect the enzymes of the constituent microbial strains not only contribute to the suppression of fungal pathogens but also promote plant growth by enhancing nutrient availability.

[0091] Moreover, the bioformulations are versatile and can be used against a range of fungal diseases in different crops. They also offer dual functionality as both disease suppressors and plant growth promoters, making them a valuable tool for sustainable agriculture. This invention thus presents a comprehensive and environmentally friendly solution to managing blister blight in tea plants, with the potential for broader applications in agriculture.

[0092] Some of the key advantages of he disclosed bioformulation and its preparation method includes:

[0093] • Highly specific to blister blight disease: The bioformulation targets the blister blight pathogen Exobasidium vexans effectively, reducing disease incidence.

[0094] • Versatility: The bioformulation can be applied to different crops and is effective against other fungal diseases.

[0095] • Dual functionality: Acts as both a disease control agent and a plant growth promoter.

[0096] • Eco-friendly and safe: The bioformulation has no harmful effects on indigenous microbial communities and poses no health risks to humans.

[0097] • Ease of application: The bioformulation can be applied through both soil and foliar spraying, making it convenient for use in various agricultural practices.This invention thus provides a robust solution to one of the most challenging diseases in tea cultivation, offering a sustainable and effective alternative to conventional chemical treatments.

[0098] EXAMPLES:

[0099] The following examples include only exemplary embodiments to illustrate the practice of this disclosure. It will be evident to those skilled in the art that the disclosure is not limited to the details of the following illustrative examples and that the present disclosure may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive.

[0100] Exampl e-1: For testing purpose two bioformulations have been developed using the disclosed strains. Bioformulation- 1 comprised of AIPL1, AIPL2, and AIPL6, while Bioformulation-2 comprises AIPL TR2, AIPL TR9, and AZPL TR19. Each of the bioformulations were a consortia of three actinobacterial isolates.

[0101] Example- 1 A: Preparation of Bioformulation-1

[0102] Material used in preparation:

[0103] GLM broth prepared by mixing malt extract: 3 g; yeast extract: 3 g; peptone: 5 g; glycerol: 10 ml; PH: 7.4; water: 1000 L. All the material used for preparation of broth were procured from local vendors.

[0104] Carbon source: Glycerol

[0105] Nitrogen source: Peptone

[0106] Actinobacterial isolates- AIPL1, AIPL2, and AIPL6

[0107] Strains AIPL1, AIPL2, and AIPL6 were grown individually in 500 ml GLM broth medium along with carbon and nitrogen source and incubated at 28 °C with continuous shaking at 180 rpm for 7 days. After the incubation period, the strains were amalgamated to a final volume of 1.5 L (bioformulation- 1).

[0108] Example- IB: Preparation of Bioformulation-2

[0109] Material used in preparation:GLM broth prepared by mixing malt extract: 3 g; yeast extract: 3 g; peptone: 5 g; glycerol: 10 ml; PH: 7.4; water: 1000 L. All the material used for preparation of broth were procured from local vendors.

[0110] Carbon source: Glycerol

[0111] Nitrogen source: Peptone

[0112] Actinob acteri al isolates- AIPL TR2, AIPL TR9, and AZPL TR19

[0113] Strains AIPL TR.2, AIPL TR9, and AIPL TR19 were grown individually in 500 ml GLM broth medium along with carbon and nitrogen source and incubated at 28 °C with continuous shaking at 180 rpm for 7 days. After the incubation period, the strains were amalgamated to a final volume of 1.5 L (bioformulation- 1).

[0114] Example-2: Evaluation of prepared bioformulation

[0115] To effectively demonstrate the efficacy of the bioformulations developed, the following field trials were conducted:

[0116] Field Trial was conducted at Tea gardens in Northeast India (Umsning, Meghalaya). Testing condition and criteria were as below:

[0117] Tea Clones Used: TV19 and Nandadevi clones

[0118] Trial Period: April to June

[0119] Spray Interval: Every 15 days

[0120] Spray Time: Early morning hours (7-8 AM)

[0121] Conditions: No spraying during rain; re-spray if rain occurred immediately after application.

[0122] Experimental Blocks:

[0123] 1. Block A (TV19 Clone)

[0124] o Dosage 1 : 5 L water + 5 L Bioformulation

[0125] o Dosage 2: 8 L water + 3 L Bioformulation

[0126] 2. Block D (Nandadevi Clone)

[0127] o Dosage 1 : 5 L water + 5 L Bioformulationo Dosage 2: 8 L water + 3 L Bioformulation

[0128] Trial Results Analysis

[0129] Results were analyzed monthly, with a final analysis conducted after 6 months to assess the efficacy of the bioformulations in reducing blister blight infection.

[0130] Table: Field Trial Results:

[0131]

[0132] Observation:

[0133] . TV19 Clone (Block A):

[0134] o Bioformulation- 1 was more effective at the 5L:5L ratio, reducing blister blight infections to 98 compared to the control (468).

[0135] o Bioformulation-2 showed superior performance at the 8L:3L ratio, reducing infections to 54 compared to the control (537).

[0136] Nandadevi Clone (Block D):

[0137] o Bioformulation-2 showed consistent performance, reducing blister blight infections to 105 (5L:5L dosage) and 156 (8L:3L dosage) compared to their respective controls.

[0138] o Bioformulation- 1 was slightly less effective in Block D, with a reduction in infections to 167 and 179 for the 5L:5L and 8L:3L dosages, respectively. These results highlight the potential of both bioformulations in controlling blister blight disease in tea plants, with specific effectiveness depending on the dosage and clone type.The data demonstrates the robustness of the formulations in different environmental conditions and their adaptability to various tea clones

[0139] The culture conditions for these bioformulations have been optimized, and they have been subjected to field trials in tea gardens in Umsning, Meghalaya. The results from these trials indicate that the bioformulations are effective in suppressing blister blight when applied preventively, without disrupting the indigenous microbial communities.

Claims

CLAIMS:

1. A microbial bioformulation for treating or eradicating blister blight disease in tea plants, comprising a:consortium of actinobacterial strains isolated from the rhizospheric soil of tea plants, wherein said actinobacterial strains include strains selected from Streptomyces sp.,a broth medium, a carbon source, and a nitrogen source,characterized in that the bioformulation exhibits enhanced broad-spectrum antifungal activity and promotes plant growth.

2. The bioformulation as claimed in claim 1, wherein the streptomyces sp. in the consortium of isolated strains is selected from Streptomyces califomicus, Streptomyces fagopyri, Streptomyces kanamyceticus, Streptomyces goshikiensis, Nocardia coffeae, Streptomyces vinaceus, and Streptomyces parvulus.

3. The bioformulation as claimed in claim 1, wherein the consortium of isolated strains when incorporated in the bioformulation has enhanced production of bioactive secondary metabolites and extracellular enzymes selected from cellulase, protease, amylase, and pectinase for providing synergistic antifungal and enhancing plant growth promotion.

4. The bioformulation as claimed in claim 1, wherein the broth medium is a GLM broth medium comprising of different formulation containing glycerol, L- asparagine, and methionine.

5. The bioformulation as claimed in claim 1, wherein the nitrogen source is selected from peptone and other nitrogen sources or combination thereof.

6. The bioformulation as claimed in claim 1, wherein the carbon source is selected from glycerol and other carbon sources or combination thereof.

7. A process for preparing an antimicrobial bioformulation for treating or eradicating blister blight disease in tea plants, comprising:a. Isolating actinobacterial strains from the rhizospheric soil of tea plants;b. Culturing the isolated actinobacterial strains, selected from streptomyces sp., in a GLM broth medium under controlled conditions of temperature and agitation for a period of at least 5 days;c. Optimizing the culture conditions to enhance the production of antifungal metabolites and extracellular enzymes, thereby ensuring the bioformulation’s effectiveness in controlling the pathogen responsible for blister blight disease in tea plants.

8. The bioformulation as claimed in claims 1 and 7, wherein the GLM broth medium used for culturing the actinobacterial strains comprises malt extract, yeast extract, nitrogen source comprises of peptone, and carbon source comprises of glycerol, with an alkaline pH of 7.1 and above.

9. The bioformulation as claimed in claims 1 and 7, wherein the actinobacterial strains are cultured at a temperature ranging from about 27°C to about 32°C, and at an agitation speed of about 150 RPM to about 200 RPM.

10. The process according to claim 2, wherein the bioformulation is applied to tea plants as a foliar spray or soil treatment at intervals optimized based on field trials to achieve maximum disease suppression and plant growth promotion.

11. The bioformulation according to claim 1, wherein the consortium of actinobacterial strains includes a combination selected from:AIPL1 andAIPL2,AIPL1 andAIPL6,AIPL2 and AIPL6,AZPL TR2 and AIPL TR9,AZPL TR2 and AIPL TR19,AZPL TR9 and AIPL TR19,AIPL1, AIPL2, and AZPL TR2,AIPL1, AIPL2 and AIPL6,AIPL1, AIPL6, and AZPL TR9,AZPL TR2, AZPL TR9, AIPL TR19,AIPL2, AIPL TR2, and AIPL TR19, andAIPL1 andAIPL_TR2,each providing synergistic effects for treating blister blight disease and promoting plant growth.

12. The bioformulation according to claim 1, wherein the actinob acteri al strains in the consortium are adaptable to diverse agro-climatic conditions, making the bioformulation particularly suitable for use in tea-growing regions with varying environmental conditions.

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