WO2025013066A1

WO2025013066A1 - A bioformulation and a method for controlling pathogen and enhanced growth in banana

Info

Publication number: WO2025013066A1
Application number: PCT/IN2024/051140
Authority: WO
Inventors: Mithilesh Kumar; Pascal FOEHN; Anup SUBHASH KARWA
Original assignee: Innoterra Bioscience Private Ltd
Current assignee: Innoterra Bioscience Private Ltd
Priority date: 2023-07-13
Filing date: 2024-07-10
Publication date: 2025-01-16
Anticipated expiration: 2026-01-13

Abstract

The invention discloses a bioformulation and a method to control pathogen and enhance growth in banana. The methods of applications for controlling, preventing, or combating fungal diseases and thrips in banana plants, plant parts or the surroundings, comprising applying to banana plants or the locus thereof, a bioformulation containing Trichoderma reesei. More specifically, the invention discloses a method for controlling all soil borne fungal pathogens causing wilts and blights, particularly caused by Fusarium oxysporum f.sp. cubense strains or F. odorotassimum or other Fusarium sp. strains, leaf spots (Sigatoka) or freckles caused by different species of Pseudocercospora, Mycosphaerella, Alternania or Cladosporium and banana flower-, rust-, or silvering- thrips by using a bioformulation comprising a fungal culture selected from pure culture of Trichoderma ressei or microsclerotia, or submerged conidia or chlamydospores or combinations thereof; at least one organic complex oligosaccharide, at least one nutrition source, and at least one acceptable carrier.

Description

A BIOFORMULATION AND A METHOD FOR CONTROLLING PATHOGEN AND ENHANCED GROWTH IN BANANA

FIELD OF THE INVENTION

[1] The present invention relates to the field of providing a bioformulation for controlling plant disease, improving plant health, and developing a method for sustainable, chemical pesticide free disease control in banana cultivation to prevent microbial and thrips induced spoilage of crop and harvests. More specifically, the invention relates to a formulation and a method for controlling all soil borne fungal pathogens causing wilts and blights, particularly caused by Fusarium oxysporum f.sp. cubense or F. odorotassimum or other Fusarium sp. and leaf spots (Sigatoka) or freckles caused by different species of Pseudocercospora, Mycosphaerella, Alternania or Cladosporium and banana flower, rust or silvering thrips.

[2] Thus, the present invention is related to providing a Trichoderma reesei based bioformulation for banana cultivation for disease control along with improving plant growth and yield and developing methods for disease control.

[3] Background of the Invention

[4] Bananas are rich source of vitamin B6, vitamin C and potassium and hailed to be common man’s nutrition. With a global production of about 160 million tons, they are highly valued agriculture commodity having great human consumption, ranking only after rice, wheat, and maize. Banana is primarily cultivated word- wide for fruits, however, used for fiber and other purposes as well. Around 70% of the world’s banana production comes from a cavendish cultivar, Grand Naine having AAA genome type.

[5] Although economically very important, banana crop is infested by many diseases, which pose significant crop loss and loss in production and quality parameters of fruits, every banana growing season. Mostly these diseases are caused by fungal or bacterial pathogens or by viruses. Insect pests are another menace to banana production. [6] Fusarium wilt or Panama wilt or Panama disease has been the most devastating disease of banana caused by soil borne pathogenic strains of Fusarium sp. of which several strains are now known to the world. By 1960s, Fusarium oxysporum f.sp. cubense (also abbreviated Foe) -strain Racel almost abolished ‘Gros Michel’ banana cultivar, the then major variety from world by spreading to all major banana growing areas, despite all chemical fungicide and phytosanitary practices efforts undertaken. Later, a new cultivar named Cavendish was found to be relatively resistant to the then pathogenic strains of Foe and most of the banana production and export industry adopted and started trading Cavendish cultivars. However, shortly after its adoption, Cavendish clones were found to be sensitive to a new strain of Foe, called Tropical Race-4 (Foc-TR4). This associated Fusarium wilt disease gradually spread to the rest of the banana growing regions of world.

[7] The Foc-TR4 is virulent to Cavendish banana as well as many other locally grown cultivars and has been recently renamed as Fusarium odoratissimum (Maryani et al., 2019; Garcia-Bastidas et al., 2019). Apart from TR4, other pathogenic strains of Foc- like STR4 and R4 have also been identified. An effective management strategy to control this deadly, soil borne disease of banana caused by Foc-TR4, STR4, R4 or R1 is lacking, and banana growers mostly resort to phytosanitary practices, isolation and removal, and a few common chemical fungicides, whereas pathogen once established in the field can survive for years and spread by farm implements, trade and transport of infected plant parts and via farm personnel (Ploetz, 2015). Chemical fungicides have shown their inefficacy towards prevention and spread of Fusarium wilt and its pathogen.

[8] After Fusarium wilt, the disease that affects banana cultivation the most is the Sigatoka disease complex. It is the most destructive foliar disease causing greatest economic losses in banana cultivation, having the potential to cause yield losses up to 50%. It is a disease complex- Mycosphaerella fijiensis anamorph Pseudocercospora fijiensis causes Black Sigatoka or Black Leaf Streak disease in banana; Mycosphaerella musicola anamorph Pseudocercospora musae causes Yellow Sigatoka and Mycosphaerella eumusae anamorph Pseudocercospora eumusae causes eumusae leaf spots in banana plants (Chang et al., 2016). [9] Initially, mineral oil and then benzimidazole and then triazole fungicides have been used to control the disease, with some success, but pathogen soon developed resistance towards it. Many other new chemical fungicides or combination thereof have been introduced in recent past, but number of spray applications must be increased to control the disease effectively, however, in field scenario, no chemical control or combination has been found completely effective for Sigatoka disease control in banana. Higher frequency of applications not only affects plant health in general, it also has impact on environment and health of banana farmers.

[10] The infestation of different insect pests limits the quantitative and qualitative aspect of banana. In India alone, about 19 insect pests have been reported to cause damage, resulting significant economic loss. Among these, red rust thrips, Chaetanophothrips signipennis affect the cosmetic value of the fruits and hence the sale value (Bisane et al., 2018). The other thrips that cause significant damage to the fruit quality and harvests are banana flower thrips (Thrips florum or Franklinella parvula) and banana silvering thrips (Elixothrips breviselis). Chemical pesticide of synthetic pyrethroids class and bro ad- spectrum activity like deltamethrin (non- systemic) or Fipronil based phenyl pyrazole (systemic) have been used by banana culture growers to inject or spray to the buds for protection against thrips. Although these offer some degree of control over thrips, but under field conditions and heavy infestation, extensive application is likely to induce insect resistance. Frequent and high dose application also invites increased cost of cultivation, and it is not eco- friendly and sustainable.

[11] As noted above the chemical control is not sustainable, and non-safe or human and environment as they are toxic and non-biodegradable. People are becoming increasingly aware of the harmful effects of chemical pesticides and more concerned about chemical residues on plant parts and fruits. Noteworthy is the fact that pests and pathogens are turning more and more resistant over the period of time to available chemical pesticides and fungicides due to repeated and non-precise application and exposure. Unintended exposure of chemical pesticides is harmful to other beneficial organisms also. An effective control of pathogens and pests, therefore, with such chemical control agents is no longer possible. Moreover, using chemical pesticides is economically expensive apart from it being not environment friendly. Further, using fertilizer is not promotive for plant growth and yield.

[12] Currently there are no single control measure that exists which can simultaneously take care of fungal pathogens, notably those causing Fusarium wilt and Sigatoka diseases as well as of thrips in banana and at the same time, which is eco-friendly, environmentally safe, sustainable, commercially viable and can bring banana cultivation, quality, and trade to improved status.

[13] Another challenging fact is the presence of combination products of biocontrol agents, where either different strains of Trichoderma species or Trichoderma strain with other, bacterial or fungal biocontrol agents are used. All selected mycopathogenic microbes used in a combination formulation must not compete and should be compatible under field conditions suitable for banana crop. Further, it has been found that direct application of Trichoderma conidia, ascospores, hyphal portions or propagules or combination thereof has been found less effective than a bioformulation where Trichoderma active constituents are present with an agriculturally acceptable carrier and other adjuvants.

[14] Most of the Trichoderma based bioformulations are applied via various means-to the soil to reach plant rhizosphere, colonize there and exert their mycopathogenic or entomopathogenic action. In only a few of the Trichoderma strains, rhizosphere competence has been demonstrated (Bailey et al., 1998). However, this is more important for crops which are seed propagated, where Trichoderma based formulation is applied as seed coating or at the time of seedling establishes, so that Trichoderma colonizes there for its activity. Banana is a crop which is not propagated via seeds. Most of the commercially available bioformulations are applied via soil drenching only as of now and lack components or methods feasibility to apply the formulation or its active constituent on banana plant aerial parts, inside of the banana vascular system or the floral parts, where it is greatly needed to control the spread of the fungal pathogens and insects. [15] To address the problem in the art, such as frequent use of chemical pesticide leading to disease resistance; lack of availability of competent strains of Trichoderma based bioformulations for banana crop; lack of bioformulations with universal applicability to aerial, below ground and internal parts of banana crop; lack of formulation which is eco-friendly, and affordable; and lack of scalable bioformulation which is growth promoting and effective against common pests in banana crop; there is a requirement of a novel and improved bioformulation with Trichoderma species strain having high activity levels and extended shelf life for the banana crop.

[16] A more severe need is to have a formulation and application methods where such effective, environment friendly, less toxic-harmful and plant growth promotant Trichoderma based formulation can be applied to banana plants in order to prevent, combat or control Fusarium wilt, Sigatoka disease and other fungal pathogens and banana thrips infestation. The applicants of present invention have identified such formulation and application methods comprising a novel Trichoderma reesei based bioformulation that is highly effective in controlling banana fungal diseases and thrips as well as to promote plant growth.

Summary of the Invention:

[17] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[18] In one aspect, the present invention relates to a bioformulation comprising: a. A fungal culture selected from pure culture of Trichoderma ressei or microsclerotia, or submerged conidia or chlamydospores or combinations thereof; b. at least one organic complex oligosaccharide, c. at least one nutrition source, and d. at least one acceptable carrier. [19] In another aspect, the present invention relates to a method of preparing the bioformulation for controlling infestation and promoting banana plant growth in terms of plant height, plant girth and number of leaves, and enhancing yield in terms of fruit bunch weight enhancing growth in the banana plant, comprising the steps of: i) mixing fungal culture selected from pure culture of Trichoderma ressei or microsclerotia, or submerged conidia or chlamydospores or combinations thereof; at least one organic complex oligosaccharide, at least one nutrition source, and at least one acceptable carrier; ii) soaking the obtained bioformulation in water for 24-36 hours; iii) filtering the wet bioformulation; and iv) applying the bioformulation to the banana plants.

[20] In another aspect, the present invention relates a bioformulation for promoting banana plant growth in terms of plant height, plant girth and number of leaves, and enhancing yield in terms of fruit bunch weight and controlling infection comprising of a fungal culture selected from pure culture of Trichoderma ressei, or micro sclerotia, or submerged conidia or chlamydospores or combinations thereof; at least one organic complex oligosaccharide, at least one nutrition source, and at least one acceptable carrier.

[21] In another aspect, the present invention relates to a use of a bioformulation comprising a fungal culture selected from pure culture of Trichoderma ressei, or microsclerotia or submerged conidia or chlamydospores or combinations thereof; at least one organic complex oligosaccharide, at least one nutrition source, and at least one an acceptable carrier for preparation of biopesticides for treatment of banana crop from infestation, promoting banana plant growth in terms of plant height, plant girth and number of leaves, and enhancing yield in terms of fruit bunch weight.

[22] In another aspect, the present invention relates to a method of treating the banana crop from infestation and enhancing its growth comprising the steps of: a. providing a bioformulation comprising a fungal culture selected from pure culture of Trichoderma ressei, or microsclerotia, or submerged conidia or chlamydospores or combinations thereof; at least one organic complex oligosaccharide, at least one nutrition source, and at least one acceptable carrier in a wettable powder or a liquid form; b. applying bioformulation to the banana plant, plant parts or surroundings via soil drenching, via foliar spray, via drone spray, or a combination thereof at defined intervals, frequencies and in concentrations at specified growth stages; via injection to pseudo-stem, or banana mat, via injection to emerging floral buds or a combination thereof at defined intervals, frequencies and in concentrations and at specified growth stages with a specified angle and process.

[23] In another aspect, the present invention relates to a method of application to the banana crop from infestation and enhancing its growth by applying bioformulation to the banana plant, plant parts or surroundings via soil drenching, via foliar spray, via drone spray, or a combination thereof at defined intervals, frequencies and in concentrations at specified growth stages; via injection to pseudo-stem, or banana mat, via injection to emerging floral buds or a combination thereof at defined intervals, frequencies and in concentrations and at specified growth stages with a specified angle and process.

[24] The method treatment with bioformulation of the present invention to the plant plants, plant parts or surroundings to effectively control fungal diseases and thrips and to promote plant growth and yield involve: a. antagonizing, preventing or combating fungal pathogen of Fusarium wilt or Panama wilt in banana, that comprises applying Trichoderma reesei based bioformulation to the banana plant, banana rhizosphere, banana pseudo-stem vascular tissues, banana leaves or the locus of the plant. b. antagonizing, preventing or combating fungal pathogens of Sigatoka disease and other leaf spot diseases, that comprises applying Trichoderma reesei based bioformulation to the banana plant, banana rhizosphere, banana pseudo-stem vascular tissues, banana leaves or the locus of the plant. c. antagonizing, preventing or combating banana thrips, that comprises applying Trichoderma reesei based bioformulation to the banana plant, emerging floral bud or parts thereof. d. applying Trichoderma reesei based bioformulation to banana plant, plant parts and surroundings, enhancing nutrient uptake, promoting plant growth in terms of plant height, plant girth and number of leaves, and increasing plant yield in terms of enhanced fruit bunch weight. e. applying T. reesei based bioformulation to the banana plant, plant parts or surroundings via soil drenching, via foliar spray, via drone spray, or a combination thereof at defined intervals, frequencies and in concentrations at specified growth stages; via injection to pseudo-stem, or banana mat, via injection to emerging floral buds or a combination thereof at defined intervals, frequencies and in concentrations and at specified growth stages with a specified angle and process. f. achieving full Fusarium Wilt and Sigatoka disease control, banana thrips control and enhanced plant growth and yield with the help of embodiments as specified in (a)- (e) and reducing the usage of synthetic, chemical fungicides to a minimal level, in most preferred embodiment, reduced to nil or so.

Detailed Description of the Invention

[25] In the following description, reference is made to the accompanying drawings where, by way of illustration, specific embodiments of the invention are shown. It is to be understood that other embodiments may be used, and other changes may be made without departing from the scope of the present invention. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not as restrictive.

[26 ] Definition

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

[28] The terms “comprising”, “comprised of’ and “comprises” when referring to recited members or method steps also include embodiments which consist of said recited members or method steps, as used herein and synonymous with “including” or “containing”, and do not exclude non-recited members or method steps. The term “composition” as used herein refers to the form in which T. reesei is applied to or used on/ in banana plants, parts thereof or surroundings. [29] The term “embodiment” when referring to a particular characteristic, feature or structure described in relation to that embodiment is included in at least one aspect of the invention and representation of phrases “in one embodiment” or “in an embodiment” at different places throughout description are not necessarily referring to the same embodiment, but they may. Further, combination of characteristic features of different embodiments are meant to be within the scope of the disclosed invention.

[30] The terms “plants” or “banana plants” as used herein refer to banana plants belonging to genus Musa is Musa acuminata, Musa balbisiana or any Musa hybrid. The term “plants” or “plant parts” include whole banana plants or parts of the banana plants such as roots, pseudo-stem, leaves, plant vasculature, corms, buds, flowers, fruits unless mentioned in contextual manner specific to particular plant part. The term “surroundings” refer to plant rhizosphere including banana plant soil microenvironment or plant phyllosphere including upper and lower sides of leaves, or both, as per the context.

[31] The term “bioformulation” as used herein in the phrase “Trichoderma reesei based bioformulation” refer to a solid form or powder form, wettable bioformulation wherein a naturally existing, non-artificially mutated, non-transgenic and competent Trichoderma reesei strain is present as active constituent, in the form of pure culture, hyphae, conidia, chlamydospores or micro sclerotia along with an agriculturally acceptable carrier such as biochar, a food source such as gum acacia and a complex oligosaccharide complex such as chitosan in any combination and concentration, advantageously capable of controlling or preventing banana fungal diseases and banana thrips and promoting healthy plant growth.

[32] As used herein, the terms “conidia”, “chlamydospores”, “microsclerotia” or “hyphae” or “pure culture” refer to the same meaning as those used in fungal biology, mycology, biology or which can be interpreted by those skilled in the art. A “CFU” is a unit used to estimate the number of viable cells per unit volume or weight. The term “viable structures” as used herein in the bioformulation refer to the cells having ability to divide and multiply. In a preferred embodiment of the present invention, Trichoderma reesei is present in the bioformulation in an amount in the range IxlO⁷ to 1 x IO¹⁰ CFU/g and at field level, Trichoderma reesei is applied in an amount in the range of 7 x IO¹⁰ to 3 x 10¹¹ CFU/Ha by the methods disclosed in the invention for effective disease and pest control and promoting plant growth.

[33] In one aspect, the present invention relates to a bioformulation comprising: i) A fungal culture selected from pure culture of Trichoderma ressei or micro sclerotia, or submerged conidia or chlamydospores or combinations thereof; ii) at least one organic complex oligosaccharide, iii) at least one nutrition source, and iv) at least one acceptable carrier.

[34] In an embodiment, Trichoderma ressei is present in the range of lx 10⁷ to lx IO¹⁰ CFU per unit weight or volume - gm or ml.

[35] In an embodiment, the fungal culture is present in the weight range of l%-4% and the other components are present in the range of 94-99%.

[36] In an embodiment, organic complex oligosaccharide is in the range of 0.15- 0.3%, the nutrient source is in the range of 2-4%, and acceptable carrier is in the range of 93-95%.

[37] In an embodiment, the organic complex oligosaccharide is chitosan; the nutrition source is gum acacia; and the acceptable carrier is selected from biochar, anoxically burnt wood, glycerol or propylene glycol.

[38] In an embodiment, the formulation is applied on the banana plants, plant parts or surroundings.

[39] In an embodiment, the bioformulation controls Fusarium Wilt caused by Fusarium oxysporum f.sp. cubense Race 1, Race 2, Race 4, STR4 or TR4 or Fusarium odoratissimum.

[40] In an embodiment, bioformulation is in a solid form- as wettable powder or dust or granules, or in liquid form - as suspension, solution, dispersion or concentrate; wherein biochar, or anoxically burnt wood is used in wettable powder or dust or granules and glycerol or propylene glycol is used in suspension, solution, dispersion or concentrate. [41] In an embodiment, bioformulation controls Sigatoka disease, infestation caused by banana thrips, other leaf spot diseases, and is effective in promoting banana plant growth, plant height, plant girth and number of leaves, and enhancing yield in terms of fruit bunch weight.

[42] In an embodiment, Sigatoka and other leaf spot disease is caused by any strain of Pseudocercospora fijiensis, P. musae, P. eumusae, Mycospherella fijiensis, M. musicola, M. eumusae or Cladosporium sp. or Alternaria alternata.

[43] In another aspect, the present invention relates to a method of preparing the bioformulation for controlling infestation and promoting banana plant growth, plant height, plant girth and number of leaves, and enhancing yield in terms of fruit bunch weight enhancing growth in the Banana plant, comprising the steps of: i) mixing fungal culture selected from pure culture of Trichoderma ressei or microsclerotia, or submerged conidia or chlamydospores or combinations thereof; at least one organic complex oligosaccharide, at least one nutrition source, and at least one acceptable carrier; ii) soaking the obtained bioformulation in water for 24-36 hours; iii) filtering the wet bioformulation; and iv) applying the bioformulation to the banana plants.

[44] In an embodiment, the fungal culture is mixed in the weight range of l%-4% and the other components are present in the range of 94-99%.

[45] In an embodiment, Trichoderma ressei is present in the range of lx 10⁷ to lx IO¹⁰ CFU per unit weight or volume - gm or ml.

[46] In an embodiment, the organic complex oligosaccharide is mixed in the range of 0.15-0.3%, the nutrient source is mixed in the range of 2-4%, and acceptable carrier mixed in the range of 93-95%.

[47] In an embodiment, the organic complex oligosaccharide is chitosan; the nutrition source is gum acacia; and the acceptable carrier is selected from biochar, anoxically burnt wood, glycerol or propylene glycol.

[48] In an embodiment, bioformulation is applied on the banana plants selected from species Musa acuminata, Musa balbisiana or any Musa hybrid. [49] In an embodiment, applying the bioformulation controls Fusarium Wilt caused by Fusarium oxysporum f.sp. cubense Race 1, Race 2, Race 4, STR4 or TR4 or Fusarium odoratissimum.

[50] In an embodiment, method provides the bioformulation in a solid form- as wettable powder or dust or granules, or in liquid form - as suspension, solution, dispersion or concentrate; wherein biochar, or anoxically burnt wood is used in wettable powder or dust or granules and glycerol or propylene glycol is used in suspension, solution, dispersion or concentrate.

[51] In an embodiment, the method controls Sigatoka disease, other leaf spot diseases, infestation caused by banana thrips.

[52] In an embodiment, the said method controls Sigatoka and other leaf spot disease is caused by any strain of Pseudocercospora fijiensis, P. musae, P eumusae, Mycospherella fijiensis, M. musicola, M. eumusae or Cladosporium sp. or Alternaria alternata.

[53] In another aspect, the present invention relates to use of a bioformulation comprising a fungal culture selected from pure culture of Trichoderma ressei, or microsclerotia or submerged conidia or chlamydospores or combinations thereof; at least one organic complex oligosaccharide, at least one nutrition source, and at least one acceptable carrier for preparation of biopesticides for treatment of banana crop from infestation, controlling promoting banana plant growth, plant height, plant girth and number of leaves, and enhancing yield in terms of fruit bunch weight.

[54] In an embodiment, the fungal culture is present in the weight range of l%-4% and the other components are present in the range of 94-99%.

[55] In an embodiment, the Trichoderma ressei is present in an amount in the range IxlO⁷ to 1 x IO¹⁰ CFU / g and applied to the banana plants in an amount in the range of 7 x IO¹⁰ to 3 x 10¹¹ CFU/Hectare.

[56] In an embodiment, organic complex oligosaccharide is in the range of 0.15- 0.3%, the nutrient source is in the range of 2-4%, and acceptable carrier is in the range of 93-95%.

[57] In an embodiment, the organic complex oligosaccharide is chitosan; the nutrition source is gum acacia; and the acceptable carrier is selected from biochar, anoxically burnt wood, glycerol or propylene glycol. [58] In an embodiment, the said formulation is applied on the banana plant parts or locus thereof and surroundings including plant rhizosphere and phyllosphere.

[59] In an embodiment, the said formulation controls infestation of Fusarium Wilt caused by Fusarium oxysporum f.sp. cubense Race 1, Race 2, Race 4, STR4 or TR4 or Fusarium odoratissimum, and infestation caused by banana thrips.

[60] In an embodiment, the said formulation is in a solid form- as wettable powder or dust or granules, or in liquid form - as suspension, solution, dispersion or concentrate wherein biochar, or anoxically burnt wood is used in wettable powder or dust or granules and glycerol or propylene glycol is used in suspension, solution, dispersion or concentrate.

[61] In an embodiment, said formulation controls of Sigatoka disease, banana thrip infestation and other leaf spot diseases.

[62] In an embodiment, Sigatoka and other leaf spot disease is caused by any strain of Pseudocercospora fijiensis, P. musae, P. eumusae, Mycospherella fijiensis, M. musicola, M. eumusae or Cladosporium sp. or Alternaria alternata.

[63] In one aspect the present invention relates to a bioformulation for use in promoting banana plant growth, plant height, plant girth and number of leaves, and enhancing yield in terms of fruit bunch weight and controlling infection comprising a fungal culture selected from pure culture of Trichoderma ressei, or microsclerotia, or submerged conidia or chlamydospores or combinations thereof; at least one organic complex oligosaccharide, at least one nutrition source, and at least one acceptable carrier.

[64] In an embodiment, said fungal culture is present in the weight range of l%-4% and the other components are present in the range of 94-99%.

[65] In an embodiment, Trichoderma reesei is present in an amount in the range of IxlO⁷ to 1 x IO¹⁰ CFU / g and applied to the banana plants in an amount in the range of 7 x IO¹⁰ to 3 x 10¹¹ CFU/Hectare.

[66] In an embodiment, the organic complex oligosaccharide is in the range of 0.15-0.3%, the nutrient source is in the range of 2-4%, and acceptable carrier is in the range of 93-95%.

[67] In an embodiment, the organic complex oligosaccharide is chitosan; the nutrition source is gum acacia; and the acceptable carrier is selected from biochar, glycerol or propylene glycol. [68] In an embodiment, the said formulation is applied on the banana crops plant parts or locus thereof and surroundings including plant rhizosphere and phyllosphere.

[69] In an embodiment, the said formulation controls infestation of Fusarium Wilt caused by Fusarium oxysporum f.sp. cubense Race 1, Race 2, Race 4, STR4 or TR4 or Fusarium odoratissimum and infestation caused by banana thrips.

[70] In an embodiment, the bioformulation is in a solid form- as wettable powder or dust or granules, or in liquid form - as suspension, solution, dispersion or concentrate wherein a. biochar, or anoxically burnt wood is used in wettable powder or dust or granules and glycerol or propylene glycol is used in suspension, solution, dispersion or concentrate.

[71] In an embodiment, the said bioformulation controls of Sigatoka disease, banana thrip infestation, other leaf spot diseases.

[72] In an embodiment, Sigatoka and other leaf spot disease is caused by any strain of Pseudocercospora fijiensis, P. musae, P. eumusae, Mycospherella fijiensis, M. musicola, M. eumusae or Cladosporium sp. or Alternaria

[73] In another aspect, the present invention relates to a method of treating the banana crop from infestation and enhancing its growth comprising the steps of: a. providing a bioformulation comprising a fungal culture selected from pure culture of Trichoderma ressei, or microsclerotia, or submerged conidia or chlamydospores or combinations thereof; at least one organic complex oligosaccharide, at least one nutrition source, and at least one an acceptable carrier in a wettable powder or a liquid form; b. applying bioformulation to the banana plant, plant parts or surroundings via soil drenching, via foliar spray, via drone spray, or a combination thereof at defined intervals, frequencies and in concentrations at specified growth stages; via injection to pseudo-stem, or banana mat, via injection to emerging floral buds or a combination thereof at defined intervals, frequencies and in concentrations and at specified growth stages with a specified angle and process.

[74] In an embodiment, said bioformulation is a wettable powder first soaked and mixed with clean water at the rate of 6-9 g per 10-250 ml water, incubated for 24-36 hours, and filtered and wherein for soil drenching and foliar spray, the water used is in the range of 200-250 ml, for pseudo stem injection the water used is in the range of 10-15 ml, and for floral bud injection the water used is in the range of 80-120 ml for application per plant.

[75] In an embodiment, the said wettable powder mixture or in liquid form is having active microorganism Trichoderma reesei present in an amount in the range IxlO⁷ to 1 x 10¹⁰ CFU / g and applied to the banana plants in an amount in the range of 7 x 10¹⁰ to 3 x 10¹¹ CFU/Hectare.

[76] In an embodiment, the bioformulation is applied either once, twice, thrice or four times, during the crop cycle, with a minimum interval period of 35- 45 days between any two applications, depending upon disease severity.

[77] In an embodiment, the bioformulation application promotes plant growth in terms of plant height, plant girth and number of leaves, and enhances yield in terms of fruit bunch weight.

[78] In an embodiment, the bioformulation is applied to the banana plant, banana rhizosphere, banana pseudo-stem vascular tissues, banana leaves or the locus of the plant.

[79] In an embodiment, banana plant is selected from species Musa acuminata, Musa balbisiana or any Musa hybrid.

[80] In an embodiment, injection is provided by an injection needle penetrating the pseudo-stem at an angle of 40-50 degrees, most preferably at an angle of 45 degrees with reference to plant vertical and horizontal planes, to attain maximum retention and spread of injected liquid to plant vasculature and internal tissues.

[81] In an embodiment, the formulation is effective in treating the banana crops from infestation caused by Fusarium oxysporum f.sp. cubense Race 1, Race 2, Race 4, STR4 or TR4 or Fusarium odoratissimum.

[82] In an embodiment, the formulation is effective in controlling Sigatoka disease, other leaf spot diseases.

[83] In an embodiment, Sigatoka and leaf spot disease is caused by any strain of Pseudocercospora fijiensis, P. musae, P. eumusae, Mycospherella fijiensis, M. musicola, M. eumusae or Cladosporium sp. or Alternaria alternata. [84] In an embodiment, the formulation is treating the banana crops from infestation caused by banana thrips by applying the formulation to the banana plant, emerging floral bud or parts thereof.

[85] In another aspect, the present invention relates to a method of application to the banana crop from infestation and enhancing its growth by applying bioformulation to the banana plant, plant parts or surroundings via soil drenching, via foliar spray, via drone spray, or a combination thereof at defined intervals, frequencies and in concentrations at specified growth stages; via injection to pseudo-stem, or banana mat, via injection to emerging floral buds or a combination thereof at defined intervals, frequencies and in concentrations and at specified growth stages with a specified angle and process.

[86] In an embodiment, the effective amount of formulation is 6-9 g per 10-250 ml water, incubated for 24-36 hours, and filtered and wherein for soil drenching and foliar spray, the water used is in the range of 200-250 ml water, for pseudo stem injection the water used is in the range 10-15 ml, and for floral bud injection the water used is in the range 80-120 ml for application per plant.

[87] In an embodiment, the bioformulation is applied either once, twice, thrice or four times, during the crop cycle, with a minimum interval period of 35- 45 days between any two applications, depending upon disease severity.

[88] In an embodiment, injection is provided by an injection needle penetrating the pseudo-stem at an angle of 40-50 degrees, most preferably at an angle of 45 degrees with reference to plant vertical and horizontal planes, to attain maximum retention and spread of injected liquid to plant vasculature and internal tissues.

[89] In an embodiment, methods of applying the bioformulation to banana plants, parts thereof, banana phyllosphere or rhizosphere prevents or control of banana fungal diseases and thrips as well as promote healthy plant growth. The bioformulations and methods of application described herein protect the plants from pathogenic fungal growth and /or infection, thus is used to treat banana plants infected with pathogens such as those associated with Fusarium wilt, Sigatoka disease complex, banana leaf spots, streaks and speckles.

[90] In an embodiment, the bioformulation is applied to banana plants, parts thereof or surroundings via soil drenching or via injection to emerging floral buds of the mature plants either prophylactically to prevent any incoming thrips infestation or to thrips infected plants so as to control and combat the disease spread and subsequent spoilage, achieving effective relief to banana fruits from the thrips, attaining thrips- damage free banana fingers, fruits and bunches.

[91] In an embodiment of the invention, bioformulation increases plant height by 30-45%, plant girth by 30-45% and plant leaves number by 15-25% in treated plants of mother plantation in comparison to untreated controls. The bioformulation of present invention preferably increases plant height by 20-25%, plant girth by 25-30% and plant leaves number by 20-25% in treated plants of follower plants in comparison to untreated controls. The bioformulation treated plants have been observed to have bunch weight increased by 10-20%.

[92] Trichoderma reesei has been used for the production of glucoamylase (WO 2006/060062), xylanase (US 6555335), cellulase (EP 91908986.2). A naturally existing strain of Transgenic reesei isolated from India was described for its efficacy in control of Foc-TR4 by Damodaran et al., 2020. The present invention discloses use of Trichoderma reesei based bioformulation in the effective biological management of not only Fusarium wilt of banana caused by all known races, but also its use in the control of Sigatoka disease complex in banana, other banana leaf spots and streaks and in controlling banana flower, silvering and rust thrips and in promoting plant growth. Trichoderma reesei can be detected by PCR amplification of small subunit ribosomal RNA gene, Internal transcribed spacer (ITS1), 5.8S ribosomal RNA gene and ITS2 as described in characteristic sequence of NCBI accession nos MK050013.1 or OP363964.1 to the identity extent of 95-100%. In one aspect of the invention, INNO- 121 is the Trichoderma reesei strain used herein, having the characteristic sequence in ITS region as provided below. However, the invention includes all Trichoderma reesei strains whose DNA sequence matches with INNO-121 in the range of 95-100% in ITS region and used for bioformulation application in the disease control and healthy plant growth in banana as provided in this invention.

[93] Trichoderma reesei strain INNO-121 ITS sequence:

[94] The novel bioformulation and methods of applications to present it to banana plants are also used to protect banana healthy plants from getting infected and to treat banana plants infected with pathogens that cause foliar diseases, such as Sigatoka disease compXex-Mycosphaerella fijiensis anamorph Pseudocercospora fijiensis causing Black Sigatoka or Black Leaf Streak disease in banana or Mycosphaerella musicola anamorph Pseudocercospora musae causing Yellow Sigatoka or Mycosphaerella eumusae anamorph Pseudocercospora eumusae causing eumusae leaf spots in banana plants, or other leaf spots, speckles and streaks caused by

[95] As different pathogenic fungal species causing any Sigatoka and other leaf spot disease cause foliar disease in banana, in yet another aspect of the invention, Trichoderma reesei based bioformulation is applied via foliar spray to banana plants either once, twice, thrice or four times, during the crop cycle, with a minimum interval period of 35- 45 days between any two applications, depending upon disease severity. In most preferred embodiment, the Trichoderma reesei based bioformulation wettable powder may be soaked and mixed with clean water at the rate of 6-9 g per 200-250 ml water, incubated for 24-36 hours, filtered, and filtered liquid bioformulation preparation containing active constituent of viable Trichoderma reesei cells and other dissolved components is applied to either side of banana leaves via foliar spray from ground or drone spray at the rate of 200-250 ml per plant. At farm level, 6-9 Kg of Trichoderma reesei based bioformulation may be sufficient for one acre banana farm having 800-1200 plants, achieving 6-9 x 10¹⁰ CPU per application to control Sigatoka, other leaf spots and other fungal foliar diseases in banana. [96] The competency of Trichoderma in controlling plant diseases, promoting plant growth, and augmenting the yield is known to be fungal isolate and plant species specific. In addition to promoting stress tolerance, competent Trichoderma also increase nutrient acquisition and growth rates of host plants and enhance water use efficiency. The antagonistic bioformulation of present invention has been found to have such competence by colonizing soil, plant rhizosphere, parts of the plant and plant tissues or plant phyllosphere in banana and facilitate and promote nutrient uptake by plants as well as depriving pathogens of nutrients and space. The stimulatory effect of Trichoderma reesei on plants is related to their participation in the crosstalk between the growth hormones synthesized by these fungi and the defense hormones, plant enzymes and antioxidants induced by them in the plant, thereby bringing better abiotic and biotic stress tolerance. The multilevel communication with roots and shoots induced by Trichoderma reesei of this invention increase the branching capacity of the roots, enhance the nutrient uptake efficiency, and thereby augment the productivity of the plant in terms of greater plant biomass and yield.

[97] The bioformulation and methods can be advantageously applied on banana plants or their floral parts to effectively protect banana bunches and other associated structures from banana rust, silvering or flower thrips. It has also been shown by present invention that such T. reesei containing bioformulation application are capable of promoting banana plant growth, particularly in conditions not ideal for growth, such as under abiotic or biotic stress. The invention disclosed will be described with respect to particular embodiments, but the invention is not limited to them, but only by claims. Any reference signs in the claims should not be deemed as limiting the scope thereof.

Technical advantages of the present invention

The bioformulation of the present invention protects/prevents the plants from pathogenic fungal infection, associated with Fusarium wilt, Sigatoka disease complex, banana leaf spots, streaks and speckles. • The bioformulation present invention treats the plant from pathogenic fungal growth associated with Fusarium wilt, Sigatoka disease complex, banana leaf spots, streaks and speckles.

• The bioformulation and methods can be advantageously applied on banana plants or their floral parts to effectively protect banana bunches and other associated structures from banana rust, silvering or flower thrips.

• The bioformulation of the present invention promotes healthy plant growth. It has also been shown by present invention that T. reesei containing bioformulation are capable of promoting banana plant growth, particularly in conditions not ideal for growth, such as under abiotic or biotic stress.

• The bioformulation and the method of the present invention has multiple roles: Apart from enhanced protection against soil borne and foliar fungal diseases and banana thrips, it aids in enhanced nutrient uptake, improves soil structure and fertility and improves healthy plant growth and quality yield.

• The bioformulation and the method of the present invention minimizes the dependence on chemical pesticide for disease control and thus results in farmers savings.

• The bioformulation and the method of the present invention promotes organic farming.

• The bioformulation and the method of the present invention promotes sustainable way to improve soil fertility and crop yield.

• The bioformulation and the method of the present invention increases microbial diversity, leading to a more resilient and productive soil ecosystem.

• The bioformulation and the method of the present invention is highly economical to use, cost of application being INR 10 per plant. In conventional method of controlling pathogens in Banana plant by using chemical control the cost of application per plant is INR 184 per plant, whereas when other commercially used biocontrol method is used it costs INR 61 per plant. Thus, the present bioformulation and the method is more economical compared to the existing methods.

• The bioformulation and the method can be easily adapted by banana growing farmers as it takes care of most of the problems, and it is compatible with current agriculture practices in banana. [98] Overall, the formulation of the present invention provides a sustainable approach to enhance plant growth, protect against diseases, and improve soil health, contributing to more productive and environmentally friendly agricultural practices.

[99] The invention will now be described in the following non-limiting examples in connection with certain preferred embodiments. These examples are intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as specified by the appended claims. These examples will serve to illustrate the practice of this invention, it being understood that the descriptions and specifications shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention only or believed to be the most useful and readily understood description of procedures as well as the principles and conceptual aspects of the invention and not to limit the invention to these particular embodiments.

Working examples of the present invention:

[100] EXAMPLE 1: Field Trials on mature banana plants showing Trichoderma reesei based bioformulations efficacy in controlling Fusarium wilt.

Filed trials were carried out utilizing methods described in present invention to assess the efficacy of Trichoderma reesei based bioformulation in the control of Fusarium wilt in banana plantations disease hotspots.

The bioformulation comprises of Trichoderma reesei viable structures to amount of 1% to 4% (w/v), most preferably 3% (w/v) along with other components consisting of gum acacia, chitosan and propylene glycol or glycerol dissolved in aqueous base in the form of suspension concentrate. In the present embodiment Trichoderma reesei 3% , Gum Acacia 3%, Chitosan 0.25% and biochar 93.75% (in solid form) or diluted Glycerol 93.75% ((in liquid form), having lx 10⁸ CFU per unit weight or volume of bioformulation.

Trial 1 was conducted in India at Kamrej, Surat (Gujarat), India, between Mar’ 2022 to Dec’ 2022 over a population of 6380 first generation follower plants, spread over 6 Acres and three farmers plots. At the time of installing the first application, the mother plants in this plantation had 9.87% of Fusarium wilt disease incidence, whereas followers were not showing any symptoms. The follower plants were selected for further growth and mother plants were discarded post-harvest. A total of 4320 follower plants in random blocks were treated with Trichoderma reesei based bioformulation via soil drenching applications in March-, May-, June- and Aug- 2022 at 45 days interval, the remaining 2060 plants were kept as control, non-treated, without any application.

Trial 2 was conducted in Bantacan, New Bataan (Davao del Norte), Philippines, between July’2022 to Jan’2023 over a population of 6000 first generation follower plants, spread over 6.5 acres. At the time of installing the first application, the mother plants in this plantation had 26.08% of Fusarium wilt disease incidence, whereas followers were not showing any symptoms. The follower plants were selected for further growth and mother plants were discarded post-harvest. A total of 4000 follower plants in random blocks were treated with Trichoderma reesei based bioformulation via soil drenching applications in July-, Sep-, Oct- and Dec- 2022 at 45 days interval, the remaining 2000 plants were kept as control, non-treated, without any application.

In both the trial setups in different agro-climatic zones, in each application, the Trichoderma reesei based bioformulation wettable powder was soaked and mixed with clean water at the rate of 7.5 Kg per 250 L water, incubated for 24-36 hours, filtered, and filtered liquid bioformulation preparation containing active constituent of viable Trichoderma reesei cells, Gum Acacia, Chitosan and carrier (biochar) is applied to the base of the banana plants via drenching either manually or via Knapsack sprayer at the rate of 200-250 ml per plant. With this rate, each application presented 7 x 10¹⁰ CFU or active microbial structures of T. reesei to banana plantation per acre with 1000-1200 plants. Observations were recorded in terms of disease incidence score in these plants based on morphological symptoms of Fusarium Wilt disease yellowing of older leaves from margins, hanging of affected leaves in skirt like appearance and splitting of pseudo-stem at the base, 15 days after each application. Representative diseased plant samples were also tested at molecular level for the presence of FW-TR4 strain via PCR. Final observations were taken before harvesting and results were summarized.

Table 1: Results of Trichoderma reesei based bioformulation field trial with banana follower population at two locations for efficacy against Fusarium Wilt (Foc-TR4).

FW - % DI = Fusarium Wilt Percent Disease Incidence.

From the results presented in Table 1, it is concluded that Trichoderma reesei based bioformulation applied via soil drenching provided good and efficient control of Fusarium Wilt (caused by oc-TR4) in the range of 71 to 92% in banana plants grown in two diverse agroclimatic regions.

[74] EXAMPLE 2: Field Trials on mature banana plants showing Trichoderma reesei based bioformulations efficacy in controlling Fusarium wilt via Pseudostem injection.

Filed trials were carried out utilizing methods described in present invention to assess the efficacy of Trichoderma reesei based bioformulation in the control of Fusarium wilt in banana plantations disease hotspots via pseudo-stem injection and to check the efficacy of Trichoderma reesei based bioformulations of present invention in recovery and revival of death-prone plants or plants heavily infested with Fusarium wilt.

This trial was conducted in San Roque, New Bataan (Davao de Oro), Philippines, between Apr ’2024 to June’2024 over a population of 100 first generation follower plants, spread over 2 acres. At the time of installing the first application, the plants selected in this plantation were showing early symptoms of Fusarium wilt. A total of 60 follower plants in random blocks were treated with Trichoderma reesei based bioformulation via pseudo-stem injection applications in Apr and May- 2024 at 20 days interval, the remaining 40 plants were kept as control, non-treated, without any application.

In each application, the Trichoderma reesei based bioformulation wettable powder was soaked and mixed with clean water at the rate of 6-9 g per 10-15 ml water, incubated for 24-36 hours, filtered, and filtered liquid bioformulation preparation containing active constituent of viable Trichoderma reesei cells and other dissolved components is applied to the pseudo-stem of banana plants via injection at the rate of 10-15 ml per plant, most preferably 9 g of bioformulation per 10 ml water. The injection was done either with market available 20 ml volume capacity disposable syringes or with animal injectors having good capacity, tough needles and adjustable volume ejector. While doing injection application to banana plant pseudo-stem, injection needle was penetrated to pseudo-stem at an angle of 40-50 degrees, most preferably at an angle of 45 degrees with reference to plant vertical and horizontal planes, so as to attain maximum retention and spread of injected liquid to plant vasculature and internal tissues. With this rate, each application presented 3 x 10⁸ CFU or active microbial structures of T. reesei to each banana plant treated banana plantation per acre with 1000-1200 plants. Observations were recorded in terms of disease incidence score in these plants based on morphological symptoms of Fusarium wilt disease-yellowing of older leaves from margins, hanging of affected leaves in skirt like appearance and splitting of pseudo-stem at the base, and signs of recovery in terms of healthy green leaves appearance and decrease of Fusarium wilt symptoms, 20 days after each application. Table 2: Results of Trichoderma reesei based bioformulation field application via Pseudo-stem Injection to Fusarium Wilt ( oe -TR4) disease-infested banana follower population for efficacy against disease and recovery of plants.

From the results presented in Table 2, it is concluded that Trichoderma reesei based bioformulation applied via injection to pseudo-stem was highly effective in controlling Fusarium wilt in highly infested, death-prone banana plants and their revival, as 43.33% of the treated plants showed full recovery in terms of appearance of healthy green leaves and subsequent plant growth, whereas 28.33% of treated plants still showed symptoms. In comparison, in control, non-treated block, no plant showed signs of recovery or disease resistance, 32.5% plants showed disease symptoms and 67.5% plants died due to disease progression and severity. EXAMPLE 3: Field Trials on young and mature banana plants showing Trichoderma reesei based bioformulations efficacy in controlling Sigatoka disease.

Filed trials were carried out utilizing methods described in present invention to assess the efficacy of Trichoderma reesei based bioformulation in the control of Sigatoka and associated leaf spot or leaf streak disease in banana plantations disease hotspots.

Trial 1 was conducted in India at Anantapur (AP), India, between Aug’ 2022 to Apr’ 2023 over a population of 5115 tissue culture raised young plants, newly planted in field spread over 3 Acres and two farmers plots. A total of 3465 plants in random blocks were treated with Trichoderma reesei based bioformulation first via soil drenching application and then via three foliar sprays in Aug-, Oct-, Dec- 2022 and Feb- 2023 at 45 days interval, the remaining 1650 plants were kept as control, nontreated, without any application. At the time of installing the first application, the young plants in this plantation had no incidence of Sigatoka disease and plants were not showing any symptoms.

Trial 2 was conducted in San Roque, New Bataan (Davao de Oro), Philippines, between Sep’2022 to May’2023 over a population of 2094 first generation follower plants, spread over 2 acres. At the time of installing the first application, the mature plants in this plantation had 50-52% of Sigatoka disease incidence. A total of 1402 follower plants in random blocks were treated with Trichoderma reesei based bioformulation first via soil drenching application and then via three foliar sprays in Sep-, Nov-2022 and Jan- and Mar- 2023 at 45 days interval, the remaining 692 plants were kept as control, non-treated, without any application.

In both the trial setups in different agro-climatic zones, in each application, the Trichoderma reesei based bioformulation wettable powder was soaked and mixed with clean water at the rate of 7.5 Kg per 250 L water, incubated for 24-36 hours, filtered, and filtered liquid bioformulation preparation containing active constituent of viable Trichoderma reesei cells and other dissolved components is applied to the base of the banana plants via drenching either manually or via Knapsack sprayer at the rate of 200-250 ml per plant or in case of foliar sprays from ground or via drone, on both sides of leaves and plant surface at the rate of 200-250 ml per plant. With this rate, each application presented 7 x 10¹⁰ CFU or active microbial structures of T. reesei to banana plantation per acre with 1000-1200 plants. Observations were recorded in terms of disease incidence in these plants based on morphological symptoms of Sigatoka disease (Stage 1 to Stage 6), 15 days after each application. The plants in any stage from stage 1 (<1% leaf area affected; streaks and until 10 spots, classified as slight symptoms) to stage 6 (>51% of the leaf area necrotic, classified as severe) were scored as infection positive. Final observations were taken before harvesting and results were summarized.

Table 3: Results of Trichoderma reesei based bioformulation field trial with banana young and mature populations at two locations for efficacy against Sigatoka leaf streak and spot disease.

From the results presented in Table 3, it is concluded that Trichoderma reesei based bioformulation applied via one soil drenching and three foliar sprays provided good and efficient control of Sigatoka leaf spot/ streak disease in the range of 36 to 41% in banana plants grown in two diverse agroclimatic regions, and disease incidence is taken as parameter to validate the efficacy of T. reesei based bioformulation of the present invention. A small group of Sigatoka infected plants spread over T. reesei based bioformulation of this invention- treated and control- untreated blocks at these two trial locations were also observed for Sigatoka disease and severity progression (through stage 1 to stage 6) increase or reduction. It was found that in India location, 13.5 % of the treated population of Sigatoka infected plants progressed through severity stages until Stage 5, whereas 92.32% of control, untreated Sigatoka infected plants progressed through advanced stages and reached to highest severity level of Stage 6. In Philippines trial location, 11.64% of the treated population of Sigatoka infected plants progressed through severity stage upto stage 5, whereas 95.05% of control, untreated Sigatoka infected plants progressed through advanced stages and reached to highest severity level of Stage 6. This observation led to the conclusion that Trichoderma reesei based bioformulation of the present invention via its soil drenching and foliar applications was able to control the Sigatoka disease progression and severity by 85- 88% in these two diverse agroclimatic zones showing its efficacy in controlling, combating, or preventing Sigatoka leaf spot and leaf streak disease in banana.

[101] EXAMPLE 4: Field Trials on mature banana plants showing Trichoderma reesei based bioformulations efficacy in controlling banana thrips.

Filed trials were carried out utilizing methods described in present invention to assess the efficacy of Trichoderma reesei based bioformulation in the control of banana thrips in banana plantations disease hotspots.

Trial 1 was conducted in India at Kamrej, Surat (Gujarat), India, between Dec’2022 to Jan’ 2023 over a population of 50 first generation follower plants having emerging floral buds of similar sizes, preferably 15-25 cm in length and with 25-50 % emergence, most preferably 30% emergence, randomly selected in a banana farm spread over 2 Acres. A total of 30 floral buds were treated with Trichoderma reesei based bioformulation via injection application once in Dec- 2022, the remaining 20 floral buds were kept as control, non-treated, without any application.

Trial 2 was conducted in San Roque, New Bataan (Davao de Oro), Philippines, between Apr’2024 to May’2024 over a population of 50 first generation follower plants having emerging floral buds of similar sizes, preferably 15-25 cm in length and with 25-50 % emergence, most preferably 30% emergence, randomly selected in a banana farm spread over 1.8 Acres. A total of 30 floral buds were treated with Trichoderma reesei based bioformulation via injection application once in Apr- 2024, the remaining 20 floral buds were kept as control, non-treated, without any application.

In both the trial setups in different agro-climatic zones, in floral bud injection application, Trichoderma reesei based bioformulation wettable powder was soaked and mixed with clean water at the rate of 6-9 g per 80-120 ml water, incubated for 24- 36 hours, filtered and filtered liquid bioformulation preparation containing active constituent of viable Trichoderma reesei cells and other dissolved components is applied to emerging floral buds via injection at the rate of 80-120 ml per plant, most preferably 100 ml. At this rate, the bud injection application presented 7 x 10⁷ CFU or active microbial structures of T. reesei to each emerging floral buds in the treated category. Observations were recorded in terms of banana thrips incidence score in the banana plants having treated T. reesei based bioformulation- treated floral buds and banana plants with untreated, control floral buds at the base and tips of floral buds as well as on the bunches after buds opening, after 20 days, 40 days and 60 days of the bud injection application, as shown in Table 4.

Table 4: Results of Trichoderma reesei based bioformulation field trial with banana mature plants’ emerging floral buds at two locations for efficacy against banana thrips.

Control

(Non-treated)

The observations and data presented in Table 4 showed that Trichoderma reesei based bioformulation applied via banana tree injector tool to emerging floral buds as injection provided good and efficient control of banana thrips in the range of 33 to 41% in banana plants grown in two diverse agroclimatic regions.

[102] EXAMPLE 5: Field Trials on young banana plants (new plantation) showing Trichoderma reesei based bioformulations efficacy in promoting plant growth.

Filed trials were carried out utilizing methods described in present invention to assess the efficacy of Trichoderma reesei based bioformulation in promoting plant growth in tissue culture raised plants field population (new plantation). Three parameters were taken to demonstrate this: plant height, plant girth and number of green healthy leaves.

Trial 1 was conducted in India at Anantapur (AP), India, between Aug’ 2022 to Apr’ 2023 over a population of 5115 tissue culture raised young plants, newly planted in field spread over 3 Acres and two farmers plots. A total of 3465 plants in random blocks were treated with Trichoderma reesei based bioformulation via soil drenching applications in Aug-, Oct-, Dec- 2022 and Feb- 2023 at 45 days interval, the remaining 1650 plants were kept as control, non-treated, without any application.

Trial 2 was conducted in Compostela valley (Davao del Norte), Philippines, between Sep’2022 to May’2023 over a population of 2000 tissue culture raised young plants, newly planted in field, spread over 2 acres. A total of 1000 follower plants in random blocks were treated with Trichoderma reesei based bioformulation via soil drenching applications in Sep-, Nov-2022 and Jan- and Mar- 2023 at 45 days interval, the remaining 1000 plants were kept as control, non-treated, without any application.

In both the trial setups in different agro-climatic zones, in each application, the Trichoderma reesei based bioformulation wettable powder was soaked and mixed with clean water at the rate of 7.5 Kg per 250 L water, incubated for 24-36 hours, filtered, and filtered liquid bioformulation preparation containing active constituent of viable Trichoderma reesei cells and other dissolved components is applied to the base of the banana plants via drenching either manually or via Knapsack sprayer at the rate of 200-250 ml per plant. With this rate, each application presented 7 x 10¹⁰ CFU or active microbial structures of T. reesei to banana plantation per acre with 1000-1200 plants. At the time of installing the trial, selected parameters of plant growth viz. plant height (in cm), plant girth (in cm) and number of healthy green plant leaves were counted plant-wise and recorded. Further observations for these plant growth parameters were recorded after 2^nd, 3^rd and 4^th application in both treated and control plots in both trial locations. After 3^rd observation, results were summarized.

Table 5: Results of Trichoderma reesei based bioformulation field trial with banana new plantation at two locations for efficacy in promoting plant growth.

From the results presented in Table 5, it is concluded that Trichoderma reesei based bioformulation applied via soil drenching provided efficient plant growth and it can act as effective plant growth promotant for newly planted, banana young plants. With reference to selected parameters, it was found that treated plants showed 28.39 - 42.07% better plant height; 26.5 - 43.05% better plant girth and 12.82 - 20.89% more healthy green leaves in the two geographically different trial locations in comparison to non-treated, control banana plants.

[103] EXAMPLE 6: Field Trials on mature banana plants showing Trichoderma reesei based bioformulations efficacy in promoting plant growth.

Filed trials were carried out utilizing methods described in present invention to assess the efficacy of Trichoderma reesei based bioformulation in promoting plant growth in first generation follower plants. Three parameters were taken to demonstrate this: plant height, plant girth and number of green healthy leaves.

Trial 1 was conducted in India at Kamrej, Surat (Gujarat), India, between Mar’ 2022 to Dec’ 2022 over a population of 6380 first generation follower plants, spread over 6 Acres and three farmers plots. A total of 4320 follower plants in random blocks were treated with Trichoderma reesei based bioformulation via soil drenching applications in March-, May-, June- and Aug- 2022 at 45 days interval, the remaining 2060 plants were kept as control, non-treated, without any application.

Trial 2 was conducted in Bantacan, New Bataan (Davao del Norte), Philippines, between July’2022 to Jan’2023 over a population of 6000 first generation follower plants, spread over 6.5 acres. A total of 4000 follower plants in random blocks were treated with Trichoderma reesei based bioformulation via soil drenching applications in July-, Sep-, Oct- and Dec- 2022 at 45 days interval, the remaining 2000 plants were kept as control, non-treated, without any application.

In both the trial setups in different agro-climatic zones, in each application, the Trichoderma reesei based bioformulation wettable powder was soaked and mixed with clean water at the rate of 7.5 Kg per 250 L water, incubated for 24-36 hours, filtered, and filtered liquid bioformulation preparation containing active constituent of viable Trichoderma reesei cells and other dissolved components is applied to the base of the banana plants via drenching either manually or via Knapsack sprayer at the rate of 200-250 ml per plant. With this rate, each application presented 7 x 10¹⁰ CFU or active microbial structures of T. reesei to banana plantation per acre with 1000-1200 plants. At the time of installing the trial, selected parameters of plant growth viz. plant height (in cm), plant girth (in cm) and number of healthy green plant leaves were counted plant wise and recorded. Further observations for these plant growth parameters were recorded after 2^nd, 3^rd and 4^th application in both treated and control plots in both trial locations. After 4^th observation, results were summarized.

Table 6: Results of Trichoderma reesei based bioformulation field trial with banana follower population at two locations for efficacy in promoting plant growth.

From the results presented in Table 6, it is concluded that Trichoderma reesei based bioformulation applied via soil drenching provided efficient plant growth and it can act as effective plant growth promotant for banana follower plants as well. With reference to selected parameters, it was found that treated plants showed 22.78- 24.26% better plant height; 24.47-26.05% better plant girth and 21.67-25.07% more healthy green leaves in the two geographically different trial locations in comparison to non-treated, control banana plants.

[104] EXAMPLE 7: Field Trials on mature banana plants showing Trichoderma reesei based bioformulations efficacy in enhancing banana yield.

Filed trials were carried out utilizing methods described in present invention to assess the efficacy of Trichoderma reesei based bioformulation of present invention in enhancing plant yield. Average bunch weight of a field trial population of Trichoderma reesei based bioformulation- treated and control, untreated groups were observed just after harvesting at packhouses to know its effect in promoting yield in terms of healthy banana fingers and their weight.

Trial conducted in India at Kamrej, Surat (Gujarat), India, between Mar ’2022 to Dec’ 2022 over a population of 6380 first generation follower plants, spread over 6 Acres and three farmers plots was used to assess efficacy of Trichoderma reesei based bioformulation in enhancing plant yield at the time of harvest in Feb-2023. A total of 4320 follower plants in random blocks were treated with Trichoderma reesei based bioformulation via soil drenching applications in March-, May-, June- and Aug- 2022 at 45 days interval, the remaining 2060 plants were kept as control, non-treated, without any application, as provided in EXAMPLE 6.

A total of 990 bunches from treated block and 910 bunches from control block of this population was observed for their bunch weight as shown in Table 7.

Table 7: Results of Trichoderma reesei based bioformulation field trial with banana follower population for efficacy in promoting plant yield.

From the results presented in Table 7, it is concluded that Trichoderma reesei based bioformulation applied via soil drenching provided an effective yield promotant for banana plants as the treated plants showed 13.85% better plant yield in terms of average bunch weight in comparison to non-treated, control banana plants.

Claims

We Claim:

1. A bioformulation comprising: i. a fungal culture selected from pure culture of Trichoderma ressei or microsclerotia, or submerged conidia or chlamydospores or combinations thereof; ii. at least one organic complex oligosaccharide, iii. at least one nutrition source, and iv. at least one acceptable carrier.

2. The bioformulation as claimed in claim 1, wherein the fungal culture is present in the weight range of l%-4% and the other components are present in the range of 94-99%.

3. The bioformulation as claimed in claim 1, wherein the Trichoderma ressei is present in the range of lx 10⁷ to lx 10¹⁰CFU per unit weight or volume- gm or ml.

4. The bioformulation as claimed in claim 1, wherein organic complex oligosaccharide is in the range of 0.15-0.3%, the nutrient source is in the range of 2-4%, and acceptable carrier is in the range of 93-95%.

5. The bioformulation as claimed in claim 1, wherein the organic complex oligosaccharide is chitosan; the nutrition source is gum acacia; and the acceptable carrier is selected from biochar, anoxically burnt wood, glycerol or propylene glycol.

6. The bioformulation as claimed in claim 1, wherein the said formulation is applied on the banana plants, plant parts or surroundings.

7. The bioformulation as claimed in claim 1, wherein the said bioformulation controls Fusarium Wilt caused by Fusarium oxysporum f.sp. cubense Race 1, Race 2, Race 4, STR4 or TR4 or Fusarium odoratissimum.

8. The bioformulation as claimed in claim 1, wherein the said bioformulation is in a solid form- as wettable powder or dust or granules, or in liquid form - as suspension, solution, dispersion or concentrate; wherein biochar, or anoxically burnt wood is used in wettable powder or dust or granules and glycerol or propylene glycol is used in suspension, solution, dispersion or concentrate.

9. The bioformulation as claimed in claim 1, wherein the said bioformulation controls Sigatoka disease, infestation caused by banana thrips, other leaf spot diseases, and is effective in promoting banana plant growth in terms of plant height, plant girth and number of leaves, and enhancing yield in terms of fruit bunch weight.

10. The bioformulation as claimed in claim 9, wherein Sigatoka and other leaf spot disease is caused by any strain of Pseudocercospora fijiensis, P. musae, P eumusae, Mycospherella fijiensis, M. musicola, M. eumusae or Cladosporium sp. or Alternaria alternata.

11. A method of preparing a bioformulation for controlling infestation and promoting banana plant growth in terms of plant height, plant girth and number of leaves, and enhancing yield in terms of fruit bunch weight in banana plant, comprising the steps of: a. mixing fungal culture selected from pure culture of Trichoderma ressei or microsclerotia, or submerged conidia or chlamydospores or combinations thereof; at least one organic complex oligosaccharide, at least one nutrition source, and at least one acceptable carrier; b. soaking the obtained bioformulation in water for 24-36 hours; c. filtering the wet bioformulation; and d. applying the bioformulation to the banana plants.

12. The method as claimed in claim 11, wherein the fungal culture is mixed in the weight range of l%-4% and the other components are present in the range of 94-99%.

13. The method as claimed in claim 11, wherein Trichoderma ressei is present in the range of lx 10⁷ to lx IO¹⁰ CFU per unit weight or volume- gm or ml.

14. The method as claimed in claim 11, wherein the organic complex oligosaccharide is mixed in the range of 0.15-0.3%, the nutrient source is mixed in the range of 2-4%, and acceptable carrier mixed in the range of 93-95%.

15. The method as claimed in claim 11, wherein the organic complex oligosaccharide is chitosan; the nutrition source is gum acacia; and the acceptable carrier is selected from biochar, anoxically burnt wood, glycerol or propylene glycol.

16. The method as claimed in claim 11, wherein the said bioformulation is applied on the banana plants selected from species Musa acuminata, Musa balbisiana or any Musa hybrid.

17. The method as claimed in claim 11, wherein applying the bioformulation controls Fusarium Wilt caused by Fusarium oxysporum f.sp. cubense Race 1, Race 2, Race 4, STR4 or TR4 or Fusarium odoratissimum.

18. The method as claimed in claim 11, wherein the said method provides the bioformulation in a solid form- as wettable powder or dust or granules, or in liquid form - as suspension, solution, dispersion or concentrate; wherein biochar, or anoxically burnt wood is used in wettable powder or dust or granules and glycerol or propylene glycol is used in suspension, solution, dispersion or concentrate.

19. The method as claimed in claim 11, wherein the said method controls Sigatoka disease, other leaf spot diseases, infestation caused by banana thrips.

20. The method as claimed in claim 19, wherein the said method controls Sigatoka and other leaf spot disease is caused by any strain of Pseudocercospora fijiensis, P. musae, P. eumusae, My cospherella fijiensis, M. musicola, M. eumusae or Cladosporium sp. or Alternaria alternata.

21. A bioformulation for use in promoting banana plant growth in terms of plant height, plant girth and number of leaves, and enhancing yield in terms of fruit bunch weight and controlling infection comprising a fungal culture selected from pure culture of Trichoderma ressei, or micro sclerotia, or submerged conidia or chlamydospores or combinations thereof; at least one organic complex oligosaccharide, at least one nutrition source, and at least one an acceptable carrier.

22. The bioformulation as claimed in claim 21, wherein the said fungal culture is present in the weight range of l%-4% and the other components are present in the range of 94-99%.

23. The bioformulation as claimed in claim 21, wherein Trichoderma reesei is present in an amount in the range of IxlO⁷ to 1 x IO¹⁰ CFU / g and applied to the banana plants in an amount in the range of 7 x IO¹⁰ to 3 x 10¹¹ CFU/Hectare.

24. The bioformulation as claimed in claim 21, wherein the organic complex oligosaccharide is in the range of 0.15-0.3%, the nutrient source is in the range of 2- 4%, and acceptable carrier is in the range of 93-95%.

25. The bioformulation as claimed in claim 21, wherein the organic complex oligosaccharide is chitosan; the nutrition source is gum acacia; and the acceptable carrier is selected from biochar, glycerol or propylene glycol.

26. The bioformulation as claimed in claim 21, wherein the said formulation is applied on the banana crops plant parts or locus thereof and surroundings including plant rhizosphere and phyllosphere.

27. The bioformulation as claimed in claim 21, wherein the said formulation controls infestation of Fusarium Wilt caused by Fusarium oxysporum f.sp. cubense Race 1, Race 2, Race 4, STR4 or TR4 or Fusarium odoratissimum and infestation caused by banana thrips.

28. The bioformulation as claimed in claim 21, wherein the bioformulation is in a solid form- as wettable powder or dust or granules, or in liquid form - as suspension, solution, dispersion or concentrate wherein biochar, or anoxically burnt wood is used in wettable powder or dust or granules and glycerol or propylene glycol is used in suspension, solution, dispersion or concentrate.

29. The bioformulation as claimed in claim 21, wherein the said bioformulation controls of Sigatoka disease, banana thrip infestation, other leaf spot diseases.

30. The bioformulation as claimed in claim 21, wherein Sigatoka and other leaf spot disease is caused by any strain of Pseudocercospora fijiensis, P. musae, P eumusae, Mycospherellafijiensis, M. musicola, M. eumusae or Cladosporium sp. or Alternaria.

31. Use of a bioformulation comprising a fungal culture selected from pure culture of Trichoderma ressei, or micro sclerotia or submerged conidia or chlamydospores or combinations thereof; at least one organic complex oligosaccharide, at least one nutrition source, and at least one acceptable carrier for preparation of biopesticides for treatment of banana crop from infestation, controlling promoting banana plant growth, plant height, plant girth and number of leaves, and enhancing yield in terms of fruit bunch weight.

32. The use as claimed in claim 31, wherein the fungal culture is present in the weight range of l%-4% and the other components are present in the range of 94-99%.

33. The use as claimed in claim 31, wherein the Trichoderma ressei is present in an amount in the range IxlO⁷ to 1 x IO¹⁰ CFU / g and applied to the banana plants in an amount in the range of 7 x IO¹⁰ to 3 x 10¹¹ CFU/Hectare.

34. The use as claimed in claim 31, wherein organic complex oligosaccharide is in the range of 0.15-0.3%, the nutrient source is in the range of 2-4%, and acceptable carrier is in the range of 93-95%.

35. The use as claimed in claim 31, wherein the organic complex oligosaccharide is chitosan; the nutrition source is gum acacia; and the acceptable carrier is selected from biochar, anoxically burnt wood, glycerol or propylene glycol.

36. The use as claimed in claim 31, wherein the said formulation is applied on the banana plant parts or locus thereof and surroundings including plant rhizosphere and phyllosphere.

37. The use as claimed in claim 31, wherein the said formulation controls infestation of Fusarium Wilt caused by Fusarium oxysporum f.sp. cubense Race 1, Race 2, Race 4, STR4 or TR4 or Fusarium odoratissimum, and infestation caused by banana thrips.

38. The use as claimed in claim 31, wherein the said formulation is in a solid form- as wettable powder or dust or granules, or in liquid form - as suspension, solution, dispersion or concentrate wherein biochar, or anoxically burnt wood is used in wettable powder or dust or granules and glycerol or propylene glycol is used in suspension, solution, dispersion or concentrate.

39. The use as claimed in claim 31, wherein the said formulation controls of Sigatoka disease, banana thrip infestation and other leaf spot diseases.

40. The formulation as claimed in claim 39, wherein Sigatoka and other leaf spot disease is caused by any strain of Pseudocercospora fijiensis, P. musae, P. eumusae, Mycospherella fijiensis, M. musicola, M. eumusae or Cladosporium sp. or Alternaria alternata.

41. A method of treating the banana crop from infestation and enhancing its growth comprising the steps of: a. providing a bioformulation comprising a fungal culture selected from pure culture of Trichoderma ressei, or microsclerotia, or submerged conidia or chlamydospores or combinations thereof; at least one organic complex oligosaccharide, at least one nutrition source, and at least one an acceptable carrier in a wettable powder or a liquid form; b. applying bioformulation to the banana plant, plant parts or surroundings via soil drenching, via foliar spray, via drone spray, or a combination thereof at defined intervals, frequencies and in concentrations at specified growth stages; via injection to pseudo-stem, or banana mat, via injection to emerging floral buds or a combination thereof at defined intervals, frequencies and in concentrations and at specified growth stages with a specified angle and process.

42. The method as claimed in claim 41, wherein the said bioformulation is a wettable powder first soaked and mixed with clean water at the rate of 6-9 g per 10-250 ml water, incubated for 24-36 hours, and filtered and wherein for soil drenching and foliar spray, the water used is in the range of 200-250 ml, for pseudo stem injection the water used is in the range 10-15 ml, and for floral bud injection the water used is in the range 80-120 ml for application per plant.

43. The method as claimed in claim 41, wherein the said wettable powder mixture or in liquid form is having active microorganism Trichoderma reesei present in an amount in the range IxlO⁷ to 1 x IO¹⁰ CFU / g and an amount in the range of 7 x IO¹⁰ to 3 x 10¹¹ CFU/Hectare.

44. The method as claimed in claim 41, wherein the bioformulation is applied either once, twice, thrice or four times, during the crop cycle, with a minimum interval period of 35- 45 days between any two applications, depending upon disease severity.

45. The method as claimed in claim 41, wherein the bioformulation application promotes plant growth in terms of plant height, plant girth and number of leaves, and enhances yield in terms of fruit bunch weight.

46. The method as claimed in claim 41, wherein the bioformulation is applied to the banana plant, banana rhizosphere, banana pseudo-stem vascular tissues, banana leaves or the locus of the plant.

47. The method as claimed in claim 41, wherein banana plant is selected from species Musa acuminata, Musa balbisiana or any Musa hybrid.

48. The method as claimed in claim 41, wherein injection is provided by an injection needle penetrating the pseudo-stem at an angle of 40-50 degrees, most preferably at an angle of 45 degrees with reference to plant vertical and horizontal planes, to attain maximum retention and spread of injected liquid to plant vasculature and internal tissues.

49. The method as claimed in claim 41, wherein the formulation is effective in treating the banana crops from infestation caused by Fusarium oxysporum f.sp. cubense Race 1, Race 2, Race 4, STR4 or TR4 or Fusarium odoratissimum.

50. The method as claimed in claim 41, wherein the formulation is effective in controlling Sigatoka disease, other leaf spot diseases and banana thrips.

51. The method as claimed in claim 50, wherein Sigatoka and leaf spot disease is caused by any strain of Pseudocercospora fijiensis, P. musae, P. eumusae, Mycospherella fijiensis, M. musicola, M. eumusae or Cladosporium sp. or Alternaria alternata.

52. The method as claimed in claim 50, wherein the formulation is treating the banana crops from infestation caused by banana thrips by applying the formulation to the banana plant, emerging floral bud or parts thereof.

53. A method of application to the banana crop from infestation and enhancing its growth comprising applying bioformulation to the banana plant, plant parts or surroundings via soil drenching, via foliar spray, via drone spray, or a combination thereof at defined intervals, frequencies and in concentrations at specified growth stages; via injection to pseudo-stem, or banana mat, via injection to emerging floral buds or a combination thereof at defined intervals, frequencies and in concentrations and at specified growth stages with a specified angle and process.

54. The method as claimed in claim 53, wherein the effective amount of formulation is 6- 9 g per 10-250 ml water, incubated for 24-36 hours, and filtered and wherein for soil drenching and foliar spray, the water used is in the range of 200-250 ml, for pseudo stem injection the water used is in the range 10-15 ml, and for floral bud injection the water used is in the range 80-120 ml for application per plant.

55. The method as claimed in claim 53, wherein the bioformulation is applied either once, twice, thrice or four times, during the crop cycle, with a minimum interval period of 35- 45 days between any two applications, depending upon disease severity.

56. The method as claimed in claim 53, wherein injection is provided by an injection needle penetrating the pseudo-stem at an angle of 40-50 degrees, most preferably at an angle of 45 degrees with reference to plant vertical and horizontal planes, to attain maximum retention and spread of injected liquid to plant vasculature and internal tissues.