CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to Singapore Provisional Patent Application

Serial No. 10202251638Q, which was filed on November 7, 2022, entitled BIOFERTILIZER COMPOSITIONS AND METHODS FOR THEIR USE, the contents of which are incorporated in their entirety by reference

BACKGROUND

[0002] Biofertilizers are alternative effective solutions for farmers to improve soil health quality through supplying plants macronutrients and micronutrients, or inhibiting plant diseases from spreading to the plant roots or leaves near soil surfaces. Organic biofertilizers are naturally desirable in all agricultural systems as they offer multiple benefits simultaneously through numerous modes of action especially where pathogen antagonisms are targeted. This activation phenomenon differentiates from conventional synthetic fertilizers and fungicides that inherently possess a narrow and limited range of nutrients which usually target pathogens with a single mode of action. In addition, using synthetic fertilizers can degrade soils in the long term as it leads to a loss of critical soil organic matter and decreased diversity in the soil microbiome.

Likewise, the use of synthetic fungicides increasingly resulted in resistant strains of pathogens in the soil. This resistance arises easily in situations where single modes of action are used. One alternative to synthetic fungicides is biological control products, which are usually based on a single microbial species in purified form.

[0003] Despite considerable advances made to date, there still exists a need for biofertilizers with plant protection properties. Conventional biological control products are rarely effective in field conditions as the microbes often fail to establish themselves effectively in field conditions as they lack the organic matter and conditions to facilitate their growth. There remains a need for chemical-free biofertilizers that offer multiple modes of action and robust establishment rates in field conditions.

SUMMARY

[0004] In one example, a biofertilizer composition is described comprising fermented insect frass; and at least one Bacillota bacteria, at least one Actinomycetota bacteria, or a combination thereof.

[0005] In another example, a liquid extract composition is described comprising an aqueous extract of a biofertilizer composition, wherein the biofertilizer composition comprises fermented insect frass; and at least one Bacillota bacteria, at least one Actinomycetota bacteria, or a combination thereof.

[0006] In a further example, a method of inhibiting at least one plant pathogen is described, the method comprising: providing a biofertilizer composition comprising fermented insect frass; and at least one Bacillota bacteria, at least one Actinomycetota bacteria, or a combination thereof; and applying the composition to a plant suspected of having at least one plant pathogen.

[0007] In an additional example, a method of preparing a biofertilizer composition is described, the method comprising: contacting fermented insect frass, and at least one Bacillota bacteria, at least one Actinomycetota bacteria, or a combination thereof to form a mixture; and aging the mixture to prepare the biofertilizer composition.

[0008] In another example, a method of preparing a liquid extract composition is described, the method comprising: contacting water and a biofertilizer composition to form a mixture; and filtering the mixture, wherein the biofertilizer composition comprises fermented insect frass; and at least one Bacillota bacteria, at least one Actinomycetota bacteria, or a combination thereof.

DEFINITIONS

[0009] ‘Biofertilizer” refers to a material which contains living micro-organisms which, when applied to seeds, plant surfaces, or soil, colonize the rhizosphere or the interior of the plant and promotes growth or protects the plant from disease.

[0010] ‘Exuviae” refers to the exoskeleton remains left after ecdysozoan insects have molted.

[0011] ‘Fermented” refers to a type of aging using a metabolic process that produces chemical changes in organic substrates through the action of microorganisms.

[0012] ‘Frass” refers to insect feces. Frass can often be accompanied by residual insect feed.

DETAILED DESCRIPTION

[0013] Use of the described methods and materials can result in a reduction or elimination of pathogen infection in a plant relative to operation of the same or similar plant without the described methods and materials. The degree of pathogen infection can generally be reduced by any amount. For example, the degree of pathogen infection can be reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, and in an ideal situation, about 100% reduction (complete elimination of pathogen infection).

[0014] This disclosure is not limited to the particular systems, devices, compositions, and methods described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope.

[0015] Biofertilizer compositions

[0016] In some examples, biofertilizer compositions are provided. The composition can comprise fermented insect frass, and at least one Bacillota bacteria, at least one Actinomycetota bacteria, or a combination thereof.

[0017] The insect frass can generally be obtained from any insect. For example, the insect frass can be black soldier fly frass. In other examples, the insect frass can be Silkworm (Bombyx mori) frass, House Cricket (Acheta domesticus) frass, Jamaican Field Cricket (Gryllus assimilis) frass, Mealworm (Tenebrio molitor) frass, Super Worm (Zophobas morio) frass, House fly Musca domestica) frass, or combinations thereof.

[0018] The fermented insect frass can generally be fermented for any length of time.

For example, it can be fermented for equal to or greater than 30 days. In some examples, it can be fermented in conditions that are open to the environment, where at least one gas can reach the surface of the insect frass.

[0019] The fermented insect frass can be present in the composition in generally any concentration. For example, the fermented insect frass can be present at about 30% w/w to about 99% w/w, about 60% w/w to about 99% w/w, or about 97% w/w to about 99% w/w. Specific examples of concentrations include about 30% w/w, about 40% w/w, about 50% w/w, about 60% w/w, about 70% w/w, about 80% w/w, about 90% w/w, about 95% w/w, about 96% w/w, about 97% w/w, about 98% w/w, about 99% w/w, or ranges between any two of these values.

[0020] The composition can further comprise one or more additional materials. For example, the composition can further comprise insect exuviae. The exuviae can generally be from any insect. For example, the exuviae can be obtained by collecting cuticles shed during ecdysis or it can be obtained through mechanical separation. Cuticle sources include puparium (pupae cuticle), whole larvae, whole imago (adult insect), insect parts (for example legs or wings). The composition can further comprise puparia (pupal exuviae), larval cuticle (from mechanical separation), whole larvae, whole imago (adult insect), insect parts (for example, legs or wings), or fermented insect exuviae. The composition can comprise chitin, chitosan, or a combination thereof. The composition can further comprise soil, fertilizers, and one or more common agricultural compounds.

[0021] The insect exuviae can generally be present in the composition at any concentration. For example, the insect exuviae can be present at a concentration of about 0.01% w/w to about 5% w/w, about 0.01% w/w to about 3% w/w, or about 0.01% w/w to about 1% w/w. Specific examples of concentrations include about 0.01 % w/w, about 0.1% w/w, about 1 % w/w, about 2% w/w, about 3% w/w, and ranges between any two of these values.

[0022] The Bacillota (phylum) bacteria can generally be any Bacillota bacteria. For example, the Bacillota bacteria can be Bacillales (order) bacteria. The Bacillota bacteria can also be a Bacillus (genus) bacteria. The Actinomycetota (phylum) bacteria can generally be any Actinomycetota bacteria. For example, the Actinomycetota bacteria can be Actinomycetales (order) bacteria. The Actinomycetota bacteria can also be a Streptomyces (genus) bacteria.

[0023] The composition can comprise one, two, or more types of bacteria. For example, the composition can comprise Bacillota bacteria but not Actinomycetota bacteria, or Actinomycetota bacteria but not Bacillota bacteria. In other examples, the composition can comprise Actinomycetota bacteria and Bacillota bacteria. [0024] The Bacillota bacteria and/or Actinomycetota bacteria can be present in the composition at generally any concentration. For example, the bacteria can be present at a concentration of at least about 10 ³ CFU/g, a concentration well above any naturally occurring concentrations of any single species of Bacillota bacteria and/or Actinomycetota bacteria in insect frass. In other examples, the bacteria can be present at a concentration of about 10 ⁴ CFU/g to about 10 ¹² CFU/g, about 10 ⁶ CFU/g to about 10 ¹² CFU/g, or about 10 ⁸ CFU/g to about 10 ¹² CFU/g. In specific examples, the concentration can be about 10 ⁴ CFU/g, about 10 ⁵ CFU/g, about 10 ⁶ CFU/g, about 10 ⁷ CFU/g, about 10 ⁸ CFU/g, about 10 ⁹ CFU/g, about IO ¹⁰ CFU/g, about 10 ¹¹ CFU/g, about 10 ¹² CFU/g, and ranges between any two of these values. If multiple types of bacteria are present, their combined concentration can be at these values or ranges

[0025] The composition can be in various forms. In some examples, it is in the form of a powder. In other examples, it is in the form of a liquid. In other examples, it is in the form of a slurry.

[0026] In some examples, the composition can further comprise water. The water can generally be at any concentration, such as about 5% w/w to about 60% w/w. Specific examples of concentrations include about 5% w/w, about 10% w/w, about 20% w/w, about 30% w/w, about 40% w/w, about 50% w/w, about 60% w/w, and ranges between any two of these values. [0027] In some examples, the composition can be characterized as not containing plant pathogens. Without being bound by theory, the use of fermented insect frass reduces or eliminates plant pathogens in the composition that could be present if unfermented insect frass were used.

[0028] Liquid extract compositions [0029] A liquid extract composition can be made as an aqueous extract of any of the above-described biofertilizer compositions. For example, a liquid extract composition can comprise an aqueous extract of a biofertilizer composition, wherein the biofertilizer composition comprises fermented insect frass; and at least one Bacillota bacteria, at least one Actinomycetota bacteria, or a combination thereof.

[0030] The fermented insect frass can generally be fermented for any length of time.

For example, it can be fermented for equal to or greater than 30 days. In some examples, it can be fermented in conditions that are open to the environment, where at least one gas can reach the surface of the insect frass.

[0031] The liquid extract composition can comprise water. The liquid extract composition can further contain one or more additional dissolved materials. Examples of which include calcium salt, calcium carbonate, ammonium hydroxide, potassium hydroxide, potassium oxide, potassium salt of ammonium salt, or combinations thereof. Additional examples include a sugar source, sucrose, glucose, fructose, molasses, biochar, antifoaming agent, humic acid, fulvic acid, gibberellic acid indole-3-acetic acid (IAA), abscisic acid (ABA), amino acid, or combinations thereof. The additional dissolved materials can be at least one plant growth promoter, at least one phytohormone, or combinations thereof.

[0032] Methods of inhibiting plant pathogens

[0033] Any of the above-described compositions can be used to inhibit plant pathogens.

For example, a method of inhibiting at least one plant pathogen can comprise providing a biofertilizer composition comprising fermented insect frass; and at least one Bacillota bacteria, at least one Actinomycetota bacteria, or a combination thereof; and applying the composition to a plant suspected of having at least one plant pathogen. The methods can be “therapeutic”, that is, to treat a plant having or suspected of having at least one plant pathogen. The methods can also be “prophylactic”, that is, to treat a plant before it has at least one plant pathogen.

[0034] The fermented insect frass can generally be fermented for any length of time.

For example, it can be fermented for equal to or greater than 30 days. In some examples, it can be fermented in conditions that are open to the environment, where at least one gas can reach the surface of the insect frass.

[0035] In some examples, the plant is already infested with at least one plant pathogen.

In other examples, the plant is at risk of being infested with at least one plant pathogen. In further examples, the composition is applied to reduce or eliminate the chance of future infestation with at least one plant pathogen.

[0036] The plant pathogen can generally be any plant pathogen, such as a fungal pathogen, or a filamentous fungal pathogen. Specific pathogens include Fusarium, Phytophthora, or Ganoderma. Additional specific pathogens include Fusarium oxysporum, Phytophthora capsica, or Ganoderma boninense. Additional specific pathogens include Rhizoctonia, Sclerotinia, Botrytis, or Altemaria. Further specific pathogens include Rhizoctonia solani, Sclerotinia sclerotiorum, Botrytis cinerea, or Alternaria solani.

[0037] The plant can have one or more plant pathogens, such as 1, 2, 3, 4, 5, 6, or more plant pathogens.

[0038] Efficacy can be measured in several ways. For example, a percent reduction can be calculated relative to the initial amount of pathogen. For example, the plant can have an initial amount of pathogen before the applying step that is greater than a final amount of pathogen after the applying step; and the final amount represents a percentage reduction relative to the initial amount. The percentage reduction can generally be any percentage, such as at least a 50% reduction. Additional percentage reduction can include at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and ranges between any two of these values. In an ideal example, the percentage reduction is 100%, that is, total reduction in plant pathogen.

[0039] Alternatively, a plant can have a lower amount of pathogen after the applying step relative to a similar plant that did not receive application of the composition. The lower amount can be represented as a percentage reduction relative to the amount of pathogen in the similar plant, and can generally be any percentage, such as at least a 50% reduction. Additional percentage reduction can include at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and ranges between any two of these values. In an ideal example, the percentage reduction is 100%, that is, total reduction in plant pathogen.

[0040] Alternatively, either of the two reductions mentioned above can be calculated using a log value (log 10). For example, a log reduction can be at least about 0.5. Specific examples of log reduction include about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, and ranges between any two of these values. Specific examples of ranges include about 0.5 to about 1, about 1 to about 2, and about 0.75 to about 2.

[0041] In yet another alternative way of describing efficacy of the method, the plant can have a lower (better) disease severity index (DSI) score after the applying step relative to a similar plant that did not receive application of the composition. A disease severity index (DSI) is a single number (0-9) for summarizing a large amount of information on disease severity. [0042] The disease severity index is usually converted from a disease severity percentage as follows: 0 = no infection; 1 = less than 1%; 2 = 1-5%; 3 = 5-10%; 4 = 10-20%; 5 = 20-40%; 6 = 40-60%; 7 = 60-80%; 8 = 80-90%; and 9 = more than 90%.

[0043] Disease severity percentage calculation is based on the number of sick plants and the wilted leaves:

[0044] DSI (%) = 100.£(f/nHv/x)

[0045] Where f = number of sick plants; n = total of plants; v = number of leaves with symptoms; and x = total number of leaves (with symptoms and healthy).

[0046] The composition can be applied in the applying step in the form of a powder or a liquid. The composition can further comprise water.

[0047] The applying step can include one or more types of application. For example, the applying step can comprise applying the composition to leaves of the plant, applying the composition to soil at the base of the plant, applying the composition to roots of the plant, or combinations thereof. In some examples, applying to soil at the base of the plant can include applying at about 2 cm to about 100 cm distance from the plant stem. The method can further comprise mixing the composition with soil before the applying step. The method can further comprise pelleting the composition before the applying step.

[0048] In some examples, the composition can be in the form of a liquid, and the applying step comprises spraying or misting the composition onto leaves, shoots, or both of the plant. In other examples, the composition can be in the form of a liquid, and the applying step comprises spraying or misting the composition onto roots of the plant or onto soil at the base of the plant. In some examples, applying to soil at the base of the plant can include applying at about 2 cm to about 100 cm distance from the plant stem. In further examples, the composition can be in the form of a liquid, the plant is a seedling having roots; and the applying step comprises dipping the roots into the composition. In yet another example, the composition can be in the form of a liquid, and the applying step comprises adding the composition into water in a hydroponics system, an aquaponics system, or an aeroponics system.

[0049] In some examples, the applying step can comprise co-applying with one or more additional materials such as carbon-rich mulch. Examples of additional materials include biochar, fly ash, peat, compost, vermiculite, perlite, rock phosphate, calcium sulfate, alginate, carrageenan, fermentation broth filtrates, and combinations thereof.

[0050] The plant can generally be any plant, such as a commercially grown plant.

Examples of lists of plants include rice, wheat, soybeans, sugarcane, grapes, cotton, cucumber, garlic, rubber, coconut, maize, cassava, oil palm, cocoa, banana, pineapple, orchids, watermelon, mango, durian, mangosteen, guava, sweet potatoes, oranges, tobacco, black pepper, tomato, eggplant, pepper, potato, brassica, cabbage, choy sum, kale, or coffee. Additional lists of plants include rice, rubber, oil palm, cocoa, banana, orchids, tomato, chili, or cucumber.

[0051] Methods of preparing biofertilizer compositions

[0052] Additional examples include methods for preparing a biofertilizer composition.

The methods can comprise contacting insect frass, and at least one Bacillota bacteria, at least one Actinomycetota bacteria, or a combination thereof to form a mixture; and fermenting the mixture to prepare the biofertilizer composition.

[0053] The insect frass can be unfermented insect frass. The mixture can generally be fermented for any length of time. For example, it can be fermented for equal to or greater than 30 days. In some examples, it can be fermented in conditions that are open to the environment, where at least one gas can reach the surface of the insect frass. [0054] The methods can further include one or more additional steps, such as adding water to the mixture before the fermenting step. The amount of added water can generally be any amount, such as adding water to give a moisture content of about 5% w/w to about 60% w/w. Specific examples of concentrations include about 5% w/w, about 10% w/w, about 20% w/w, about 30% w/w, about 40% w/w, about 50% w/w, about 60% w/w, and ranges between any two of these values.

[0055] The fermenting step can include solid state fermenting or composting.

[0056] The fermenting step can be performed at various times and temperatures. For example, the fermenting step can comprise storing the mixture for a time of at least about 15 days, about 15 days to about 1 year, about 1 month to about 3 months, or about 1 month to about 6 months. In some examples, the fermenting step can comprise storing the mixture for about 3 months. Specific examples of storing times include about 15 days, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, or ranges between any two of these values.

[0057] The fermenting step can be performed at generally any temperature of the mixture, such as a temperature of at least about 10 degrees Celsius above ambient temperature. For example, if ambient temperature is 30 degrees Celsius, the mixture can be at least about 40 degrees Celsius. This increased temperature comes from microbes in the mixture and is sometimes referred to as “biological heat” or “self-heating”.

[0058] Methods of preparing liquid extract compositions

[0059] Methods of preparing a liquid extract composition can comprise forming an aqueous extract of a biofertilizer composition. For example, the method can comprise contacting water and a biofertilizer composition to form a mixture, and filtering the mixture to form the liquid extract composition. The biofertilizer can be any of the above-described biofertilizer compositions. For example, the biofertilizer composition can comprise fermented insect frass; and at least one Bacillota bacteria, at least one Actinomycetota bacteria, or a combination thereof. The biofertilizer composition can further comprise insect exuviae.

[0060] The fermented insect frass can generally be fermented for any length of time.

For example, it can be fermented for equal to or greater than 30 days. In some examples, it can be fermented in conditions that are open to the environment, where at least one gas can reach the surface of the insect frass.

[0061] The amount of water added to the biofertilizer composition can generally be any amount. For example, water can be added at an amount at least about 3: 1 (w/w). Ranges of amounts include about 3: 1 (w/w) to about 10:1 (w/w), about 4: 1 (w/w) to about 8: 1 (w/w), or about 4: 1 (w/w) to about 5:1 (w/w). Specific examples include about 3:1 (w/w), about 4:1 (w/w), about 5: 1 (w/w), about 6: 1 (w/w), about 7:1 (w/w), about 8: 1 (w/w), about 9: 1 (w/w), about 10: 1 (w/w), and ranges between any two of these values.

[0062] The method can further comprise one or more steps after the contacting step and before the filtering step. For example, the method can further comprise aerating the mixture.

The method can further comprise mixing, churning, or blending the mixture to improve dissolution. The method can further include evaporating, vacuum concentrating, distilling, or other methods of concentrating the mixture before or after the filtering step. The method can further comprise one or more steps after the filtering step. For example, the method can further comprise aging the liquid extract composition after the filtering step, and sealing the liquid extract composition in a container after the aging step. At least one advantage of aging before sealing is to allow gases dissolved in the liquid extract composition to fully or partially outgas.

The aging step can include exposing the filtered mixture to the atmosphere. In some examples, pests or insects can be kept away from the filtered mixture by using a mesh or gauze over the container.

[0063] The method can further comprise placing the filtered mixture in at least one bottle. The bottle can generally be any volume, such as 0.25 liter, 0.5 liter, 1 liter, 5 liters, or 25 liters. The bottle can be equipped with a mechanism to allow off-gassing and to avoid swelling of the bottle. For example, the mechanism can be a vent liner in a bottle cap, or an inner plug in a bottle cap. The method can further include capping the bottle after the placing step.

[0064] EXAMPLES

[0065] Example 1 : Preparation of insect frass-based biofertilizer

[0066] Black Soldier Fly (BSF) larvae frass was first screened in a rotary sieve to remove impurities and large particles. Within 24 hours of harvest the frass was loaded into a 0.5 cubic meter bulk bag, where the bag material allows gas permeability. The frass was fermented for 91 days in a well-ventilated space. The temperature and moisture content of the frass was monitored throughout the fermentation process. If the temperature of the frass dropped below 40 Celsius in the first 14 days the material was removed from the bag, water was added via a fine spray and the material was mixed then reloaded into the bag.

[0067] At the end of the fermentation process the frass was screened once more in a rotary sieve to remove clumps of material. Separately a liquid inoculum of Bacillus halotolerans at a concentration of 10 ⁶ CFU / mF was prepared. The fermented frass was then loaded into a ribbon blade mixer and the Bacillus inoculum was added by way of spraying during mixing. The inoculum was added at a rate of 1% (w/w) of the frass. After mixing the material was conveyed into piles of approximately one cubic meter and allowed to dry for 48 hours. The material was then moved into a hopper which conveyed it into a bagging line where it was loaded into 20kg polypropylene woven bags.

[0068] Example 2: Preparation of biofertilizer containing insect exuviae

[0069] Black Soldier Fly Larvae Frass was first screened in a rotary sieve to remove impurities and large particles. Within 24 hours of harvest the frass was loaded into a 0.5 cubic meter bulk bag, where the bag material allows gas permeability. The frass was fermented for 91 days in a well-ventilated space. The temperature and moisture content of the frass was monitored throughout the fermentation process. If the temperature of the frass dropped below 40 Celsius in the first 14 days the material was removed from the bag, water was added via a fine spray and the material was mixed then reloaded into the bag.

[0070] At the end of the fermentation process the frass was screened once more in a rotary sieve to remove clumps of material. Separately insect exuviae in the form of Black Soldier Fly puparium was collected and ground using a blade grinder until the particle size was below 3 mm. Separately a liquid inoculum of Bacillus halotolerans at a concentration of 10 ⁶ CFU / mL was prepared. The fermented frass and exuviae was then loaded into a ribbon blade mixer. The material was mixed while the Bacillus inoculum was added by way of spraying. The exuviae and inoculum each were added at a rate 1% (w/w) of the frass. After mixing the material was conveyed into piles of approximately one cubic meter and allowed to dry for 48 hours. The material was then moved into a hopper which conveyed it into a bagging line where it was loaded into 20 kg Polypropylene woven bags.

[0071] Example 3 : Preparation of biofertilizer containing Actinomycetes [0072] Black Soldier Fly Larvae Frass will be first screened in a rotary sieve to remove impurities and large particles. The frass product will then be introduced to ribbon blade mixer along with an inoculum of Streptomyces violaceusniger at a rate of 0.5- 1.0 % w/w dry cell mass of inoculum. The time between harvesting the insect frass and inoculation will not exceed 3 hours and the temperature of the frass prior to inoculation below 40 degrees Celsius. After inoculation the material will be loaded into a 0.5 cubic meter bulk bag, where the bag material allows gas permeability. The frass will be fermented for 91 days in a well-ventilated space. The temperature and moisture content of the frass will be monitored throughout the fermentation process. If the temperature of the frass drops below 40 degrees Celsius in the first 14 days the material will be removed from the bag, water is added via a fine spray and the material is mixed then reloaded into the bag. The material will be monitored for the presence of Streptomyces mycelium in the material with the exception of the top 5 cm layer.

[0073] At the end of the fermentation process the frass will be screened once more in a rotary sieve to remove clumps of material. The material will then be moved into a hopper which conveys it into a bagging line where it will be loaded into 20 kg polypropylene woven bags. [0074] Example 4: Evaluation of solid biofertilizer (in vitro)

[0075] Solid biofertilizers were evaluated using four different methods.

[0076] A ) Antifungal test using disk diffusion method.

[0077] The solid biofertilizer was added to sterile distilled water at ratio 1: 10 (w/v) and incubated in an incubator shaker at room temperature and 120 rpm for 7 days. The fermented broth was filtered using sterile filter paper to remove the solid particles and only the liquid was collected. Sterile filter disk with a diameter of 0.6 cm is soaked with the liquid and placed on top of a petri plate containing Mueller Hinton agar (MHA), 1.5 cm away from the edge. A 0.5 cm mycelium plugs of phytopathogenic fungi were placed at the center of the MHA plates, separately, approximately 2.5 cm from the liquid-soaked filter disks. The plates were incubated at 27 degrees Celsius until the control plate (without soaked disks) was fully covered with fungal mycelium. The mycelium inhibition percentage is calculated according to the following equation:

[0078] Mycelium inhibition percentage = (A-B)/A * 100%

[0079] Where A = length of mycelium in control plates, and B = length of mycelium in treatment plates.

[0080] B) Antifungal test using sample-amended medium method.

[0081] 3 grams of solid biofertilizer was weighed and added into a sterile petri plate.

The solid biofertilizer was arranged on the sides of the plate, forming a ring with empty space at the center of the plate. 20 mL of warm molten potato dextrose agar was added into the plate, covering the solid biofertilizer completely and left to solidify. Then, 0.5 cm of fungal mycelium plug was placed on the center of the plate. The inoculated petri dishes were incubated at 27 degrees Celsius until the negative control plate was fully covered with fungal mycelial growth. The mycelium inhibition percentage is calculated according to the following equation [0082] Mycelium inhibition percentage = (A-B)/A * 100%

[0083] Where A = length of mycelium in control plates, and B = length of mycelium in treatment plates.

[0084] C) Antifungal test using inverted plate method (for volatile organic compounds

(VOCs)).

[0085] In one sterile petri plate, 1 g of the solid biofertilizer was mixed with 20 mL warm molten nutrient agar and left to solidify. Another sterile petri plate containing 20 mL potato dextrose agar was inoculated with the fungal mycelium plug. Then, the two base plates were sealed together with parafilm (one plate was inverted) and incubated at 27oC, until the negative control plate (without solid biofertilizer) was fully covered with fungal mycelial growth. The mycelium inhibition percentage is calculated according to the following equation: [0086] Mycelium inhibition percentage = (A-B)/A * 100%

[0087] Where A = length of mycelium in control plates, and B = length of mycelium in treatment plates.

[0088] D) Antifungal test using dual layer agar testing method.

[0089] 1 gram of the solid biofertilizer was weighed and added to sterile petri plates.

Then, 20 ml of warm molten potato dextrose agar was added to the plates and mixed thoroughly with the samples. The plates were then incubated at 27 degrees Celsius for 48 hours. Then, another 10 mL of warm molten potato dextrose agar (without the solid biofertilizer) was added on top of the first potato dextrose agar layer. The plates were sealed with parafilm and kept in the fridge at 4 degrees Celsius for 24 hours. Next, a 0.5 cm fungal mycelium plug was placed at the center of the plate. The petri dish was incubated at 27 degrees Celsius until the negative control plate was fully covered with fungal mycelial growth. The mycelium inhibition percentage is calculated according to the following equation:

[0090] Mycelium inhibition percentage = (A-B)/A * 100%

[0091] Where A = length of mycelium in control plates, and B = length of mycelium in treatment plates.

[0092] E) Evaluation of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of solid biofertilizer against the spores of Fusarium oxysporum sp. Cubense TR4. [0093] The solid biofertilizer extract was prepared by mixing with sterile normal saline (0.85% NaCl) at ratio 1:2. The mixture was incubated and shaken at 27°C, 300 rpm for 1 hour. The mixture was then filtered with a sterile coffee filter to remove solid particles. The extract was prepared at different dilutions (50%, 40%, 25% and 10%). The extract was tested in two conditions: sterile and non-sterile. For the sterile extract, the extract was filtered again using 0.22 um syringe filter, to remove bacterial cells. 20 pL of each of the dilutions (both sterile and non-sterile) were added into the wells of the 96-wells plate, containing 100 L of fungal spore suspensions (10 ⁶ spores/mL) and 80 pL of potato dextrose broth (PDB). The wells were prepared in 6 replicates for each dilution. For negative control wells, the fungal spore suspensions were only mixed with PDB, without the extract. For the positive control, the fungal spore suspension was mixed with PDB and antifungal solution (nystatin, 30 mg/L). The plates were incubated at 27 °C for 5 days. Then, 100 pL of each of the mixtures were spread onto plates containing PDA and antibiotic (Streptomycin, 100 pg/mL) to prevent the growth of bacteria. The plates were then incubated for 3 days at 27 °C. The number of fungal colonies were counted, and percentage of inhibition was calculated using this formula:

[0094] Spores inhibition percentage = (A-B)/A * 100%

[0095] Where, A is the number of fungal colonies for negative control plates and B is the number of fungal colonies for treatment plates.

[0096] The minimum concentration of the extract that produces at least 60% inhibitory activity against TR4 was selected as the MIC of the solid biofertilizer, while the minimum concentration of the extract that could kill 99% of the fungal spores was selected as the MFC of the solid biofertilizer. [0097] F) Evaluation of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of solid biofertilizer against the mycelium of Ganoderma boninense

[0098] 500 p L of each of the solid biofertilizer extract dilutions (both sterile and non-sterile) were added into the wells of the 26-wells plate, containing 500 p L of 2x potato dextrose broth (PDB). A fungal mycelium plug (4 x 4 mm) was added to each of the wells. The wells were prepared in 6 replicates for each extract dilution. For negative control wells, the mycelium plugs were only mixed with PDB, without extract. For the positive control, the mycelium plugs were mixed with PDB and antifungal solution (nystatin, 50 mg/L). The plates were incubated at 27 °C for 5 days. The MFC was taken as the lowest concentration of extract at which there was no visible growth of the fungus after incubation. The mycelium of fungus was collected and transferred to a filter paper in a petri dish, then dried at 45 °C for 1 night. The dried mycelium was weighed, and the inhibition percentage was calculated using this formula:

[0099] Mycelium inhibition percentage = (A-B)/A * 100%

[00100] Where, A is the weight of fungal mycelium for negative control and B is the weight of fungal mycelium for treatment plates

[00101] The minimum concentration of the extract that produces at least 60% inhibitory activity against TR4 was selected as the MIC of the solid biofertilizer, while the minimum concentration of the extract that could kill 99% of the fungal mycelium was selected as the MFC of the solid biofertilizer.

[00102] Example 5 : Preparation of liquid biofertilizer

[00103] Insect frass and exuviae were separately placed into cloth bags and submerged in water in a barrel drum. To produce a more concentrated frass tea, the ratio of the exuviae to the frass and water was 1:4: 16 (w/w/v). Bacillus halotolerans inoculant was also added at 0.1%, with the inoculant concentration of 10 ⁶ CFU/mL. The aeration at a flow rate of 1 vvm (volume of air sparged in aerobic cultures per unit volume of growth medium per minute) was provided to the mixtures using a water pump, connected to a tube and air stone, for a better air diffusion.

The barrel drum was tightly closed with a lid (to avoid pests and insects) with a small vent hole. The aerated extraction was conducted for 3 days. The bag was agitated for 2-5 minutes every day to encourage the material to dissolve.

[00104] After 3 days, the cloth bag was removed from the barrel drum and the frass tea was filtered using a filter cloth. Physical and chemical factors of the aerobic fermentation of the liquid fertilizer are taken into consideration, therefore, temperatures should range between 25-35 degrees Celsius; and the desired pH is 7 to 8.

[00105] The liquid extract of this process was subsequently put at rest for 1 -3 days at room temperature to demonstrate chemical and microbial stability and storability, especially to avoid excessive off gassing behavior which may cause a swelling bottle condition prior to bottling. For a sedimentation possibility during the resting period of the liquid extract, the amount of settling solids or insoluble matters is minor with no compact solids on the bottom of the resting tank. During the resting period, a clean fabric cloth covers the top of the tank to avoid any possible contamination. The resting tank was agitated for 5-10 minutes every day to encourage off gassing.

[00106] For bottling, the liquid extract underwent a series of operations from pumping, filling and vent liner or inner plug fitting and capping into 1-L, 5-L and/or 25-L HDPE bottles. [00107] Example 6: Evaluation of liquid biofertilizer (in vitro) [00108] Antifungal test using disk diffusion method [00109] Sterile filter disk with a diameter of 0.6 cm is soaked with the liquid biofertilizer and placed on top of a petri plate containing Mueller Hinton agar (MHA), 1.5 cm away from the edge. A 0.5 cm mycelium plugs of phytopathogenic fungi were placed at the center of the MHA plates, separately, approximately 2.5 cm from the liquid-soaked filter disks. The plates were incubated at 27 degrees Celsius until the control plate (without soaked disks) was fully covered with fungal mycelium. The mycelium inhibition percentage is calculated according to the following equation:

[00110] Mycelium inhibition percentage = (A-B)/A * 100%

[00111] Where A = length of mycelium in control plates, and B = length of mycelium in treatment plates.

[00112] Antifungal test using inverted plate method (for volatile organic compounds (VOCs))

[00113] In one sterile petri plate, 1 mL of the liquid biofertilizer was mixed with 20 mL warm molten nutrient agar and left to solidify. Another sterile petri plate containing 20 mL potato dextrose agar was inoculated with the fungal mycelium plug. Then, the two base plates were sealed together with parafilm (one plate was inverted) and incubated at 27 degrees Celsius, until the negative control plate (without the liquid biofertilizer) was fully covered with fungal mycelial growth. The mycelium inhibition percentage is calculated according to the following equation:

[00114] Mycelium inhibition percentage = (A-B)/A * 100%

[00115] Where A = length of mycelium in control plates, and B = length of mycelium in treatment plates. [00116] Evaluation of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of liquid biofertilizer against the spores of Fusarium oxysporum sp. Cubense TR4.

[00117] The liquid biofertilizer was prepared at different dilutions (50%, 40%, 25% and 10%). The liquid biofertilizer was tested in two conditions: sterile and non-sterile. For the sterile liquid, the liquid was filtered using a 0.22 um syringe filter, to remove bacterial cells. 20 L of each of the dilutions (both sterile and non-sterile) were added into the wells of the 96-wells plate, containing 100 L of fungal spore suspensions (10 ⁶ spores/mL) and 80 pL of potato dextrose broth (PDB). The wells were prepared in 6 replicates for each dilution. For negative control wells, the fungal spore suspensions were only mixed with PDB, without the liquid fertilizer. For the positive control, the fungal spore suspension was mixed with PDB and antifungal solution (nystatin, 30 mg/L). The plates were incubated at 27 °C for 5 days. Then, 100 pL of each of the mixtures were spread onto plates containing PDA and antibiotic (Streptomycin, 100 pg/mL) to prevent the growth of bacteria. The plates were then incubated for 3 days at 27 °C. The number of fungal colonies were counted, and percentage of inhibition was calculated using this formula:

[00118] Spores inhibition percentage = (A-B)/A * 100%

[00119] Where, A is the number of fungal colonies for negative control plates and B is the number of fungal colonies for treatment plates.

[00120] The minimum concentration of the liquid that produces at least 60% inhibitory activity against TR4 was selected as the MIC of the liquid biofertilizer, while the minimum concentration of the liquid that could kill 99% of the fungal spores was selected as the MFC of the liquid biofertilizer. [00121] F) Evaluation of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of liquid biofertilizer against the mycelium of Ganoderma boninense

[00122] 500 p L of each of the liquid biofertilizer dilutions (both sterile and non-sterile) were added into the wells of the 26-wells plate, containing 500 p L of 2x potato dextrose broth (PDB). A fungal mycelium plug (4 x 4 mm) was added to each of the wells. The wells were prepared in 6 replicates for each dilution. For negative control wells, the mycelium plugs were only mixed with PDB, without liquid fertilizer. For the positive control, the mycelium plugs were mixed with PDB and antifungal solution (nystatin, 50 mg/L). The plates were incubated at 27 °C for 5 days. The MFC was taken as the lowest concentration of liquid fertilizer at which there was no visible growth of the fungus after incubation. The mycelium of fungus was collected and transferred to a filter paper in a petri dish, then dried at 45 °C for 1 night. The dried mycelium was weighed, and the inhibition percentage was calculated using this formula:

[00123] Mycelium inhibition percentage = (A-B)/A * 100%

[00124] Where, A is the weight of fungal mycelium for negative control and B is the weight of fungal mycelium for treatment plates

[00125] The minimum concentration of the liquid that produces at least 60% inhibitory activity against TR4 was selected as the MIC of the liquid biofertilizer, while the minimum concentration of the liquid that could kill 99% of the fungal mycelium was selected as the MFC of the liquid biofertilizer.

[00126] Example 7: Evaluation of solid and liquid biofertilizer (in vivo)

[00127] The liquid and solid biofertilizer were mixed with the soil prior to planting the banana plantlets. The dosage of the solid and liquid fertilizer are 1.6% (w/w) and 5% (v/w), respectively. The soil mixture was then immediately transferred to individual IL plastic pots, and banana plantlets were transferred to the pot ( 1 plant per pot) A total of 200ml of Fusarium oxysporum sp. cubense TR4 (focTR4) spore suspension at a concentration of 10 ^A 6/mL was poured into each pot. The negative control plants received dechlorinated water, while the positive control plants received fungicide (benomyl) at a concentration of 5 x 10’ ⁴ g/mL. Another set of control plants only received focTR4 suspension. The treatment with solid and liquid biofertilizer was repeated every 3 weeks following the first treatment as a top dressing around the base of the banana plants but avoiding contact with the corm and foliar spray, respectively. The total dosage of solid and liquid biofertilizer were 2.76% (w/w) and 12.5% (v/w). respectively. The disease symptoms were analyzed at the end of the observation period (6 weeks) based on the formula and protocol below:

[00128] External symptoms: The percentage of yellowing/wilting leaves was scored following a 1-4 class scale in which: 1 = 0 > x < 25%, 2 = 25 < x < 50%, 3 = 50 < x < 75% and 4 = 75 < x < 100%.

[00129] Internal symptoms: The individual plant was uprooted, cleaned, and cut longitudinally or horizontally at the rhizome (corm) of each plant. T Disease severity was visually assessed following a 1-6 scale where: 1 = No discoloration in the corm, 2 = isolated points x < 5%, 3 = 5 < x < 30%, 4 = 30 < x < 50%, 5 = 50 < x < 90% and 6 = plant totally decayed x < 90%.

[00130] Disease index = E(Nl-5 x Sl-5)/(N x S)] x 100%, where Nl-5: number of banana plants with wilt symptoms, SI -5: value of the score of symptoms, N: total number of tested banana plants, and S: the highest value of the score of symptoms. The following DI values were used to rank varieties: 0 - Immune, > 0 & < 5% - Resistant, >5 < 20% - Intermediate resistant, > 20 < 50% - Susceptible, > 50 % - Highly susceptible. A lower DI value is better.

[00131] Example 8: In-vitro fungal pathogen inhibition by solid biofertilizer [00132] i) By using the disk diffusion method, the antifungal activity of solid biofertilizer was tested against 3 phytopathogenic fungi Phytophthora capsici, Fusarium oxysporum and Fusarium oxysporum sp. Cubense TR4). The unprocessed frass was compared with the fermented frass. Table 1 below shows that the same inhibition percentage by unprocessed and fermented frass was observed for Phytophthora capsici. The fungus Fusarium oxysporum is more inhibited by the fresh frass, compared to the fermented frass. Meanwhile, the Fusarium oxysporum sp. Cubense TR4 was more inhibited by the fermented frass compared to the unprocessed frass. The results showed that the inhibition of the fungi is related to the susceptibility level of the phytopathogenic fungi to the frass microbes at the different ages of the frass, as the microbial communities in the frass would differ at different fermentation stages.

[00133] Table 1: Phytopathogenic fungi inhibition by the fresh and fermented frass.

[00134] ii) By using the sample-amended medium method, the antifungal activity of the solid biofertilizer (fermented frass, supplemented with 1% (w/w) exuviae and 1% (v/w) Bacillus) was tested against Fusarium oxysporum sp. Cubense TR4 and Ganoderma boninense. Table 2 shows the fungal inhibition percentage by the solid biofertilizer. [00135] Table 2: Fusarium oxysporum sp. Cubense TR4 and Ganoderma boninense inhibition by the solid biofertilizer.

[00136] iii) By using the sample-amended medium method, the antifungal activity of the solid biofertilizers (Fermented frass supplemented with 1 % (w/w) exuviae and different concentrations of Bacillus) were tested against Fusarium oxysporum sp. Cubense TR4.

[00137] Table 3: Fusarium oxysporum sp. Cubense TR4 inhibition by the solid biofertilizer with different inclusion percentage of Bacillus by using the sample-amended medium method.

[00138] iv) By using the disk diffusion method and the dual layer agar method, the antifungal activity of the solid biofertilizers (Fermented frass supplemented with 1 % (w/w) exuviae and different concentrations of Bacillus) were tested against Fusarium oxysporum sp. Cubense TR4. [00139] Table 4: Fusarium oxysporum sp. Cubense TR4 inhibition by the solid biofertilizer with different inclusion percentage of Bacillus by using the disk diffusion and dual layer agar methods.

[00140] v) By using the inverted plate method, the effects of volatile organic compounds (VOCs) from the solid biofertilizer (fermented frass supplemented with 1% exuviae and 1% Bacillus) were tested against Ganoderma boninense. The VOCs from the solid biofertilizer could inhibit 53% of the Ganoderma boninense.

[00141] vi) Different concentrations of solid biofertilizer extract were tested against the spores of Fusarium oxysporum sp. Cubense TR4, to determine the MIF and MFC of the solid biofertilizer. The minimum concentration of the extract that produces at least 60% inhibitory activity against TR4 was selected as the MIC of the solid biofertilizer, while the minimum concentration of the extract that could kill 99% of the fungal spores was selected as the MFC of the solid biofertilizer.

[00142] Table 5: Fusarium oxysporum sp. Cubense TR4 spores inhibition by the extract of the solid biofertilizer at different concentrations.

[00143] vii) Different concentrations of sterile solid biofertilizer extract were tested against the spores of Fusarium oxysporum sp. Cubense TR4, to determine the MIF and MFC of the solid biofertilizer. The minimum concentration of the extract that produces at least 60% inhibitory activity against TR4 was selected as the MIC of the solid biofertilizer, while the minimum concentration of the extract that could kill 99% of the fungal spores was selected as the MFC of the solid biofertilizer.

[00144] Table 6: Fusarium oxysporum sp. Cubense TR4 spores inhibition by the sterile extract of the solid biofertilizer at different concentrations.

[00145] viii) Different concentrations of solid biofertilizer extract were tested against the mycelium of Ganoderma boninense, to determine the MIF and MFC of the solid biofertilizer. The minimum concentration of the extract that produces at least 60% inhibitory activity against G. boninense was selected as the MIC of the solid biofertilizer, while the minimum concentration of the extract that could kill 99% of the fungal mycelium was selected as the MFC of the solid biofertilizer.

[00146] Table 7: G. boninense mycelium inhibition by the extract of the solid biofertilizer at different concentrations.

[00147] vii) Different concentrations of sterile solid biofertilizer extract were tested against the mycelium of Ganoderma boninense, to determine the MIF and MFC of the solid biofertilizer. The minimum concentration of the sterile extract that produces at least 60% inhibitory activity against G. boninense was selected as the MIC of the solid biofertilizer, while the minimum concentration of the sterile extract that could kill 99% of the fungal mycelium was selected as the MFC of the solid biofertilizer.

[00148] Table 8: G. boninense mycelium inhibition by the sterile extract of the solid biofertilizer at different concentrations.

[00149] Example 9: In-vitro fungal pathogen inhibition by liquid biofertilizer

[00150] i) By using the disk diffusion method, the antifungal activity of the liquid biofertilizer was tested against Fusarium oxysporum sp. Cubense TR4 and Ganoderma boninense. The liquid biofertilizer consists of exuviae, fermented frass and water at a ratio of 1:4: 16 (w/w/v) and 0.1 % of Bacillus.

[00151] Table 9: Phytopathogenic fungi inhibition by the liquid biofertilizer.

[00152] ii) By using the inverted plate method, the effects of volatile organic compounds (VOCs) from the liquid biofertilizer (consist of exuviae, fermented frass and water at ratio of 1:4: 16 (w/w/v) and 0.1 % of Bacillus) were tested against Ganoderma boninense. The VOCs from the liquid biofertilizer could inhibit 24% of the Ganoderma boninense. [00153] iii) Different concentrations of liquid biofertilizer were tested against the spores of Fusarium oxysporum sp. Cubense TR4, to determine the MIF and MFC of the liquid biofertilizer. The minimum concentration of the liquid biofertilizer that produces at least 60% inhibitory activity against TR4 was selected as the MIC of the liquid biofertilizer, while the minimum concentration that could kill 99% of the fungal spores was selected as the MFC of the liquid biofertilizer.

[00154] Table 10: Fusarium oxysporum sp. Cubense TR4 spores inhibition by the liquid biofertilizer at different concentrations.

[00155] vii) Different concentrations of sterile liquid biofertilizer were tested against the spores of Fusarium oxysporum sp. Cubense TR4, to determine the MIF and MFC of the liquid biofertilizer. The minimum concentration that produces at least 60% inhibitory activity against TR4 was selected as the MIC of the liquid biofertilizer, while the minimum concentration that could kill 99% of the fungal spores was selected as the MFC of the liquid biofertilizer.

[00156] Table 11: Fusarium oxysporum sp. Cubense TR4 spores inhibition by the sterile extract of the liquid biofertilizer at different concentrations.

[00157] viii) Different concentrations of liquid biofertilizer were tested against the mycelium of Ganoderma boninense, to determine the MIF and MFC of the liquid biofertilizer. The minimum concentration that produces at least 60% inhibitory activity against G. boninense was selected as the MIC of the liquid biofertilizer, while the minimum concentration that could kill 99% of the fungal mycelium was selected as the MFC of the liquid biofertilizer.

[00158] Table 12: G. boninense mycelium inhibition by the liquid biofertilizer at different concentrations. [00159] vii) Different concentrations of sterile liquid biofertilizer were tested against the mycelium of Ganoderma boninense, to determine the MIF and MFC of the liquid biofertilizer. The minimum concentration that produces at least 60% inhibitory activity against G. boninense was selected as the MIC of the liquid biofertilizer, while the minimum concentration that could kill 99% of the fungal mycelium was selected as the MFC of the liquid biofertilizer.

[00160] Table 13: G. boninense mycelium inhibition by the sterile liquid biofertilizer at different concentrations.

[00161] Example 10: Field trials

[00162] Cavendish banana cultivars were used for this trial for the resistance against Fusarium oxysporum f.sp. cubense Tropical Race 4. Foe Race 4 is found affecting Cavendish plants in cooler subtropical climates or in plants suffering from some form of stress. A more aggressive Foe race strain could cause severe disease in Cavendish plants even under optimal growing conditions in the tropics

[00163] The trial design for this test was as follows: [00164] Treatment 1: The soil was mixed with 1.6% (w/w) of solid biofertilizer and 5% (v/w) of liquid biofertilizer before plantlet planting. Then, another 0.2% (w/w) of solid biofertilizer and 2.5% (v/w) of liquid biofertilizer were applied as top dressing and soil drenching, respectively, two times within a 3 weeks interval.

[00165] Treatment 2: The soil was mixed with 2% (w/w) of solid biofertilizer and 5% (v/w) of liquid biofertilizer before plantlet planting. Then, another 0.38% (w/w) of solid biofertilizer and 2.5% (v/w) of liquid biofertilizer were applied as top dressing and soil drenching, respectively, two times within a 3 weeks interval.

[00166] Treatment 3: The soil was mixed with 2% (w/w) of solid biofertilizer and 5% (v/w) of liquid biofertilizer before plantlet planting. Then, another 0.38% (w/w) of solid biofertilizer and 2.5% (v/w) of liquid biofertilizer were applied as top dressing and soil drenching, respectively, two times within a 3 weeks interval.

[00167] The soil was not plowed or raked after frass application, as it is not preferred by the farmers (further plowing will make the soil become more compact and the banana seedlings roots may not grow well in compacted soil).

[00168] Treatment 4: The soil was treated with fungicide(Benomyl) at a concentration of 5 x 10’ ⁴ g/mL as positive control.

[00169] Treatment 5: The soil was inoculated with focTR4 spores, but there was no treatment for the soil.

[00170] Treatment 6: The soil was not inoculated with focTR4 spores, and no treatment for the soil.

[00171] The field trial was performed for 6 weeks with external and internal were observed and scored for disease index determination. [00172] Table 14: Leaf symptom index (LSI) and Rhizome discoloration index (RDI).

[00173] Table 15: Disease Index (DI) (%) based on RDI and the corresponding susceptibility ranking.

[00174] Example 11: Use of solid biofertilizer product in field conditions

[00175] The biofertilizer can be mixed with soil at a rate from 350 kg to 1000 kg per acres prior to planting African Oil Palm seedlings. After planting the mixture will be applied directly or as pellets to the surface layer of soil at the base of the plant once every two weeks. It can be performed by using hand shovels, spreaders or an aerator machine that spreads the biofertilizer uniformly and consistently over the soil or turf.

[00176] The seedlings treated with the biofertilizer product are expected over the course of three years to have a 60-90% lower rate of basal stem rot (BSR) disease relative to control plants. For those plants affected by BSR the disease severity index is expected to be lower by a statistically significant degree than plants that did not receive the biofertilizer product. [00177] Example 12: Use of the liquid product in field conditions

[00178] The liquid extract of the biofertilizer can be prepared by mixing in the ratio of 1:30 (product: dechlorinated water) followed by filling in a backpack sprayer. The applying step can include spraying or misting the liquid extract onto leaves, shoots, or roots of a tomato crop (Solarium lycopersicum) or spraying at soil layer at the base of the plant once every two weeks in the vegetative stage and once every week during the fruiting stage.

[00179] It is expected that the plants receiving the liquid biofertilizer product will have a 60-90%lower incidence of Fusarium Wilt disease over the course of a growing season relative to control plants. For those plants that received the liquid biofertilizer treatment but were infected by fusarium wilt it is expected that the disease severity index for those plants would be lower by a statistically significant degree than plants that did not receive the liquid biofertilizer product.

[00180] Example 13: Use of biofertilizer for solid product in field conditions

[00181] The biofertilizer was applied to at the base of Cucumber (Cucumis sativus) seedlings approximately 11 days after germination at the time the plants are transplanted to the field. The product was applied at the base of seedlings as a top dressing with an initial application of 20 g of product per plant. Thereafter 40 g of product was top dressed to the field plot on a weekly basis for 5 weeks. A control group of plants used 5 g of fermented chicken manure per plant as the initial dose and 10 g per week thereafter. The plants were monitored for stem rot diseases (typically caused by Fusarium sp. ) on a weekly basis a simple measure of the percentage of plants with stem rot disease was recorded.

[00182] One week 2 the treatment group had a stem rot disease incidence of 0% compared to the control which had an incidence of 50%. On week 3 conditions of both groups had not changed. On week 4 the treatment group had a 20% disease incidence compared to the control which had 70% incidence. On the fifth and final week of the trial the treatment group had a 70% incidence of stem rot disease, and the control group had an incidence of 100%.

[00183] These results showed dramatic improvement in resistance to plant pathogen disease in plants using the biofertilizer versus untreated control plants.

[00184] Example 14: Use of the liquid product in field conditions

[00185] The liquid extract of the biofertilizer was used in field conditions to cultivate

Amaranth (Amaranthus dubius). The amaranth was sown, and the liquid product was applied to the young plants from the 15th day after sowing until the 43rd day. The liquid extract of the biofertilizer was diluted for field application by mixing in the ratio of 1: 10 (product: dechlorinated water) followed by filling in a backpack sprayer. The backpack sprayer was then used to spray the liquid extract onto leaves, shoots, and the root zone of the amaranth on a daily basis. Root lengths, stem girth, leaf size and pest incidence were recorded for the treatment and for a control without product application.

[00186] The plants were harvested 43 days after initial sowing. The treatment plants had a higher stem girth than the control (0.92 ± 0.04cm vs 0.88 ± 0.04cm) and a larger leaf size (6.68± 0.29 cm diameter vs 6.44± 0.37 cm diameter in the control). Additionally, the treatment plants had longer roots compared to the control (21.33 ± 2.0cm vs 18.39 ± 1.28). Finally, the qualitative assessment revealed that no pests or disease symptoms were found in treatment plants whereas 15% of control plants exhibited unidentified disease symptoms.

[00187] These results show significant improvement in plant health and disease resistance in plants using the liquid biofertilizer versus untreated control plants.

[00188] Example 15: Use of the liquid product in field conditions [00189] Moris pineapple cultivar will be used for this trial for the resistance against Chalara paradoxa, also known as black rot of pineapple. C. paradoxa is found affecting pineapple plants in warm and wet weather. This fungus commonly infects plants through fresh wounds occurring where the planting material has been detached from the parent plant.

[00190] The liquid extract of the biofertilizer will be diluted for field application by mixing 30 mL of product in 1000 mL dechlorinated water, followed by filling in a backpack sprayer. The backpack sprayer will be used to spray the liquid extract onto leaves, shoots, and the root zone of the pineapple plants.

[00191] The plants treated with the biofertilizer product are expected over the course of two years to have a 60-90% lower rate of black rot or soft rot disease relative to control plants. For those plants affected, the disease severity index is expected to be lower by a statistically significant degree than plants that did not receive the biofertilizer product.

[00192] In the above detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be used, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein. [00193] The present disclosure is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of various aspects. Many modifications and variations can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[00194] As used in this document, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Nothing in this disclosure is to be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by virtue of prior invention. As used in this document, the term “comprising” means “including, but not limited to.”

[00195] While various compositions, methods, and devices are described in terms of "comprising" various components or steps (interpreted as meaning "including, but not limited to"), the compositions, methods, and devices can also "consist essentially of" or "consist of" the various components and steps, and such terminology should be interpreted as defining essentially closed-member groups.

[00196] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

[00197] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (for example, bodies of the appended claims) are generally intended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (for example, “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (for example, the bare recitation of "two recitations," without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (for example, “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.” [00198] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. [00199] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[00200] Various of the above-disclosed and other features and functions, or alternatives thereof, may be combined into many other different systems or applications. Various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art, each of which is also intended to be encompassed by the disclosed embodiments.