BR112019009924B1 - METHOD AND COMPOSITION FOR CONTROLLING PLANT NEMATOID PESTS - Google Patents

Abstract

The present invention relates to materials and method for controlling pests, in particular, nematodes. The invention also relates to compositions comprising biosurfactants such as pesticides.

Description

CROSS REFERENCE WITH RELATED APPLICATION

[001] This application claims the benefit of the United States provisional patent application, Serial No. U.S. 62/422,918, filed November 16, 2016, which is incorporated herein into this patent application by reference in its entirety.

BACKGROUND OF THE INVENTION

[002] In order to increase yields and protect crops against pathogens, pests, and disease, farmers have relied heavily on the use of synthetic chemicals and chemical fertilizers; however, when used excessively or when applied inappropriately, these substances can run into surface waters, run off into groundwater, and evaporate into the air. These substances are increasingly scrutinized as sources of air and water pollution, making their responsible use an ecological and commercial imperative. Even when used appropriately, excessive dependence and long-term use of certain chemical fertilizers and pesticides can deleterious changes to the soil ecosystem, reduce stress tolerance, increase pest resistance, and impede the growth and vitality of plants. plants and animals.

[003] Nematodes are a class of roundworms or filiform worms of the phylum Nemtoda. Examples in the class are the cyst-forming nematodes of the genera Heterodera (e.g., H. glycines, H. avenae, and H. shachtii) and Globodera (e.g., G. grossochiens and G. pallida), the stubby root nematodes of genus Trichodorus, the bulb and stem nematodes of the genus Ditylenchus, the golden nematode, Heterodera Roschiensis, the root knot nematodes of the genus Meloidogyne (e.g., M. javanica, M. hapla, M. arenaria, and M. incognita ), the root lesion nematodes of the genus Pratylenchus (e.g., P. goodeyi, P. penetrans, Pos bractrvurus, P. zeae, P. coffeae, P. bractrvurus, and P. thornei), the citrus nematodes of the genus Tylenchulus, and the stinger nematodes of the genus Belonolaimus.

[004] Nematodes are known to infect both plants and animals. These microscopic worms can be found in almost any type of environment. When resident in the soil, nematodes feed on plant roots, causing significant damage to the root structure and inadequate plant development. Usually the damage is manifested by the growth of galls, root knots, and other abnormalities. The formation of galls leads to a reduction in root size and ineffectiveness of the root system, which in turn seriously affects other parts of the plant. As a result, the weakened plant becomes vulnerable to attack by other pathogens. Without proper treatment, the plant dies. Nematodes cause millions of dollars in damage each year to turf grasses, ornamental plants, and food crops.

[005] Root knot nematodes (Meloidogyne spp.) are one of the three genera of plant parasitic nematodes most economically damaging to horticultural and field crops. Root knot nematodes are distributed worldwide and are obligate parasites of the roots of thousands of plant species, including monocotyledonous and dicotyledonous, herbaceous and woody plants. Vegetable crops growing in hot climates can experience severe losses due to root knot nematodes, and are often routinely treated with a chemical nematicide. Root knot nematode damage results in poor growth, a decline in crop quality and yield, and reduced resistance to other stresses (e.g., drought, other diseases). A high level of damage can lead to total crop loss. For example, approximately 1.5 and a half billion dollars per year is lost due to soybean cyst nematodes alone.

[006] Conventional nematicides used to control nematodes are applied to the seed furrow at the time of planting. Due to toxicity to nearby animals such as birds, aerial center pivots with liquid applications of toxic compounds such as Nemacur, Temik, Furadan, Dazinat and Mocap have fallen into disuse.

[007] Since the sixties, methyl bromide has been used by producers to effectively sterilize fields before planting, essentially to control nematodes, as well as to treat diseases and weeds; However, as this toxic compound is used in gas form, more than half of the amount injected into the soil may eventually end up in the atmosphere and contribute to the depletion of the ozone layer. In 2005, developed countries banned methyl bromide under the Montreal Protocol, which is an international treaty signed in 1987 to protect the stratospheric ozone layer.

[008] In accordance with the prohibition, the treaty allows the limited use of methyl bromide on strawberries, almonds, and other crops which lack alternatives for both effective and affordable control of nematodes, disease, and weeds. The extent of authorized use decreases each year and will likely end soon. Therefore, it is a priority for producers and regulatory agencies to discover alternatives to methyl bromide; however, no single product provides the broad spectrum of control offered by methyl bromides.

[009] The assembly of regulatory mandates governing the availability and use of chemicals, as well as consumer demands for waste-free and sustainably grown food is impacting the industry and causing an evolution of thinking regarding how to treat a myriad of of challenges. Although the indiscriminate elimination of chemicals is not feasible at this time, farmers are increasingly embracing the use of biological measures as viable components of Integrated Nutrient Management and Integrated Pest Management programs.

[0010] Due to the disadvantages of the main approaches described above, the demand for safer pesticides and alternative pest control strategies is growing. Particularly in recent years, biological control of nematodes has gained great interest. This method uses biological agents, such as live microbes, bio-products derived from these microbes, and combinations thereof such as pesticides. These biological pesticides have important advantages over other conventional pesticides. For example, they are less harmful compared to conventional chemical pesticides. They are more efficient and specific, and often biodegrade quickly, leading to less environmental pollution.

[0011] The use of biopesticides and other biological agents has been greatly limited due to difficulties in production, transportation, administration, evaluation and effectiveness. For example, many microbes are difficult to cultivate and subsequently deploy into agricultural and forestry production systems in sufficient quantities to be useful. This problem is exacerbated by losses in viability and/or activity due to processing, formulation, storage, stabilization prior to distribution, sporulation of vegetative cells as a means of stabilization, transportation, and application. Furthermore, once applied, biologics may fail to develop for any number of reasons including, for example, insufficient initial cell densities, the inability to compete effectively with existing microflora at a particular location, and being introduced. in soil and/or other environmental conditions in which the microbe cannot develop or even survive.

[0012] Therefore, there is an urgent need to develop methods and materials for the production of effective biopesticides for nematode control.

BRIEF SUMMARY

[0013] The present invention provides compositions and methods for controlling nematodes. Furthermore, the present invention provides methods and compositions for preventing crop damage by nematodes, thereby resulting in increased production. Advantageously, the pesticides according to the present invention use non-toxic substances, such as microbes and by-products of microbial cultivation.

[0014] In one embodiment, the present invention provides methods for preventing nematode damage and for controlling nematodes, comprising the steps of: culturing a microorganism that produces a byproduct of microbial growth and contacting the nematodes, or their environment, with an effective amount of the microbe and/or the byproduct of microbial growth.

[0015] In one embodiment, the byproduct of microbial growth is a biosurfactant. In one embodiment, the biosurfactant is a glycolipid such as a rhamnolipid, a sophorolipid (SLP), a trehalose lipid or a mannosylerythritol lipid (MEL). In particularly preferred embodiments the biosurfactant is an SLP and/or a MEL.

[0016] The microbe-based nematicidal compositions of the present invention can be obtained through cultivation processes ranging from small scale to large scale. These cultivation processes include, but are not limited to, submerged cultivation/fermentation, surface cultivation, solid state fermentation (SSF), and combinations thereof. The nematicidal composition can be, for example, the fermentation broth and/or purified biosurfactants.

[0017] In some embodiments, the biosurfactant-producing microorganisms in the composition can be cultivated on site and produce the biosurfactants for direct use to control nematodes. Consequently, a high concentration of biosurfactant and/or biosurfactant-producing microorganisms at a treatment site (e.g. in soil) can be obtained easily and continuously.

[0018] In one embodiment, the system according to the present invention comprises an additional aspect providing a field application of microbe-based products. The microbial-based products of the present invention can be applied, for example, via an irrigation system, as a spray, as a seed treatment, to the soil surface, to plant surfaces, and/or to pest surfaces. . Mechanical application through conventional implements or robotic application through aerial or land-based "drones" is also facilitated.

[0019] The nematicidal composition can be used to protect plants, humans, or animals by controlling nematodes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 shows the reduction in nematode motility after 3 days of treatment under various conditions: SD10 at 0.05% (in vol/vol), 0.125% (in vol/vol), 0.25% (in vol/vol), 0.25% (in vol/vol), 0.25% (in vol/vol), 0.25% (in vol/vol), 0.25% (in vol/vol), in vol/vol), and 0.5% (in vol/vol), respectively; SD12 at 0.025% (by vol/vol), 0.05% (by vol/vol), 0.1% (by vol/vol), and 0.2% (by vol/vol), respectively; Nimitz® at 10 ppm ai; and unhandled verification. SD10 refers to SLP treatment. SD12 refers to MEL treatment.

[0021] Figure 2 shows the mortality rate of nematodes after 3 days of treatment under various conditions: SD10 at 0.05% (in vol/vol), 0.125% (in vol/vol), 0.25% (in vol/vol) in vol/vol), and 0.5% (in vol/vol), respectively; SD12 at 0.025% (by vol/vol), 0.05% (by vol/vol), 0.1% (by vol/vol), and 0.2% (by vol/vol), respectively; Nimitz® at 10 ppm ai; and unhandled verification.

[0022] Figure 3 shows live nematode counts after 3, 14, and 23 days of various soil treatments in pathology laboratory flask tests: SD10 at 0.25% (in vol/vol) and 0. 5% (in vol/vol); SD12 at 0.1% (by vol/vol) and 0.2% (by vol/vol); SD10 (0.25% vol / vol/) + SD12 (0.1% vol / vol); Nimitz® at 10 ppm pr; and unhandled verification.

[0023] Figure 4 shows control of live nematodes (i.e., nematode death) after 3, 14, and 23 days of various soil treatments in pathology laboratory vial tests: SD10 at 0.25% (in vol/vol) and 0.5% (in vol/vol); SD12 at 0.1% (by vol/vol) and 0.2% (by vol/vol); SD10 (0.25% vol / vol/) + SD12 (0.1% vol / vol); Nimitz® at 10 ppm pr; and unhandled verification.

[0024] Figure 5 shows non-motile nematode counts after 3, 14, and 23 days of various soil treatments in pathology laboratory flask tests: SD10 at 0.25% (in vol/vol) and 0 .5% (in vol/vol); SD12 at 0.1% (by vol/vol) and 0.2% (by vol/vol); SD10 (0.25% vol / vol/) + SD12 (0.1% vol / vol); Nimitz® at 10 ppm pr; and unhandled verification.

[0025] Figure 6 shows live non-motile nematode counts after 3, 14, and 23 days of various soil treatments in pathology laboratory flask tests: SD10 at 0.25% (in vol/vol) and 0.5% (in vol/vol); SD12 at 0.1% (by vol/vol) and 0.2% (by vol/vol); SD10 (0.25% vol / vol/) + SD12 (0.1% vol / vol); Nimitz® at 10 ppm pr; and unhandled verification.

[0026] Figure 7 shows the rate of gall formation at harvest for a microplot of cucumbers treated with 5 different treatments: untreated check; Nimitz® at 5 pt/a; SD12 A 0.2% (ai/v) single treatment; SD12 A 0.2% (ai/v) double treatment; SD12 A 0.2% (ai/v) triple treatment.

[0027] Figure 8 shows the nematode count at harvest for a microplot of cucumbers treated with 5 different treatments: untreated check; Nimitz® at 5 pt/a; SD12 A 0.2% (ai/v) single treatment; SD12 A 0.2% (ai/v) double treatment; SD12 A 0.2% (ai/v) triple treatment.

[0028] Figure 9 shows a plot configuration for evaluating the effectiveness of the nematode attractant, including locations of the nematode inoculation zone and attractant application zone.

[0029] Figure 10 shows the percentage of infestation (of the total portion of the nematode population) in three different locations in plots for evaluating nematode attraction. "Center" refers to the center of the inoculation zone, "attraction" refers to the attraction zone, and "untreated" refers to the areas of all other plots.

DETAILED DESCRIPTION

[0030] The present invention provides materials and methods for controlling nematodes. The present invention also provides compositions that can be used as pesticides for controlling nematodes. The present invention further provides methods for preparing similar compositions. Furthermore, the present invention provides methods and compositions for preventing crop damage by nematodes, thereby resulting in increases in production.

[0031] The microbe-based products of the present invention can be used in scenarios including, but not limited to, crops, livestock, forestry, turf management, pastures, and human and animal health.

[0032] In one embodiment, the composition for controlling nematodes according to the present invention comprises an effective amount of a microbial biosurfactant and/or a microorganism producing said biosurfactant.

[0033] In one embodiment, the nematode control method comprises the steps of obtaining a microbial biosurfactant, and applying an effective amount of the microbial biosurfactant to nematodes or their location.

[0034] The compositions according to the present invention can be obtained, for example, by cultivation processes ranging from small scale to large scale. These cultivation processes include, but are not limited to, submerged cultivation/fermentation, surface cultivation, solid state fermentation (SSF), and combinations thereof.

[0035] The fermentation product containing the microbial biosurfactant can be used directly for nematode treatments without extraction or purification. If desired, extraction and purification of biosurfactants can be carried out as described here, in this patent application. Advantageously, the cultivation method according to the invention is capable of producing a high concentration of microorganisms and a high concentration of biosurfactant.

[0036] In certain embodiments, the methods and compositions according to the present invention reduce damage caused by nematodes, compared to an untreated plant, by about 5%, 10%, 20%, 30%, 40%, 50 %, 60% 70%, 80%, or 90% or more.

[0037] In one embodiment, the present invention provides a method for increasing crop or plant yields. In certain embodiments, the methods and compositions according to the present invention increase crop production by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. % or more compared to an untreated plant.

[0038] In one embodiment, the methods of the present invention reduce the number of nematode eggs in the roots of a plant by about 5%, 10%, 20%, 30%, 40%, 50%, 60% 70%, 80%, or 90% or more compared to an untreated plant.

[0039] In another embodiment, the present invention provides a method for increasing the weight or size of the plant relative to an untreated plant.

Selected Definitions

[0040] As used herein in this patent application, the term "control" used in reference to the activity produced by biosurfactants or biosurfactant-producing microorganisms extends to the act of killing, incapacitating or immobilizing pests or otherwise rendering pests pests substantially incapable of causing harm.

[0041] As used herein in this patent application, "nematicicide" means having the ability to control nematodes. Therefore, for example, killing nematodes, reducing their motility, and reducing egg counts are all examples of nematicidal activity.

[0042] As used herein in this patent application, reference to a "microbe-based composition" means a composition comprising components that have been produced as a result of the growth of microorganisms or other cell cultures. In this way, the microbe-based composition may comprise the microbes themselves and/or by-products of microbial growth. Microbes can be in a vegetative state, in spore form, in mycelial form, in any other propagule form, or a mixture of these. Microbes can be planktonic or in the form of a biofilm, or a mixture of both. Growth byproducts may be, for example, metabolites, cell membrane components, expressed proteins, and/or other cellular components. The microbes may be intact or may be lysed. In preferred embodiments, the microbes are present, with broth in which they were cultivated, in the microbe-based composition. The cells may be present at, for example, a concentration of 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x 1010, or 1 x 1011 or more propagules per milliliter of the composition. As used herein in this patent application, a propagule is any portion of a microorganism from which a new and/or mature organism can develop, including, but not limited to, cells, spores, mycelia, buds and seeds .

[0043] The present invention additionally provides "microbe-based products," which are products that must be applied in practice to obtain a desired result. The microbe-based product may simply be the microbe-based composition harvested from the microbe cultivation process. Alternatively, the microbe-based product may comprise additional ingredients that have been added. These additional ingredients may include, for example, stabilizers, buffers, appropriate carriers such as water, saline solutions, or any other appropriate carrier, nutrients added to support additional microbial growth, non-nutrient growth enhancers such as plant hormones, and/or agents that facilitate the tracking of microbes and/or composition in the environment to which it is applied. The microbe-based product may also comprise mixtures of microbe-based compositions. The microbial-based product may also comprise one or more components of the microbial-based composition that have been processed in some manner such as, but not limited to, filtration, centrifugation, lysis, drying, purification and the like.

Microorganisms

[0044] Microbial biosurfactants are compounds produced by a variety of microorganisms such as bacteria, fungi, and yeast.

[0045] In one embodiment, the microorganisms are bacteria including gram-positive and gram-negative bacteria. These bacteria may include, but are not limited to, for example, Escherichia coli, Rhizobium (e.g., Rhizobium japonicum, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium leguminosarum biovar trifolii, and Rhizobium etli), Bradyrhizobium (e.g., B. japanicum, and B. parasponia), Bacillus (e.g., B. subtilis, B. firmus, B. laterosporus, B. pumillus, B. cereus, B. licheniformis, B. megaterium, and B. amyloliquifaciens), Azobacter (e.g., A. vinelandii, and A. chroococcum), Arthrobacter (e.g., A. pascens), Agrobacterium (e.g., A. radiobacter), Pseudomonas (e.g., P. chlororaphis subsp. aureofaciens (Kluyver), P. aeruginosa, P. .putida, P. florescens, P. fragi, and P. syringae), Flavobacterium spp.; Azospirillium (e.g. A. brasiliensis), Azomonas, Derxia, Beijerinckia, Nocardia, Klebsiella, Clavibacter (e.g. C. xyli subsp. xyli and C. xyli subsp. cynodontis), Cyanobacteria, Pantoea (e.g. P.agglomerans ), Sphingomonas (e.g., S. paucimobilis), Streptomyces (e.g., S. griseochromogenes, S. qriseus, S. cacaoi, S. aureus, and S. kasugaenis), Streptoverticillium (e.g., S. rimofaciens), Ralslonia (e.g. R. eulropha), Rhodospirillum (e.g. R.rubrum), Xanthomonas (e.g. X. campestris), Erwinia (e.g. E. carotovora), Clostridium (e.g. C. butyricum, C. tyrobutyricum, C. acetobutyricum, C. beijerinckii, C. bravidaciens, and C. malacusomae), Rhodococcus, Campylobacter, Cornybacterium, and combinations thereof.

[0046] In another embodiment, the microorganism is a yeast. A number of yeast species are suitable for production in accordance with the present invention, including, but not limited to, Saccharomyces (e.g., S. cerevisiae, S. boulardii sequelae and S. torula), Debaromyces, Issalchenkia, Candida (C albicans, C. rugosa, C. tropicalis, C. lipolytica, and C. torulopsis), Kluyveromyces (e.g., K. lactis, K. fragilis), Pichia (e.g., Pichia pastoris), Wickerhamomyces, Starmerella (e.g. , Starmerella bombicola), and/or combinations thereof.

[0047] In one embodiment, the yeast is a killer yeast. As used herein, in this patent application, "killer yeast" means a strain of yeast characterized by its secretion of toxic proteins or glycoproteins, to which the strain itself is immune. The exotoxins secreted by killer yeasts have the ability to destroy other strains of yeast, fungi, or bacteria. For example, microorganisms that can be controlled by killer yeast include Fusarium and other filamentous fungi. Examples of killer yeasts according to the present invention are those that can be used safely in the food and fermentation industries, for example, beer, wine, and bread making; those that can be used to control other microorganisms that can contaminate the aforementioned production processes; those that can be used in biocontrol for food preservation; those that can be used to treat fungal infections in both humans and plants; and those that can be used in recombinant DNA technology. Said yeasts may include, but are not limited to, Wickerhamomyces, Pichia (e.g., Pichia anomala, Pichia guielliermondii, Pichia kudrizvzevii) Hansenula, Saccharomyces, Hanseniaspora, such as Hanseniaspora uvarum, Ustilago maydis, Debaryomyces hansenii, Candida, Cryptococcus, Kluyveromyces , Torulopsis, Ustilago, Williopsis, Zygosaccharomyces, such as Zygosaccharomyces bailii, and others.

[0048] In one embodiment, the microorganisms are fungi, including, but are not limited to, Mycorrhiza (e.g., vesicular-arbuscular mycorrhizal (VAM), arbuscular mycorrhizal (AM)), Mortierella, Phycomyces, Blakeslea, Thraustochytrium, Penicillium, Phythium, Entomophthora, Aureobasidium pullulans, Fusarium venenalum, Aspergillus, Trichoderma (e.g. T. reesei, T. harzianum, T. viride and T. hamatum), Rhizopus spp, endophytic fungi (e.g. Piriformis indica) and combinations of the same.

[0049] In an additional embodiment, the microorganisms are mycorrhizal fungi such as Glomus spp. and Acaulospora spp. In one embodiment, the microorganism is arbuscular mycorrhizal fungus (AMF).

[0050] In specific embodiments, microbes for producing SLPs can be Candida sp., Starmerella sp., Cryptococcus sp., Cyberlindnera samutprakarnensis JP52 (T), Pichia sp., Rhodotorula sp., or Wickerhamiella sp.

[0051] In additionally specific embodiments, microbes for producing MELs can be Pseudozyma sp., Candida sp., Ustilago sp., Schizonella sp., or Kurtzmanomyces sp.

[0052] In preferred embodiments, the microbes are yeast. In specific embodiments, the yeasts are Starmerella, Pichia, and/or Pseudozyma. Specifically exemplified here, in this patent application, are Starmerella bombicola, Pichia anomala (Wickerhamomyces anomalus) and Pseudozyma aphidis.

Biosurfactants

[0053] Biosurfactants form an important class of secondary metabolites that occur in many microorganisms. Biosurfactants are biodegradable and can be produced easily and cheaply using selected organisms on renewable substrates. Most biosurfactant-producing organisms produce biosurfactants in response to the presence of a hydrocarbon source (e.g., oils, sugar, glycerol, etc.) in the growth medium. Other components of the medium can also significantly affect the production of biosurfactants. For example, the production of rhamnolipids by the bacteria Pseudomonas aeruginosa can be increased if nitrate is used as a nitrogen source instead of ammonium. Likewise, the concentration of iron, magnesium, sodium, and potassium; the ratio of carbon : phosphorus; and agitation can greatly affect rhamnolipid production.

[0054] Biosurfactants include low molecular weight glycolipids (GLs), lipopeptides (LPs), flavolipids (FLs), phospholipids, and high molecular weight polymers such as lipoproteins, lipopolysaccharide and protein complexes, and polysaccharide-protein complexes. fatty acids. The common lipophilic portion of a biosurfactant molecule is the hydrocarbon chain of a fatty acid, whereas the hydrophilic portion is formed by ester or alcohol groups of neutral lipids, the carboxylate group of fatty acids, or amino acids (or peptides). , organic acid in the case of flavolipids, or, in the case of glycolipids, by the carbohydrate.

[0055] According to embodiments of this invention, the biosurfactant has the ability to penetrate through the tissues of pests and is effective to reduce quantities without the use of adjuvants. It was seen that at concentrations above the critical micelle concentration, biosurfactants have the ability to penetrate treated objects more effectively.

[0056] The development of the natural biopesticides of the present invention, which can be produced in high quantities, represents a significant advance in the art. Pests can be controlled using either biosurfactant-producing organisms as a biocontrol agent or by biosurfactants themselves. Furthermore, pest control can be carried out through the use of specific substrates to support the growth of biosurfactant-producing organisms as well as to produce biosurfactant pesticide agents. Advantageously, natural biosurfactants have the ability to inhibit the growth of competing organisms and to enhance the growth of organisms producing specific biosurfactants.

[0057] Furthermore, these biosurfactants can play important roles in the treatment of animal and human diseases. Animals can be treated, for example, by dipping or bathing in an isolated biosurfactant solution, with or without microbial cell mass, and/or in the presence of other compounds such as copper or zinc.

Methods for Cultivating Microbes

[0058] The present invention provides methods for cultivating microorganisms and producing biosurfactants and/or other byproducts of microbial growth. These cultivation processes include, but are not limited to, submerged cultivation/fermentation, surface cultivation, solid state fermentation (SSF), and combinations thereof.

[0059] Microbial cultivation systems will typically use submerged cultivation fermentation; however, hybrid and surface cropping systems can also be used. As used herein in this patent application, "fermentation" refers to the growth of cells, spores, mycelia and/or other microbial propagules under controlled conditions. Growth can be aerobic or anaerobic.

[0060] The microbial growth vessel used according to the present invention can be any fermenter or cultivation reactor for industrial use. The cultivation process is carried out in a vessel that can be, for example, conical or tubular. In one embodiment, the vessel may optionally have functional controls/sensors or may be connected to functional controls/sensors to measure important factors in the cultivation process, such as pH, oxygen, pressure, temperature, agitator shaft power, humidity, viscosity and /or microbial density and/or metabolite concentration.

[0061] In an additional embodiment, the vessel may also have the ability to monitor the growth of microorganisms within the vessel (for example, measuring the number of cells and growth phases). Alternatively, a daily sample may be taken from the vessel and subjected to enumeration by techniques known in the art, such as the plate dilution technique. Plaque dilution is a simple technique used to estimate the number of bacteria in a sample. The technique can also provide an index by which different environments or treatments can be compared.

[0062] In one embodiment, the method includes supplementing the crop with a source of nitrogen. The nitrogen source can be, for example, potassium nitrate, ammonium nitrate, ammonium sulfate, ammonium phosphate, ammonia, urea, and/or ammonium chloride. These nitrogen sources can be used independently or in a combination of two or more.

[0063] The method can provide oxygenation of the growth culture. One embodiment uses slow air movement to remove air containing low levels of oxygen and introduce oxygenated air. Oxygenated air can be ambient air supplemented daily through mechanisms including impellers for mechanical agitation of the liquid, and air sprinklers for supplying gas bubbles to the liquid for dissolving oxygen within the liquid.

[0064] The method may additionally comprise supplementing the crop with a carbon source. The carbon source is typically a carbohydrate, such as glucose, sucrose, lactose, fructose, trehalose, mannose, mannitol, and/or maltose; organic acids, such as acetic acid, fumaric acid, citric acid, propionic acid, malic acid, malonic acid, and/or pyruvic acid; alcohols, such as ethanol, propanol, butanol, pentanol, hexanol, isobutanol, and/or glycerol; fats and oils such as soybean oil, canola oil, coconut oil, rice bran oil, olive oil, corn oil, sesame oil, and/or linseed oil; etc. These carbon sources can be used independently or in a combination of two or more.

[0065] In one embodiment, growth factors and trace nutrients for microorganisms are included in the medium. This is particularly preferred when cultivating microbes, which are unable to produce all the vitamins they require. Inorganic nutrients, including trace elements such as iron, zinc, copper, manganese, molybdenum and/or cobalt may also be included in the medium.

[0066] In one embodiment, inorganic salts can also be included. Useful inorganic salts may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, manganese chloride, zinc sulfate, lead chloride, lead sulfate. copper, calcium chloride, calcium carbonate, and/or sodium carbonate. These inorganic salts can be used independently or in a combination of two or more.

[0067] In some embodiments, the method for cultivation may further comprise adding additional acids and/or antimicrobials to the liquid medium before, and/or during the cultivation process. Antimicrobial agents or antibiotics are used to protect the culture from contamination. Additionally, antifoaming agents can also be added to prevent the formation and/or accumulation of foam when gas is produced during cultivation.

[0068] The pH of the mixture must be suitable for the microorganism of interest. Buffers and pH regulators, such as carbonates and phosphates, can be used to stabilize the pH near a preferred value. When metal ions are present in high concentrations, it may be necessary to use a chelating agent in the liquid medium.

[0069] The method and equipment for cultivating microorganisms and producing microbial byproducts can be carried out in some batch, almost continuous, or continuous processes.

[0070] Microbes can be cultivated in planktonic form or as biofilm. In the case of biofilm, the vessel may have a substrate within it on which microbes can be grown in a biofilm state. The system may also have, for example, the ability to apply stimuli (such as shear stress) that stimulate and/or improve biofilm growth characteristics.

[0071] In one embodiment, the method for culturing microorganisms is carried out at about 5° to about 100° C, preferably 15 to 60° C, more preferably 25 to 50° C. In In an additional embodiment, cultivation can be carried out continuously at a constant temperature. In another modality, cultivation may be subject to temperature changes.

[0072] In one embodiment, the equipment used in the cultivation method and process is sterile. Cultivation equipment, such as the reactor/vessel, may be separate from, but connected to, a sterilization unit, for example, an autoclave. The cultivation equipment may also have a sterilization unit that sterilizes in situ before inoculating. Air may be sterilized by methods known in the art. For example, ambient air can pass through at least one filter before being introduced into the vessel. In other embodiments, the medium may be pasteurized or, optionally, no heat added, where the use of low water activity and low pH may be exploited to control bacterial growth.

[0073] The biomass content of the fermentation broth can be, for example, from 5 g/l to 180 g/l or more. In one embodiment, the solids content of the broth is from 10 g/l to 150 g/l.

[0074] In one embodiment, the method for producing a microbial metabolite according to the present invention comprises the steps of: 1) mixing hydrophobic and hydrophilic particles to form a matrix-forming solid substance; 2) contacting said matrix-forming solid substance with a medium inoculated with a microorganism of interest, thus creating a matrix of microreactors; and 3) growth of said microorganism within said micro-reactors.

[0075] The byproduct of microbial growth produced by microorganisms of interest can be retained in the microorganisms or secreted into the liquid medium. In another embodiment, the method for producing microbial growth byproduct may additionally comprise concentration and purification steps of the microbial growth byproduct of interest. In a further embodiment, the liquid medium may contain compounds that stabilize the activity of the microbial growth byproduct.

[0076] In one embodiment, the entire microbial cultivation composition is removed after completion of cultivation (e.g., after, for example, reaching a desired cell density, or a density of a specified metabolite in the broth). In this batch procedure, an entirely new batch is started after harvesting the first batch.

[0077] In another embodiment, only a portion of the fermentation product is removed at any time. In this modality, biomass with viable cells remains in the field as an inoculant for a new batch of cultivation. The composition that is removed may be a cell-free broth or may contain cells. In this way, an almost continuous system is created.

[0078] In one embodiment, the microbes are grown within 100, 50, 25, 10, 5, 1, or fewer miles of where the microbe-based product will be used.

Preparation of Microbe-Based Products

[0079] A microbe-based product of the present invention is simply the fermentation broth containing the microorganism and/or microbial metabolites (such as biosurfactants) produced by the microorganism and/or any residual nutrients. The fermentation product can be used directly without extraction, isolation, or purification. If desired, extraction, isolation and/or purification can be easily performed using routine methods or techniques described herein, in this patent application, and/or in the literature.

[0080] The microorganisms in the microbe-based product can be in an active or inactive form, in cell form, in spore form, and/or in mycelial form. Microbe-based products can be used without additional stabilization, preservation, and storage. Advantageously, the direct use of these microbe-based products preserves high viability of microorganisms, reduces the possibility of contamination by foreign agents and undesirable microorganisms, and maintains the activity of byproducts of microbial growth.

[0081] The microbes and/or broth (including distinct layers or fractions) resulting from microbial growth can be removed from the growth vessel and transferred, for example, through plumbing for immediate use.

[0082] In other embodiments, the composition (microbes, broth, or microbes and broth) may be placed within containers of appropriate size, taking into account, for example, the intended use, the method of application contemplated, the size of the tank of fermentation, and any mode of transportation from the microbial growth facility to the location of use. Thus, the containers into which the microbe-based composition is placed may be, for example, from 1 gallon to 1,000 gallons or more. In other embodiments, the containers are 2 gallons, 5 gallons, 25 gallons, or larger.

[0083] As used herein in this patent application, "broth" includes the entire broth or fractions of the entire broth.

[0084] After harvesting the microbe-based composition from the growth vessels, additional components may be added as the harvested product is placed into containers and/or piped (or otherwise transported for use). Additives may be, for example, buffers, carriers, other microbial-based compositions produced in the same or a different facility, viscosity modifiers, preservatives, pH modifiers, microbe growth nutrients, plant growth nutrients , tracking agents, pesticides, herbicides, animal feed, food products and other ingredients specific to an intended use.

[0085] Advantageously, according to the present invention, the microbe-based product can comprise broth in which the microbes have been cultivated. The product may be, for example, at least, by weight, 1%, 5%, 10%, 25%, 50%, 75%, or 100% broth. The amount of biomass in the product, by weight, can be, for example, anything from 0% to 100% inclusive of all percentages in between.

[0086] Optionally, the product can be stored before use. Storage time is short, so preferably. Therefore, the storage time can be less than 60 days, 45 days, 30 days, 20 days, 15 days, 10 days, 7 days, 5 days, 3 days, 2 days, 1 day, or 12 hours. In a preferred embodiment, if living cells are present in the product, the product is stored at a cold temperature such as, for example, less than 20° C, 15° C, 10° C, or 5° C. On the other hand, a biosurfactant composition typically can be stored for longer periods of time and at room temperature.

[0087] In one embodiment, the cultivation products can be prepared as a spray-dried biomass product. Biomass can be separated by known methods such as centrifugation, filtration, separation, decantation, a combination of separation and decantation, ultrafiltration or microfiltration. The biomass product can be separated from the growing medium and spray dried.

[0088] Microbe-based products can be formulated in a variety of ways, including liquid, solids, granular, powder, or slow release products by means that will be understood by those skilled in the art, having the benefit of the present disclosure.

[0089] For applications in humans or animals, the formulations can be prepared in liquid, paste, ointment, suppository, capsule or tablet forms and used in a manner similar to drugs used in the medicinal pharmaceutical industry. Formulations can be encapsulated using components known in the pharmaceutical industry. Encapsulation protects the components against undesirable reactions and helps the ingredients to resist adverse conditions in the environment or in the treated object or body e.g. the stomach.

[0090] The solid formulations of the invention can have different shapes and forms, such as cylinders, rods, blocks, capsules, tablets, pills, pellets, strips, staples, etc. Solid formulations can also be ground, granulated or pulverized. The granulated or pulverized material can be pressed into tablets or used to fill pre-made gelatin capsules or shells. Semi-solid formulations can be prepared in paste, wax, gel, or cream preparations.

[0091] The solid or semi-solid compositions of the invention can be coated using film coating compounds used in the pharmaceutical industry, such as polyethylene glycol, gelatin, sorbitol, gum, sugar or polyvinyl alcohol. This is particularly essential for tablets or capsules used in pesticide formulations. Film coating can protect the handler from coming into direct contact with the active ingredient in the formulations. Additionally, a bittering agent, such as denatonium benzoate or quassin, may also be incorporated into pesticide formulations, the coating, or both.

[0092] The compositions of the invention can also be prepared in powder formulations and filled into pre-made gelatin capsules.

[0093] The concentrations of ingredients in the formulations and the rate of application of the compositions can be widely varied depending on the pest, the plant or area treated, or the method of application.

[0094] In preferred embodiments, the composition for controlling nematodes according to the present invention comprises an effective amount of a microbial biosurfactant.

[0095] In one embodiment, the microbial biosurfactant is a glycolipid such as a rhamnolipid, sophorolipids (SLP), trehalose lipid and/or mannosylerythritol lipid (MEL).

[0096] In one embodiment, the composition for controlling nematodes according to the present invention comprises an effective amount of a single microbial biosurfactant and/or a single biosurfactant-producing microorganism. In another embodiment, the composition for controlling nematodes may comprise a mixture of microbial biosurfactants and/or a mixture of microorganisms producing these biosurfactants.

[0097] In one embodiment, the composition for controlling nematodes comprises SLP. SLP can be in a purified form or in a mixture of fermentation products. The composition preferably contains the active components, such as SLP, in concentrations of 0.01 to 90 weight % (wt%), preferably 0.1 to 50 wt %, and most preferably from 0.1 to 20% by weight. In another embodiment, purified SLP may be in combination with an acceptable carrier, wherein SLP may be presented in concentrations of 0.001 to 50% (by vol/vol), preferably 0.01 to 20% (by vol/vol). vol), more preferably from 0.02 to 5% (in vol/vol).

[0098] In one embodiment, the composition for controlling nematodes comprises MEL. HONEY can be in a purified form or in a fermentation product mixture. The composition preferably contains the active components, such as MEL, in concentrations of 0.01 to 90 weight % (wt%), preferably 0.1 to 50 wt %, and more preferably from 0.1 to 20% by weight. In another embodiment, purified MEL may be in combination with an acceptable carrier, wherein MEL may be presented in concentrations of 0.0001 to 50% (by vol/vol), preferably 0.005 to 20% (by vol/vol). vol), more preferably from 0.001 to 5% (in vol/vol).

[0099] In another embodiment, the composition for controlling nematodes comprises a mixture of SLP and MEL. SLP and MEL may be mixed in any proportion as long as the composition contains the active components, the combination of SLP and MEL, in a concentration of 0.01 to 90 weight % (wt%), preferably 0.1 to 50% by weight, and more preferably from 0.1 to 20% by weight. In another embodiment, purified MEL and SLP may be in combination with an accepted carrier, wherein SLP may be presented in concentrations of 0.0001 to 50% (in vol/vol), preferably 0.005 to 20% (in vol/vol). vol/vol), more preferably from 0.001 to 5% (in vol/vol).

[00100] In an additional embodiment, the composition comprises SLP and/or MEL in combination with a strain of SLP and/or MEL-producing microorganism, such as, for example, Starmerella, Wickerhamomyces anomalus or Pseudozymas species.

[00101] Biosurfactants can be used either alone or combined with other acceptable active or inactive components. These components may be, for example, an oil component such as cinnamon oil, clove oil, cottonseed oil, garlic oil, or rosemary oil; another natural surfactant, such as Yucca or Quillaja saponins; or the component may be an aldehyde such as cinnamic aldehyde. Other oils that can be used as a pesticide component or adjuvant include: almond oil, camphor oil, canola oil, castor oil, cedar oil, citronella oil, citrus oil, coconut oil, corn oil , eucalyptus oil, fish oil, geranium oil, lecithin, lemongrass oil, linseed oil, mineral oil, mint or peppermint oil, olive oil, pine oil, kola oil, safflower oil, sage oils, sesame seed oil, sweet orange oil, thyme oil, vegetable oil, and wintergreen oil.

[00102] Other acceptable components may be, for example, nematode-attracting substances, such as soluble and gaseous substances produced by the roots of host plants or by microorganisms from the rhizosphere present. An example of a nematode attractant according to the present invention is Valerian root (Valeriana officianalis). Particularly, solutions comprising Valerian root extract, as well as any other compound or by-product associated with roots of the Valerian plant, can be used as an attractant in accordance with the present compositions and methods.

[00103] Other suitable additives, which may be contained in the formulations according to the invention, include substances that are commonly used for similar preparations. Examples of such additives include adjuvants, surfactants, emulsifying agents, plant nutrients, fillers, plasticizers, lubricants, glidants, colorants, pigments, bittering agents, buffering agents, solubility control agents, pH adjusting agents, preservatives, stabilizers and agents resistant to ultraviolet light. Hardening or hardening agents may also be incorporated to strengthen the formulations and make them strong enough to withstand pressure or force in certain applications such as tablets for injection into soil, root flares or trees.

[00104] In one embodiment, the composition may additionally comprise buffering agents including organic acids and amino acids or salts thereof. Suitable buffers include citrate, gluconate, tartrate, malate, acetate, lactate, oxalate, aspartate, malonate, glucoheptonate, pyruvate, galactarate, glucarate, tartaronate, glutamate, glycine, lysine, glutamine, methionine, cysteine, arginine and a mixture thereof. Phosphoric and phosphorous acids or their salts can also be used. Synthetic buffers are suitable to be used, but it is preferable to use natural buffers such as organic acids and amino acids or their salts listed above.

[00105] In a further embodiment, pH adjusting agents include potassium hydroxide, ammonium hydroxide, potassium carbonate or bicarbonate, hydrochloric acid, nitric acid, sulfuric acid or a mixture.

[00106] In one embodiment, additional components, such as sodium bicarbonate or carbonate, sodium sulfate, sodium phosphate, sodium bisphosphate, may be included in the formulation.

[00107] In one embodiment, the compositions may include one or more chemical compounds with nematicidal activity. These include antibiotic nematicides such as abamectin; carbamate nematicides such as benomyl, carbofuran, carbosulfan, and cleotocard; oxime carbamate nematicides such as alanicarb, aldicarb, aldoxycarb, oxamyl; organophosphorus nematicides such as diamidaphos, fenamifos, fostietan, phosfamidon, cadusaphos, chlorpyrifos, diclofenthion, dimethoate, etoprophos, fensulfothion, fostiaZate, heterophos, isamidophos, isaZofos, methomyl, phorate, phosphocarb, terbufos, thiodicarb, thionazin, triaZofos, imicia fos, and mecarfon . Other compounds with nematicidal activity include acetoprol, benclothiaz, chloropicrin, dazomet, DB CP, DCIP, 1,2-dichloropropane, 1,3-dichloropropene, furfural, iodomethane, metam, methyl bromide, methyl isothiocyanate, and xylenols.

[00108] In one embodiment, the compositions may additionally comprise an effective amount of at least one insecticide. Suitable insecticides include, but are not limited to, non-nematicidal neonicotinoid insecticides, such as 1-(6-chloro-3-pyridylmethyl)-N-nitroimidazolidin-2-ylideneamine (imidacloprid); 3-(6-chloro-3-pyridylmethyl)-1,3-thiazolidin-2-ylidenecyamide (thiacloprid); 1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine (clothianidin); nitempyran;N1-[(6-chloro-3-pyridyl)methyl]-N2-cyano-N1-methylacetamidine (acetamipride); 3-(2-chloro-1,3-thiazol-5-ylmethyl)-5-methyl-1,3,5-oxadiazinan-4-ylidene(nitro)amine (thiamethoxam); and 1-methyl-2-nitro-3-(tetrahydro-3-furylmethyl)guanidine (dinotefuran).

Application of Microbe-Based Products

[00109] In yet another aspect, the present invention includes methods, systems, and devices for applying microbe-based products.

[00110] In one embodiment, the present invention provides a method for improving plant health and/or increasing crop production by applying the composition disclosed herein, in this patent application, to soil, seed, or parts of plants. In another embodiment, the present invention provides a method for increasing crop or plant production comprising multiple applications of the composition described herein in this patent application.

[00111] Advantageously, the method can effectively control nematodes, and corresponding diseases caused by pests while increasing production is achieved and side effects and additional costs are avoided.

[00112] In one embodiment, the composition can be applied to an already germinated and/or growing plant including roots, stems, and leaves. The composition can also be applied as a seed treatment. Use as a seed treatment is beneficial because the application can be carried out easily, and the amount used for the treatment can be reduced, further reducing potential toxicity, if any.

[00113] In one embodiment, microorganisms producing biosurfactants and/or biosurfactants can be added to the soil, plant growth medium, plants, aquatic environment, or any area to be treated to prevent pest damage. Microorganisms can grow in situ and produce biosurfactants on site to control nematodes. Furthermore, natural organisms producing biosurfactants applied at the application site will be able to grow and produce the biosurfactant. Consequently, a high concentration of biosurfactant and biosurfactant-producing microorganisms can be obtained at the treatment site (e.g. in the soil) easily and continuously.

[00114] Substances that enhance the growth of microorganisms and the production of biosurfactants can also be added to the composition and/or treatment site to support the growth of biosurfactant-producing organisms as well as to produce the desired biosurfactants at the site to obtain the objective of the invention. These substances include, but are not limited to, carbon, or organic substrates such as oil, glycerol, sugar, or other nutrients. For example, biosurfactant-producing organisms can grow on the substrate to produce biosurfactant on site and control nematodes. Natural organisms producing biosurfactants at the application site (soil, aquatic system, plant parts, etc.) will produce biosurfactants while using the carbon substrate. During this process, the biosurfactant produced will destroy or paralyze pests in the targeted area. According to the invention, it is not necessary to pre-inoculate the target site or the mixture of carbon substrate with biosurfactant-producing organisms.

[00115] Carbon substrates may include, but are not limited to, organic carbon sources such as natural or synthetic oil including used frying oil; fat; lipid; wax (natural or paraffin); fatty acids such as lauric; myristic, etc.; fatty acid alcohol, such as lauryl alcohol; amphiphilic esters of fatty acids with glycerol, such as glyceryl monolaurate; fatty acid glycolic esters, such as polyethylene monostearate; fatty acid amines such as lauryl amine; fatty acid amides; hexanes; glycerol; glucose; etc. It is preferable to use a water-insoluble carbon substrate to stimulate the production of biosurfactants.

[00116] One embodiment involves spiking or altering the carbon substrate with a sufficient amount of specific biosurfactant to initiate the emulsification process and to inhibit or reduce the growth of other organisms competitive to the biosurfactant-producing organism and to control nematodes. Pseudomonas syringae and Bacillus subtilis, for example, produce a series of lipopeptide biosurfactants referred to as porens. These lipopeptides include pseudomycin, syringomycin, tabotoxin, phaseolotoxin, and surfactin. Some lipopeptides are capable of creating holes in cell membranes, cells, and tissues.

[00117] Pseudomycin can be applied as a pre-plant treatment to control nematodes in crop production. If it is desirable to stimulate the growth of Bacillus subtilis, a small amount of the biosurfactant surfactin is added to the carbon substrate medium to aid in the establishment of the B. subtilis population and the production of more surfactin locally.

[00118] In one embodiment, the composition can be applied by spraying, pouring, dipping, in the form of concentrated or diluted liquids, solutions, suspensions, powders, and the like, containing said concentrations of the active agent as is most convenient for a particular purpose at hand. They can be applied as is or reconstituted before use. For example, they can be applied by direct injection into trees or root enlargements.

[00119] In one embodiment, the composition according to the present invention can be applied at about (0.0001 pounds/acre to about 10 pounds/acre, about 0.001 pounds/acre to about 5 pounds/acre, about 0.01 pound/acre to about 1 pound/acre, about 0.01 pound/acre to about 0.1 pound/acre, or about 0.01 pound/acre to about 0.05 pound / acre).

[00120] In one embodiment, the composition according to the present invention is applied to the plant or crop from about 1 to about 100 days, about 2 to about 50 days, about 10 to about 40 days , about 20 to about 30 days after initial application to the soil or seed.

[00121] In specific embodiments, the compositions may be, for example, introduced into an irrigation system, sprayed from a backpack or similar devices, applied by a ground-based or airborne robotic device, such as a drone. , and/or applied with a seed.

[00122] Application to seeds can be, for example, by seed coating or by applying the composition to the soil at the same time as planting the seeds. This can be automated, for example, by providing a device or irrigation system that applies the microbial-based composition along with the seeds, and/or adjacent to them at or near the time of planting the seeds. In this way, the microbe-based composition can be applied within, for example, 5, 4, 3, 2, or 1 day before or after the time of planting or simultaneously with planting the seeds.

[00123] In certain embodiments, the compositions provided here, in this patent application, are applied to the soil surface without mechanical incorporation. The beneficial effect of soil application can be activated by rain, sprinkler, flood, or drip irrigation, and subsequently released to, for example, target pests to bring their population levels to acceptable thresholds or to the roots of plants to influencing the root microbiome or facilitating the uptake of the microbial product within the vascular system of the crop or plant to which the microbial product is applied. In an exemplary embodiment, the compositions provided herein in this patent application can be effectively applied through a center pivot irrigation system or with a sprayer over the seed furrow.

[00124] Reference here, in this patent application, to administering the composition "on or near" a pest or a plant, or to the "environment" of a pest or plant, means that the administration is such that the composition is sufficiently in contact with the pest or plant in such a way that the desired result is obtained (e.g., destruction of the pest, increased production, prevention of damage to the plant, regulation of genes and/or hormones, etc.). This typically may be within, for example, 10, 5, 3, 2, or 1 feet or less from the pest, plant, weed, or other desired target.

[00125] The microbe-based product can also be applied in order to promote colonization of the roots and/or rhizosphere as well as the plant's vascular system in order to promote the health and vitality of plants. In this way, nutrient-fixing microbes such as rhizohium and/or mycorrhzae can be stimulated as well as other endogenous microbes (already present in the soil), as well as exogenous microbes, or their by-products, that combat pests, disease, or otherwise stimulate the growth, health and/or production of the crop. The microbe-based product may also support the vascular system of a plant, for example, by penetrating and colonizing said vascular system and contributing metabolites, and nutrients important to the health and productivity of the plant or metabolites with pest control properties.

[00126] Advantageously, the method does not require complicated equipment or high energy consumption. The microorganisms of interest can be cultivated on a small or large scale on site and used, even while still being mixed with their media. Similarly, microbial metabolites can also be produced in large quantities at the site of need.

[00127] Advantageously, microbe-based products can be produced in remote locations. In one embodiment, the microbe-based products can be used for human nutrition and/or disease prevention and/or treatment.

Target pests

[00128] In preferred, but not limiting, embodiments of the invention, the controlled nematode is chosen from:

[00129] (1) a nematode that is a plant pathogenic nematode, such as, but not limited to: Root-knot nematodes (Meloidogyne spp.) in rice (e.g., M. incognita, M. javanica or M. graminicola) , in soybeans (e.g. M. incognita or M. arenaria), in cotton (e.g. M. incognita), in potatoes (e.g. M. chitwoodi or M. hapla), in tomatoes (e.g. M. chitwoodi), tobacco (e.g., M. incognita, M. javanica, or M. arenaria), and corn (e.g., M. incognita); Cyst nematodes (Heterodera spp.) in rice (e.g., H. oryzae), soybeans (e.g., H. glycines), and corn (e.g., H. zeae); Cyst nematodes (Globodera spp.) in potato (e.g. G. pallida or G. grossochiensis); Reniform Nematodes (Rotylenchulus spp.) in cotton (e.g. R. reniformis); Root lesion nematodes (Pratylenchus spp.) in banana (e.g. P. coffeae or P. goodeyi); Cave Nematodes (Radopholus spp.) in banana (e.g. R. similis); and other rice damaging nematodes such as rice root nematode (Hirschmaniella spp., e.g., H. oryzae);

[00130] (2) a nematode capable of infesting humans such as, but not limited to: Enterobius vermicularis, the pinworm that causes enterobiasis; Ascaris lumbridoides, the large intestinal roundworm that causes ascariasis; Necator and Ancylostoma, two types of hookworms that cause hookworm; Trichuris trichiura, the whipworm that causes trichuriasis; Strongyloides stercoralis which causes strongyloidiasis; and Trichonella spirae which causes trichinosis; Brugia malayi and Wuchereria bancrofti, the filarial nematodes associated with the worm infections known as lymphatic filariasis and its gross manifestation, elephantiasis, and Onchocerca volvulus which causes river blindness (onchocerciasis);

[00131] (3) a nematode capable of infesting animals such as, but not limited to: dogs (Hookworms, e.g., Ancylostoma caninum or Uncinaria stenocephala, Ascarids, e.g., Toxocara canis or Toxascaris leonina, or Whipworms, e.g. Trichuris vulpis), cats (Hookworms, e.g. Ancylostoma tubaeforme, Ascarids, e.g. Toxocara cati), fish (herring worms or cod worms, e.g. Anisakid, or tapeworm, e.g. Diphyllobothrium), sheep (Wire worms e.g. Haemonchus contortus) and livestock (gastrointestinal worms e.g. Ostertagia ostertagi, Cooperia oncophora);

[00132] (4) a nematode that causes unwanted damage to substrates or materials, such as nematodes that attack foodstuffs, seeds, wood, paint, plastic, clothing, etc. Examples of similar nematodes include, but are not limited to: Meloidogyne spp. (e.g. M. incognita, M. javanica, M. arenaria, M. graminicola, M. chitwoodi or M. hapla); Heterodera spp. (e.g. H. oryzae, H. glycines, H. zeae or H. schachtii); Globodera spp. (e.g. G. pallida or G. grossochiensis); Ditylenchus spp. (e.g. D. dipsaci, D. destructor or D. angustus); Belonolaimus spp.; Rotylenchulus spp. (e.g. R. reniformis); Pratylenchus spp. (e.g. P. coffeae, P. goodeyi or P. zeae); Radopholus spp. (e.g. R. Similis); Hirschmaniella spp. (e.g. H. oryzae); Aphelenchoides spp. (e.g. A. besseyi); Criconemoides spp.; Longidorus spp.; Helicotylenchus spp.; Hoplolaimus spp.; Xiphinema spp.; Paratrichodorus spp. (e.g. P. minor); Tylenchorhynchus spp;

[00133] (5) virus-transmitting nematodes (e.g., Longidorus macrosoma: transmits prunus necrotic ring spot virus, Xiphinema americanum: transmits tobacco ring spot virus, Paratrichadorus teres: transmits early browning virus of the pea, or Trichodorus similis: transmits the tobacco rattle virus).

[00134] Specific nematode pests include:

[00135] Dracunculus medinensis, the roundworm that causes Dracunculiasis (Guinea worm disease); nematodes Loa loa (the African eye worm), Mansonella streptocerca and Onchocerca volvulus, which cause Cutaneous Filariasis; Mansonella perstans and Mansonella ozzardi, which cause Body Cavity Filariasis; Trichinella, including T. pseudospiralis (which infects mammals and birds worldwide), T. native (which infects Arctic bears), T. nelsoni (which infects African predators and scavengers), and T. britovi (which infects carnivores in Europe and from Western Asia), which cause Trichinellosis; Angiostrongylus cantonensis (mouse lungworm), which is the most common cause of human eosinophilic meningitis; Angiostrongylus costaricensis, which causes abdominal (or intestinal) angiostrongyliasis; Toxocara, which causes human toxocariasis; Gnathostoma spinigerum, and rarely G. hispidum, which cause Gnotostomiasis; and Anisakis simplex, or Pseudoterranova decipiens, which causes Anisakiasis.

[00136] In specific embodiments, the methods and compositions of the present invention are used to control root-knot nematode (Meloidogyne incognital), stinger nematode (Belonolaimus longicaudatus), soybean cyst nematode (Heterodera glycines), lesion nematode (Pratylenchus sp .), dagger nematode (Xiphinema sp.), and/or citrus nematode (Tylenchulus semipenetrans). Plantasalvo

[00137] Plants that may benefit from the application of the products and methods of the present invention include: Leira Crops (e.g., Corn, Soybeans, Sorghum, Peanuts, Potatoes, etc.), Field Crops (e.g., Alfalfa, Wheat , Grains, etc.), Tree Crops (e.g. Walnuts, Almonds, Pecans, Hazelnuts, Pistachios, etc.), Citrus Crops (e.g. Orange, Lemon, Grapefruit, etc.), Fruit Crops ( e.g. apples, pears, etc.), Grass Crops, Ornamental Crops (e.g. Flowers, vines, etc.), Vegetables (e.g. tomatoes, carrots, etc.), Vine Crops (e.g. Grapes , Strawberries, Blueberries, Blackberries, etc.), Forestry (e.g., pine, spruce, eucalyptus, poplar, etc.), Managed Pastures (any mixture of plants used to support grazing animals).

[00138] The benefit may be in the form of, for example, increased production, quality, resistance to diseases and pests, reduced stress (e.g., from salt, drought, heat, etc.) and better use of water.

[00139] Additional plants that can benefit from the products and methods of the invention include all plants that belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including forage or forage legumes, ornamental plants, food crops, trees or shrubs selected from Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp. ., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp ., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g., Elaeis guineensis , Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g., Hordeum vulgare), Ipomoea potatoes, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g., Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g., Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis , Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g., Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., among others.

[00140] Additional examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g. B. napus, B. rapa, B. juncea), particularly Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum ( Sorghum bicolor, Sorghum vulgare), millet (e.g. pearl millet (Pennisetum glaucum), millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), saffron (Carthamus tinctorius), wheat (Triticum aestivum), soybeans (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea Batatus) , cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp. .), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia ), almonds (Prunus amygdalus), sugar beets (Beta vulgaris), sugar cane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

[00141] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation ( Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum. Conifers that can be used in the practice of the modalities include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine ( Pinus radiata); Douglas-fir (Pseudotsuga menziesii); western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); Brazilwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as western red cedar (Thuja plicata) and Alaskan yellow cedar (Chamaecyparis nootkatensis). Plants of the embodiments include crop plants (e.g., corn, alfalfa, sunflower, Brassica, soybeans, cotton, safflower, peanuts, sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

[00142] Lawns include, but are not limited to: annual bluegrass (Poa annua); annual ryegrass (Lolium multiflorum); Canadian bluegrass (Poa compressa); chewing fescue (Festuca rubra); colonial bentgrass (Agrostis tenuis); creeping bentgrass (Agrostis palustris); crested wheatgrass (Agropyron desertum); fairway wheatgrass (Agropyron cristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poa pratensis); orchardgrass (Dactylis glomerate); perennial ryegrass (Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep fescue (Festuca ovine); smooth bromegrass (Bromus inermis); tall fescue (Festuca arundinacea); meadow grass (Phleum pretense); velvet bentgrass (Agrostis canine); weeping alkaligrass (Puccinellia distans); western wheatgrass (Agropyron smithii); Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrum secundatum); zoysia grass (Zoysia spp.); Bahia grass (Paspalum Notatum); carpet grass (Axonopus affinis); centipede grass (Eremochloa ophiuroides); kikuyu grass (Pennisetum clandesinum); seashore paspalum (Paspalum vaginatum); blue grass (Bouteloua gracilis); buffalo grass (Buchloe dactyloids); Grass sideoats (Bouteloua curtipendula).

[00143] Additional plants of interest include Cannabis (e.g., sativa, indica, and ruderalis) and industrial hemp.

[00144] Plants of interest include grain plants that provide seeds of interest, oilseed plants, and leguminous plants. Seeds of interest include grain seeds such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oilseed plants include cotton, soybean, safflower, sunflower, Brassica, corn, alfalfa, palm, coconut, flax, castor, olive, etc. Legume plants include beans and peas. Beans include guar, carob beans, fenugreek, soybeans, garden beans, string beans, mungbean, lima beans, fava beans, lentils, chickpeas, etc.

[00145] All plants and parts of plants can be treated in accordance with the invention. In this context, plants are understood to mean all plants and populations of plants, such as desired or unwanted wild plants or crop plants (including naturally occurring crop plants). Crop plants may be plants that can be obtained by traditional breeding and optimization methods or by biotechnological and recombinant methods, or combinations of these methods, including transgenic plants and plant varieties.

[00146] Parts of plants are understood to mean all parts and organs of aerial and underground plants such as shoot, leaf, flower and root, examples of which may be mentioned being leaves, needles, stems, stems, flowers, fruiting bodies, fruits and seeds, but also roots, tubers and rhizomes. Plant parts also include cultural material and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, grafts and seeds.

EXAMPLES

[00147] It should be understood that the examples and embodiments described herein in this patent application are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to those skilled in the art and should be included within the spirit and scope of this request.

EXAMPLE 1 - FERMENTATION OF S. BOMBICOLA FOR PRODUCTION OF SOPHOROLIPIDS (SLP) IN A 110 L distributable REACTOR

[00148] A portable, air transport type, fully enclosed reactor operated by PLC with water filtration, temperature control unit, and an onboard air fan is used. The reactor has a working volume of 90 L when cultivating Starmerella bombicola for SLP production.

[00149] In preferred embodiments, the nutrients for producing SLP are glucose, urea, yeast extract, canola oil, magnesium sulfate, and potassium phosphate.

[00150] The reactor is inoculated with 8 liters of culture liquid grown in flasks. The duration of the cultivation cycle for SLP production is 7 to 8 days, at 25° C and pH 3.5, with sampling carried out twice a day.

[00151] The final concentration of SLP is roughly 10% of the working volume, in this case around 9 L of product, containing 300 to 400 grams of SLP per liter.

EXAMPLE 2 - FERMENTATION OF S. BOMBICOLA FOR PRODUCTION OF SLP IN A 14 L PORTABLE DISPENSABLE REACTOR

[00152] This reactor is an autoclavable jacketed glass vessel with air spurge and impeller. It is equipped with dissolved oxygen, pH, temperature, and foam probe; has an integrated control station with a color touchscreen interface, built-in pumps, gas flow controllers, and pH/DO foam/level controllers. The working volume of the reactor is 10 liters.

[00153] The nutrient medium contains glucose, yeast extract, urea, and vegetable oil. The inoculum can be a 1 to 2 day old culture of Starmerella bombicola at about 5 to 10% of the volume of the total culture. The duration of cultivation and compilation of common product continues for 5 to 14 days. Final sophorolipid production can reach 1 to 2 kilograms per cycle.

EXAMPLE 3 - FERMENTATION OF WICKERHAMOMYCES YEAST AND/OR PICHIA FOR PRODUCTION OF SLP IN A DISPENSABLE REACTOR

[00154] A PLC-operated mobile air transport reactor with water filtration, temperature control unit, and an air fan for sufficient aeration is used. The process can be carried out as a batch cultivation process. The reactor has a working volume of 400 L when growing Wickerhamomyces or Pichia for SLP production.

[00155] In preferred embodiments, the nutrients for producing SLP are glucose, urea, yeast extract, canola oil, magnesium sulfate, and potassium phosphate.

[00156] Inoculation of this reactor requires up to 5% working volume liquid seed culture. The duration of the cultivation cycle is 7 days, at a temperature of 25° C and pH 3.5, with sampling carried out twice a day.

[00157] The final concentration of SLP is roughly 20 to 25% of the working volume, in this case it forms more than 90 L of product.

EXAMPLE 4 - FERMENTATION OF WICKERHAMOMYCES YEAST AND/OR PICHIA FOR PRODUCTION OF CELLS AND SINGLE CELL PROTEINS IN A 900 L DISPENSABLE REACTOR

[00158] A portable reactor divided into two tanks operated by a central airlift is used to help mix the two tanks simultaneously. The reactor has a working volume of 600 L when growing Wickerhamomyces and/or Pichia for cell production.

[00159] In a preferred embodiment, the nutrients for cell production are glucose or icing sugar, urea, yeast extract, magnesium sulfate, and potassium phosphate.

[00160] The reactor is inoculated with 2% seed culture. Fermentation continues for 48 to 72 hours without pH stabilization, and at a temperature of 26 to 32° C.

[00161] The final cell concentration will be 100 g wet weight per liter. Wet biomass concentration can reach 90 kilograms per cycle with protein concentration up to 45 kilograms.

EXAMPLE 5 - FERMENTATION OF WICKERHAMOMYCES YEAST AND/OR PICHIA FOR PRODUCTION OF CELLS AND SINGLE CELL PROTEINS IN A 2000 L DISPENSABLE REACTOR

[00162] A portable reactor is used divided into two square tanks accompanied by 2 handles for exchanging mass between them. The reactor has a working volume of 2000 L when growing Wickerhamomyces and/or Pichia for cell production.

[00163] In a preferred embodiment, the nutrients for cell production are glucose or icing sugar, urea, yeast extract, magnesium sulfate, and potassium phosphate.

[00164] The reactor is inoculated with 2% seed culture. Fermentation continues for 48 to 72 hours without pH stabilization, and at a temperature of 26 to 32° C.

[00165] The final cell concentration will be 100 g wet weight per liter. Wet biomass concentration can reach up to 200 kilograms per cycle with protein concentration up to 100 kilograms.

EXAMPLE 6 - FERMENTATION OF PSEUDOZYMA APHIDIS FOR PRODUCTION OF THE LIPID MANNOSYLERYTHRITOL (HONEY) IN A 14 L PORTABLE DISPENSABLE REACTOR

[00166] This is a steam autoclavable jacketed glass vessel with air spurge and Rushton impeller. It is equipped with DO, pH, temperature, and foam probe. It has an integrated control station with a color touchscreen interface, built-in pumps, gas flow controllers, and pH/DO foam/level controllers. The working volume of the reactor is 10 liters. color touch screen display, built-in pumps, gas flow controllers, and pH/foam controllers

[00167] Composition of the nutrient medium: Sodium nitrate, Potassium phosphate, Magnesium sulfate, Yeast extract, and vegetable oil. The inoculum can be a 1- to 2-day-old culture of Pseudozyma aphidis, at about 5 to 10% of the total culture volume. Duration of cultivation and sample compilation: 9 to 15 days. Final HONEY production: 800 to 1000 grams.

EXAMPLE 7 - USE OF SLP TO REDUCE ROOT NEMATOID EGGS ON TOMATO PLANTS

[00168] Starmerella bombicola was cultivated as described above. At the end of the fermentation culture, the broth settles and most of the SLP separates as a dark brown layer (concentration about 500 gm/L). This brown layer essentially contains SLP and some cells as well. The layer is collected and the pH is adjusted to pH 7.0. The solution is then diluted with water to obtain the desired concentrations.

[00169] Day 1 Germinate tomato seeds (Moneymaker)

[00170] Day 14 Prepare sand / turface (1:1) with 1% fertilizer (osmocote), transplant 2 week old tomato seedlings

[00171] Day 28 Inoculate microbes by soaking the soil to 4-week-old plants. For each 10 cm x 10 cm square bridge, 80 to 100 ml of culture was enough to moisten the soil.

[00172] Day 32 Four days later, after inoculating microbes, apply RKN eggs (4000 eggs per pot) to each plant.

[00173] Day 76 Six weeks after egg inoculation, harvest the plants.

[00174] After harvesting, the plants were washed under running water to remove the sand / turf attached to the roots. The fresh weight of the roots was recorded. Then, the roots were cut into small pieces and placed inside a small plastic snack bag. After 200 ml of 5% bleach was added, the bag was incubated and shaken gently for 15 min. Then, eggs were counted in 10 ul samples in triplicate under Stereo Microscope. The total number of eggs on each root was calculated. Finally, the number of eggs in each gram of root biomass was calculated.

[00175] SLP at 0.25% also shows activity against Root Root Nematode (RNK) with a reduction in eggs by about 52%.

EXAMPLE 8 - COMPARISON OF SLP, HONEY, AND NIMITZ® IN THE ABILITY TO CONTROL KICK, STING AND LESION NEMATOIDS

[00176] Nimitz® is the first new chemical nematicide to be developed in more than 20 years. Nimitz® causes rapid and irreversible nematicidal activity immediately after application. Within an hour of contact, the nematodes stop feeding and quickly become paralyzed. Within 24 to 48 hours, pest mortality occurs rather than temporary nematostatic (immobilizing) activity as seen with organophosphate and carbamate nematicides.

[00177] Pseudozyma aphidis was cultivated as described above. At the end of fermentation, all the culture broth was used, as HONEY is very hard to separate without using any solvent. Then MEL is diluted with water to the desired concentrations immediately before application.

[00178] Starmerella bombicola was cultivated as described above. At the end of the fermentation culture, the broth settles and most of the SLP separates as a dark brown layer (concentration about 500 gm/L). This brown layer essentially contains SLP and some cells as well. The layer is collected and the pH is adjusted to pH 7.0. The solution is then diluted with water to obtain the desired concentrations.

[00179] The following bioassay was conducted: Fill glass well slides with appropriate amounts of test product. Add a known population of target young nematodes to the nematicide-containing solution. Keep the cavity slides at room temperature. Nematode mortality can be assessed by counting the number of nematodes at 24, 48 and 72 hours. The percentage of mortality was calculated as: [average number of young people dying in treatment / total number of young people in treatment] x 100.

A. Root Root Nematode

[00180] Mortality (i.e., how many of the nematodes were killed) and lack of motility (i.e., how many of the nematodes were fatally ill) of root-knot nematodes (Meloidogyne incognita) were determined after 3 days of SLP treatment ( SD10) or MEL (SD12) at various concentrations and compared with Nimitz® treatment and untreated control (FIGURES 1 and 2). The percentage of mortality was calculated by the number of dead nematodes / the number of total nematode counts before testing. The percentage of non-motile nematodes was calculated as the number of non-motile nematodes / the number of live nematodes. Therefore, motility is calculated as the percentage of nematodes that were not dead but are determined to be non-motile.

[00181] The high level of MELs (0.1% vol/vol) was the most effective treatment in the test. It was significantly more effective than Nimitz® with regard to mortality. A clear dose response was observed. The results also show that SLP was as effective as Nimitz® on mortality and lack of motility of root-knot nematodes.

B. Sting Nematode

[00182] Mortality and reduced motility rates of stinger nematode (Belonolaimus longicaudatus) were determined after 3 days of SLP or MEL treatment at various concentrations and compared with Nimitz® treatment and untreated control (FIGURES 1 and 2).

[00183] The results show that both SLP and MEL increase the mortality and rate of reduced motility of sting nematodes in a dose-dependent manner. High levels of MEL (0.1% or 0.2% vol/vol) were the most effective treatment in the trial. They were as effective as Nimitz® with regard to mortality and more effective than Nimitz® with regard to the rate of reduction in motility.

C. Lesion Nematode

[00184] Mortality and reduced motility rates of lesion nematodes (Pratylenchus sp.) were determined after 3 days of SLP or MEL treatment at various concentrations and compared with Nimitz® treatment and untreated control (FIGURES 1 and 2).

[00185] The results show that both SLP and MEL increase the mortality and rate of reduced motility of lesion nematodes in a dose-dependent manner. MEL was more effective than SLP and comparable to Nimitz® in the test.

EXAMPLE 9 - EVALUATION OF VARIABLE TREATMENTS AGAINST SOUTHERN ROOT NEMATOIDS IN PATHOLOGY LABORATORY VIAL TESTS

[00186] SLP treatment (SD10) and MEL treatment (SD12) applied directly to nematode-infected soil were tested at two rates, alone or together, and compared with Nimitz® and untreated control to reduce Nematode populations from Galhas do Sul alive. Counts were taken at three different time points after treatment.

[00187] Six replicates of 7 treatments were carried out in glass bottles containing 350 cm3 of 99% sandy soil. The flasks were inoculated with 70 adult Southern Root-knot Nematodes (Meloidogyne incognita). A treatment application was made shortly after nematode inoculation, where 85 mL of the treatment was manually poured into each bottle. Treatment mixtures were prepared according to Table 1 (below). Counts of live and dead nematodes were made on day 3, day 14, and day 23 post-treatment. Nonmotile nematode counts were also tabulated as a percentage of total live nematodes. Table 1. Preparation of Treatment Mixtures Reps: 6; Spray volume: 85 mL/item; Installments; 4 x 20 ft.; Mix size: 1 L (0 L calculated mix size).

Results

[00188] The vial test results for the SD10 and SD12 products suggest a positive dose response for activity against Southern Root Root Nematodes. SD10 and SD12 applied together at a low rate were not as effective as either product alone at a higher rate. Nimitz® provided superior control.

[00189] FIG. 3 and Table 2 (below) show live nematode counts for each treatment. FIG. 4 and Table 3 (below) show live nematode control (i.e., nematode kill rate) for each treatment. Table 2. Live Nematode Count

[00190] There was significantly greater control recorded as early as day 3 (3 DA-A) in bottles treated with Nimitz® (10 ppm). For both SD10 and SD12, there were important dose responses observed for each at 23 DA-A, with samples treated at higher rates having lower nematode counts than samples with lower rates. When both SD10 and SD12 were applied at low rate in combination, nematode mortality was not significantly enhanced to levels seen in higher rate treatments. Table 3. Live nematode control (%)

[00191] FIG. 5 and Table 4 (below) show non-motile nematode counts. FIG. 6 and Table 5 (below) show the live non-mobile nematode count (%).Table 4. Non-mobile Nematode Count

[00192] Non-motile nematode counts were lower in samples treated with Nimitz®. At 3 DA-A there was a higher percentage of non-motile nematodes at rates of or increasing SD10 or SD12, suggesting that early pesticide activity did not result in immediate fatality. Around 14 DA- A, the percentage of live nematodes classified as non-motile was not statistically different between the flasks treated with SD. Table 5. Count of Live Non-mobile Nematodes (%)

EXAMPLE 10 - USE OF HONEY AS A NEMATICIDE IN A MICRO-PARTMENT SYSTEM OF CUCUMBER PLANTS

[00193] An outdoor microplot located in Thonotosassa, Florida, USA, was used to study MEL nematode treatment for cucumber plants compared with Nimitz® and an untreated control. Six different treatment groups were studied, each consisting of 8 plants. For groups receiving treatment with SD12 (0.2% ai/v), the treatment was applied to the soil once, twice or three times. Both nematode counts and gall formation rates were collected at harvest. Plant vigor, production and plant weight were also measured.

[00194] Plants receiving treatment with Nimitz® (5 pt/a) were planted in treated soil 7 days before planting (the chemical treatment is too phytotoxic to be applied in the growing season). For all plants receiving treatment with SD12, the first application of SD12 to the soil (60 mL for each plant) occurred at planting. For plants receiving double and triple treatments with SD12, the second application of 60 mL occurred two weeks after the first treatment. For plants receiving triple treatment with SD12, the third application of 60 mL occurred four weeks after the first treatment.

[00195] Nematode counts and gall formation rates were collected for all groups 31 days after the final treatment with SD12 in the triple treatment group. Plant vigor (index/scale 0 to 10), yield (in pounds), and weight (in pounds) were measured the next day.

Results

[00196] The response rate was observed in gall formation (FIG. 7 and Table 6 (below)) and in post-treatment nematode counts (FIG. 8 and Table 7 (below)). SD12 performed at commercial standards. There was no significant separation between the SD12 treatments and the Nimitz® treatment, but significant differences were observed between the SD12 treatments and the untreated check. Plant vigor, production and weight at harvest are reported in Table 8 below. Table 6. Gall Formation Rate (index / scale from 1 to 10) LSD (P=0.05) = 0.88; SD = 0.86; CV = 69.39; Bartlett's X2 = 11.754; P (Bartlett's X2) = 0.019*; Replicate F = 3.712; Replicate Prob(F) = 0.0061; Treatment F = 11.133; Treatment Prob(F) = 0.0001.Table 7. Nematode count LSD (P=0.05) = 17.33; SD = 16.3; CV = 63.8 8; Bartlett's X2 = 17.263; P (Bartlett's X2) = 0.002*; Replicate F = 2.774; Replicate Prob(F) = 0.0261; Treatment F = 10.44; Treatment Prob(F) = 0.0001.Table 8. Measurements of plant vigor (0 to 10), production (in pounds), and weight (in pounds) at plant harvest.

EXAMPLE 11 - EVALUATION OF THE EFFECTIVENESS OF NEMATOID ATTRACTION

[00197] Counts and infestation percentages of Southern Root-knot Nematodes were carried out in four 11.6-inch x 7.6-inch sealed chambers containing fine lake sand soil spiked with an attractive material. Pre-made Valerian root extract was mixed with water, vegetable glycerin and 20% grain alcohol to produce the attractant.

[00198] Each plot was inoculated with nematodes in a 2 cm diameter zone. 10 mL of nematode attractant was added in a zone measuring 3 cm (height) x 1 cm (width), 2 cm from the inoculation zone (FIG. 9).

[00199] Nematode counts and infestation percentages were performed at three locations, 3 days after treatment and 8 days after treatment. The three locations tested included the center of the inoculation zone, the attraction zone, and the untreated area.

Results

[00200] The results are summarized in FIG. 10 and Table 9 (below). Migrations to the attractant or untreated area were significantly different in relation to the inoculation area as a percentage of the total population. In 24- and 48-hour sampling events, there were more nematodes counted in the attraction zone than in the untreated areas of the chamber by more than 14%; however, the central zone where the nematodes were inoculated held the greatest number of nematodes. Table 9. Nematode Count and Infestation Percentage in Attractant Assessment

[00201] All patents, patent applications, provisional applications, and publications referred to or cited herein, in this patent application, are incorporated by reference in their entirety, including all figures and tables, to the extent that they are not inconsistent with the explicit lessons of this report.

[00202] The description herein in this patent application of any aspect or embodiment of the invention using terms such as "comprising", "having", "including" or "containing" with reference to an element or elements is intended to provide support for a similar aspect or embodiment of the invention that "consists of", "consists essentially of", or "substantially comprises" that particular element or elements, unless otherwise stated or clearly negated by the context.

Claims (7)

1. Method for controlling plant nematode pests, characterized in that said method comprises preparing a microbe-based nematicidal composition by culturing a microorganism Pseudozyma aphidis under conditions such that the microorganism Pseudozyma aphidis_produces a lipid biosurfactant mannosylerythritol (MEL), cultivate a Starmerella bombicola microorganism under conditions such that the Starmerella bombicola microorganism produces a sophorolipid biosurfactant (SLP); and mixing the cultivated Pseudozyma aphidis and MEL with the cultivated Starmerella bombicola and SLP, wherein said nematicidal composition comprises the microorganism Pseudozyma aphidis, the MEL, a broth in which the microorganism Pseudozyma aphidis was cultivated, as well as the microorganism Starmerella bombicola, the SLP is a broth in which the Starmerella bombicola microorganism has been cultivated, and in which said method further comprises mixing the nematicidal composition with water and applying the nematicidal composition to nematodes, soil and/or a seed. 2. Method according to claim 1, characterized in that it is used to control a nematode selected from Meloidogyne incognita, Meloidogyne hapla, Meloidogyne arenaria, Belonolaimus longicaudatus, Heterodera glycines, Pratylenchus sp., Xiphinema sp., and Tylenchulus semipenetrans. 3. Method according to claim 1, characterized by the fact that the plants are selected from tomatoes, soybeans, corn, citrus fruits and lawns. 4. Method according to claim 1, characterized by the fact that the method additionally comprises applying a nematode attractant. 5. Composition for controlling nematodes, characterized by the fact that said composition essentially consists of a fermentation broth obtained from the culture of Pseudozyma aphidis, wherein said fermentation broth comprises Pseudozyma aphidis, water and a lipid mannosylerythritol (HONEY); and a second fermentation broth obtained from a culture of Starmerella bombicola, wherein said second fermentation broth comprises Starmerella bombicola, water and a sophorolipid (SLP). 6. Composition, according to claim 5, characterized by the fact that it additionally comprises a nematode-attracting substance. 7. Composition, according to claim 6, characterized by the fact that the nematode-attracting substance comprises valerian root extract.