EA039980B1 - METHODS FOR COMBAT PLANT NEMOTODES - Google Patents

Description

Background of the Invention

To boost yields and protect crops from pathogens, pests and disease, farmers rely heavily on the use of synthetic chemicals and chemical fertilizers; however, if used excessively or incorrectly, these substances can enter surface water, leach into groundwater and evaporate into the air. As sources of air and water pollution, these substances are increasingly scrutinized, making their responsible use an environmental and commercial imperative. Even when properly applied, overdependence and long-term use of certain chemical fertilizers and pesticides can adversely alter the soil ecosystem, reduce stress tolerance, increase pest resistance, and interfere with the growth and vitality of plants and animals.

Nematodes belong to the class of roundworms or filamentous worms of the Nematoda type. Examples in this class are the cyst nematodes of the genus Heterodera (e.g. H. glycines, H. avenae and H. shachtii) and Globodera (e.g. G. rostochiens and G. pallida), root blunt nematodes of the genus Trichodorus, bulbous and stem nematodes of the genus Ditylenchus, potato nematode, Heterodera rostochiensis, root-knot nematodes of the genus Meloidogyne (e.g. M. javanica, M. hapla, M. arenaria and M. incognita), root nematodes of the genus Pratylenchus (e.g. P. goodeyi, P. penetrans, P. bractrvurus , P. zeae, P. coffeae, P. bractrvurus and P. thornei), citrus nematodes of the genus Tylenchulus and stinging nematodes of the genus Belonalaimus.

Nematodes are known to infect both plants and animals. These microscopic worms can be found in almost every type of environment. While in the soil, the nematodes feed on plant roots, causing significant damage to the root structure and improper plant development. Damage usually manifests itself in the form of growth of galls, root nodes and other anomalies. The formation of galls leads to a decrease in the size of the roots and inefficiency 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. Each year, nematodes cause millions of dollars of damage to lawn grasses, ornamental plants and food crops.

Gall nematodes (Meloidogyne spp.) are one of the three most economically damaging species of horticultural and field crop parasitic nematodes. Gall nematodes are distributed throughout the world and are obligate parasites of the roots of thousands of plant species, including monocots and dicots, herbaceous and woody plants. Vegetable crops grown in warm climates can suffer severe losses from root-knot nematodes and are often treated regularly with chemical nematocides. Injury from root-knot nematodes results in poor growth, reduced crop quality and yield, and decreased resistance to other stresses (eg, drought, other diseases). A high level of damage can lead to a complete loss of the crop. For example, approximately $1.5 billion per year is lost to soybean cyst nematodes alone.

Conventional nematocides used to control nematodes are applied into the seed furrow at sowing. Due to toxicity to nearby animals such as birds, topping off liquid toxic compounds in a circular motion such as Nemacur, Temik, Furadan, Dazinat and Mosar has fallen out of favor.

Since the 1960s, growers have used methyl bromide to effectively clear fields before planting, mainly for nematode control, but also for disease and weed control; however, since this toxic compound is used in gaseous form, more than half of the amount introduced into the soil may end up in the atmosphere and contribute to ozone depletion. 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.

Against the background of the ban, the treaty allows limited use of methyl bromide for strawberries, almonds and other crops for which there is no alternative for both effective and affordable control of nematodes, diseases and weeds. The degree of permitted use is decreasing every year and is likely to end soon. Therefore, finding an alternative to methyl bromide is a priority for manufacturers and regulators; however, no single product provides the broad spectrum of action offered by methyl bromide.

The creation of regulations governing the availability and use of chemicals, as well as consumer demand for residue-free, sustainably grown food, are impacting the industry and evolving the way we think about how to deal with many problems. Although mass elimination of chemicals is currently not possible, farmers are increasingly using

- 1 039980 list biological measures as viable components of integrated nutrient management and integrated pest management programs.

Due to shortcomings in the basic approaches described above, there is an increasing need for safer pesticides and alternative pest management strategies. In particular, in recent years, the biological control of nematodes has attracted great interest. In this method, biological agents such as live microorganisms, bioproducts derived from these microorganisms, and combinations thereof are used as pesticides. These biological pesticides offer important advantages over other conventional pesticides. For example, they are less harmful than conventional chemical pesticides. They are more efficient and specific, and they often biodegrade quickly, resulting in less environmental pollution.

The use of biopesticides and other biological agents has been largely limited by the difficulties of production, transportation, application, pricing and efficiency. For example, many microorganisms are difficult to grow and then usefully use in sufficient quantities in agricultural and forestry production systems. This problem is exacerbated by loss of viability and/or activity due to handling, formulation, storage, stabilization prior to distribution, sporulation of vegetative cells as a means of stabilization, transport and use. In addition, after application, biological products may not develop for a number of reasons, including, for example, insufficient initial cell density, inability to effectively compete with existing microflora in a particular location, and entry into soil and/or other environmental conditions in which the microorganism cannot thrive. or even survive.

Therefore, there is an urgent need to develop methods and materials for obtaining effective biopesticides for nematode control.

Brief summary of the present invention

The claimed invention provides compositions and methods for controlling nematodes. In addition, the claimed invention provides methods and compositions for preventing damage to crops by nematodes, which leads to increased yields. Preferably, pesticides according to the claimed invention use non-toxic substances, such as microorganisms and by-products of the cultivation of microorganisms.

According to one embodiment, the claimed invention provides methods for preventing nematode damage to crops and controlling nematodes, comprising the steps of: cultivating microorganisms that produce a by-product of growing microorganisms, and bringing the nematodes or their environment into contact with an effective amount of microorganisms and/or by-product cultivation of microorganisms.

In one embodiment, the by-product of growing the microorganisms is a biosurfactant. In one embodiment, the biosurfactant is a glycolipid such as rhamnolipid, sophorolipid (SLP), trehalose lipid, or mannosylerythritol lipid (MEL). In particularly preferred embodiments, the biosurfactant is SLP and/or MEL.

The microbial nematicidal composition according to the claimed invention can be obtained through cultivation processes ranging from small to large scale. These culture processes include, but are not limited to, deep culture/fermentation, surface culture, solid phase fermentation (SSF), and combinations thereof. The pesticide composition may be, for example, a fermentation broth and/or purified biosurfactants.

In some embodiments, the biosurfactant-producing microorganisms in the composition can be grown in situ to produce biosurfactants for direct use in nematode control. Therefore, it is possible to easily and continuously obtain a high concentration of biosurfactant and/or biosurfactant-producing microorganisms at the treatment site (eg soil).

According to one embodiment, the system according to the claimed invention contains the additional aspect of providing products based on microorganisms for field use. The microbial products according to the invention can be applied, for example, through an irrigation system, as a spray, as a seed treatment, on the soil surface, on the surface of plants and/or on the surface of pests. It also facilitates mechanical application with conventional guns or robotic application with ground or air-based drones.

The nematocidal composition can be used to protect plants, humans or animals by controlling nematodes.

Brief description of the figures

In FIG. 1 shows the decrease in nematode motility 3 days after treatment under different conditions: SD10 at 0.05% (v/v), 0.125% (v/v), 0.25% (v/v) and 0.5% (v/v) /o) respectively; SD12 at 0.025% (v/v),

- 2 039980

0.05% (v/v), 0.1% (v/v) and 0.2% (v/v), respectively; Nimitz® at 10 ppm au; and control without processing. SD10 refers to SLP processing. SD12 refers to MEL processing.

In FIG. 2 shows the death rate of nematodes 3 days after treatment under different conditions:

SD10 at 0.05% (v/v), 0.125% (v/v), 0.25% (v/v) and 0.5% (v/v), respectively;

SD12 at 0.025% (v/v), 0.05% (v/v), 0.1% (v/v) and 0.2% (v/v), respectively;

Nimitz® 10 (ppm) au;

and control without processing.

In FIG. 3 shows the number of live nematodes after 3, 14 and 23 days after different soil treatments in tests in vessels in a phytopathological laboratory: SD10 at 0.25% (v/v) and 0.5% (v/v); SD12 at 0.1% (v/v) and 0.2% (v/v); SD10 (0.25% o/o)+SD12 (0.1% o/o);

Nimitz® 10 (ppm) pr;

and control without processing.

In FIG. 4 shows the control of live nematodes (i.e. the death of nematodes) after 3, 14 and 23 days after different soil treatments in tests in vessels in a phytopathological laboratory: SD10 at 0.25% (v/v) and 0.5% (v /O); SD12 at 0.1% (v/v) and 0.2% (v/v); SD10 (0.25% o/o)+SD12 (0.1% o/o); Nimitz® 10 (ppm) pr; and control without processing.

In FIG. 5 shows the number of immobile nematodes after 3, 14 and 23 days after different soil treatments in tests in vessels in a phytopathological laboratory: SD10 at 0.25% (v/v) and 0.5% (v/v); SD12 at 0.1% (v/v) and 0.2% (v/v); SD10 (0.25% o/o)+SD12 (0.1% o/o); Nimitz® 10 (ppm) pr; and control without processing.

In FIG. 6 shows the number of immobile living nematodes after 3, 14 and 23 days after different soil treatments in tests in vessels in a phytopathological laboratory: SD10 at 0.25% (v/v) and 0.5% (v/v); SD12 at 0.1% (v/v) and 0.2% (v/v); SD10 (0.25% o/o)+SD12 (0.1% o/o); Nimitz® 10 (ppm) pr; and control without processing.

In FIG. 7 shows the galling rate at harvest for a micro-plot of cucumbers treated with 5 different treatments: control without treatment; Nimitz® 5 pt/a; SD12 0.2% (ai/v) single treatment; SD12 0.2% (ai/v) double treatment; SD12 0.2% (ai/v) triple treatment.

In FIG. 8 shows the number of nematodes at harvest for a microplot of cucumbers treated with 5 different types of treatment: control without treatment; Nimitz® 5 pt/a; SD12 0.2% (ai/v) single treatment; SD12 0.2% (ai/v) double treatment; SD12 0.2% (ai/v) triple treatment.

In FIG. 9 shows a plot setup for evaluating the effectiveness of nematode baiting, including the locations of the nematode infestation area and the baiting area.

In FIG. 10 shows the percentage infestation (of the total nematode population in the plot) at three different locations in the plots to evaluate nematode bait. Center refers to the center of the infestation zone, bait refers to the bait zone, and untreated refers to all other zones in the plot.

Detailed disclosure

The claimed invention provides materials and methods for combating nematodes. The claimed invention also provides a composition that can be used as pesticides to control nematodes. The claimed invention further provides methods for preparing such compositions. In addition, the claimed invention provides methods and compositions for preventing nematode damage to crops, resulting in increased yield.

Microorganism-based products of the claimed invention can be used in the field including, but not limited to, crops, livestock, forestry, lawn management, grazing, and human and animal health.

In one embodiment, the nematode control composition of the invention comprises an effective amount of a microorganism-produced biosurfactant and/or microorganisms producing such a biosurfactant.

In one embodiment, a method for controlling nematodes includes the steps of obtaining a microbial-produced biosurfactant and applying an effective amount of the microbial-produced biosurfactant to or at the location of the nematodes.

The composition according to the claimed invention can be obtained, for example, using culture methods ranging from small to large scale. These culture methods include, but are not limited to, deep culture/fermentation, surface culture, solid phase fermentation (SSF), and combinations thereof.

A fermentation product containing a microorganism-produced biosurfactant can be used directly for nematode treatments without extraction or purification. If necessary, extraction and purification of biosurfactants can be performed as described herein. Preferably, in the cultivation method according to the invention, a high concentration of microorganisms and a high concentration of biosurfactant can be obtained.

In certain embodiments, methods and compositions as claimed

- 3 039980 of the invention reduce the damage caused by nematodes, compared with an untreated plant, by about 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90% or more.

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

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

According to another embodiment, the claimed invention provides a method for increasing the weight or size of a plant relative to an untreated plant.

Selected definitions

Within the scope of the present invention, the term control as used in relation to the activity obtained by biosurfactants or biosurfactant-producing microorganisms extends to the action of killing, rendering harmless or immobilizing pests or otherwise renders pests essentially incapable of causing harm.

In the context of the present invention, nematicidal means having the ability to control nematodes. Thus, for example, killing nematodes, reducing their motility, and reducing the number of eggs are all examples of nematocidal activity.

In the context of the present invention, reference to a microbial composition means a composition that contains components that have been obtained from the cultivation of microorganisms or other cell cultures. Thus, the microbial composition may contain the microorganisms themselves and/or by-products of the growth of the microorganisms. The microorganisms may be in the vegetative state, in the form of spores, in the form of mycelium, in any other form of propagules, or mixtures thereof. Microorganisms can be in the form of plankton, or biofilm, or a mixture of both. Growth by-products can be, for example, metabolites, cell membrane components, expressed proteins, and/or other cellular components. Microorganisms may be whole or lysed. In preferred embodiments, the microorganisms are present in the microbial composition along with the broth in which they were grown. Cells may be present, for example, at a concentration of 1x10, 1x105, 1x106, 1x10 ⁷ , 1x108, 1x109, 1x10 ¹⁰ or 1x1011 or more propagules per ml of composition. In the context of the present invention, a propagule is any part of a microorganism from which a new and/or mature organism can develop, including, but not limited to, cells, spores, mycelium, buds, and seeds.

The claimed invention additionally provides products based on microorganisms, which are products that must be applied in practice to obtain the desired result. The microbial product may simply be a microbial composition collected during the microbial culture process. Alternatively, the microbial product may contain additional added ingredients. These additional ingredients may include, for example, stabilizers, buffers, suitable carriers such as water, saline solutions or any other suitable carrier, added nutrients to support further microbial growth, non-nutrient growth enhancers such as plant hormones and/ or means that facilitate the tracking of microorganisms and/or compositions in the environment in which they are applied. The microbial product may also contain mixtures of microbial compositions. The microbial product may also contain one or more components of the microbial composition that have been processed in some way, such as, but not limited to, filtration, centrifugation, lysing, drying, purification, and the like.

Microorganisms

Microbial biosurfactants are compounds produced by various microorganisms such as bacteria, fungi and yeasts.

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 (for example, Rhizobium japonicum, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium leguminosarum biovar trifolii and Rhizobium etli), Bradyrhizobium (for example, 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 species; 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. griseochro

- 4 039980 mogenes, 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.

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

In one embodiment, the yeast is a killer yeast. In the context of the present invention, killer yeast means a strain of yeast characterized by the secretion of toxic proteins or glycoproteins, for which the strain itself is immune. The exotoxins produced by killer yeast are capable of killing other strains of yeast, fungi, or bacteria. For example, microorganisms that can be controlled with killer yeast include Fusarium and other filamentous fungi. Examples of killer yeasts according to the present invention are killer yeasts that can be safely used in the food industry and in fermentation, for example, in the production of beer, wine and bread; killer yeasts that can be used to control other microorganisms that can contaminate such manufacturing processes; killer yeasts that can be used for biocontrol of food safety; killer yeast, which can be used to treat fungal infections in humans and plants; and killer yeasts that can be used in recombinant DNA technology. Such 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.

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

In a further embodiment, the microorganisms are mycorrhizal fungi such as Glomus spp. and Acaulospora spp. In one embodiment, the microorganisms are arbuscular mycorrhizal fungi (AMF).

In specific embodiments, the SLP producing microorganisms may be Candida spp., Starmerella spp., Cryptococcus spp., Cyberlindnera samutprakarnensis JP52 (T), Pichia spp., Rhodotorula spp., or Wickerhamiella spp.

In additional specific embodiments, the microorganisms for producing MEL can be Pseudozyma spp., Candida spp., Ustilago spp., Schizonella spp., or Kurtzmanomyces spp.

In preferred embodiments, the microorganisms are yeasts. In specific embodiments, the yeast is Starmerella, Pichia and/or Pseudozyma. Specifically, Starmerella bombicola, Pichia anomala (Wickerhamomyces anomalus) and Pseudozyma aphidis are given as examples herein.

Biosurfactants

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 (eg, oils, sugar, glycerol, etc.) in nutrient media. Other environmental components can also significantly influence the production of biosurfactants. For example, the production of rhamnolipids by Pseudomonas aeruginosa bacteria can be increased if nitrate is used instead of ammonium as a nitrogen source. Also, the production of rhamnolipid can be largely influenced by the concentration of iron, magnesium, sodium and potassium; carbon:phosphorus ratio; and churning.

Biosurfactants include low molecular weight glycolipids (GL), lipopeptides (LPs), flavolipids (FLs), phospholipids, and high molecular weight polymers such as lipoproteins, lipopolysaccharide-protein complexes, and polysaccharide-protein-fatty acid complexes. The usual lipophilic fragment of a biosurfactant molecule is the fatty acid hydrocarbon chain, while the hydrophilic part is formed by ester or alcohol groups of neutral lipids, a carboxylate group of fatty acids or amino acids (or peptides), an organic acid in the case of flavolipi

- 5 039980 dov or, in the case of glycolipids, carbohydrates.

According to embodiments of the present invention, the biosurfactant is able to penetrate pest tissues and is effective in smaller amounts without the use of adjuvants. It has been found that at concentrations above the critical micelle concentration, biosurfactants are able to more effectively penetrate the treated objects.

The development of natural biopesticides according to the claimed invention, which can be obtained in large quantities, represents a significant advance in this field. As biocontrol agents for pest control, either biosurfactant-producing organisms or the biosurfactants themselves can be used. In addition, pest control can be carried out by using specific substrates to support the growth of biosurfactant-producing organisms, as well as for the production of pesticides in the form of biosurfactants. Advantageously, natural biosurfactants are capable of inhibiting the growth of competing organisms and enhancing the growth of specific biosurfactant-producing organisms.

In addition, these biosurfactants may play an important role in the treatment of animal and human diseases. Animals can be treated, for example, by immersion or bathing in a biosurfactant solution alone, with or without microbial cell mass, and/or in the presence of other compounds such as copper or zinc.

Methods for cultivating microorganisms

The claimed invention provides methods for cultivating microorganisms and producing biosurfactants and/or other by-products of microorganism cultivation. These culture methods include, but are not limited to, deep culture/fermentation, surface culture, solid phase fermentation (SSF), and combinations thereof.

Microbial culture systems typically use deep fermentation of the culture; however, surface culture and hybrid systems can also be used. Within the scope of the present invention, fermentation refers to the growth of cells, spores, mycelium and/or other propagules of microorganisms under controlled conditions. Growth can be aerobic or anaerobic.

The microorganism growth vessel used according to the claimed invention may be any fermenter or bioreactor for industrial use. The cultivation process takes place in a vessel, which may be, for example, conical or tubular. According to one embodiment, the vessel may optionally have functional controls/sensors or may be connected to functional controls/sensors to measure important factors in the culture process such as pH, oxygen, pressure, temperature, agitator power, humidity, viscosity, and /or the density of microorganisms and/or the concentration of metabolites.

According to a further embodiment, the vessel may also be capable of monitoring the growth of microorganisms within the vessel (eg, measuring the number of cells and growth phases). Alternatively, the vessel can be sampled daily and counted using techniques known in the art, such as the dilution seeding technique. Dilution inoculation is a simple technique used to estimate the amount of bacteria in a sample. The technique may also provide an index against which different environmental conditions or treatments can be compared.

According to one embodiment, the method includes supplementing the culture 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 may be used independently or in combination of two or more.

The method can provide oxygenation to a growing culture. In one embodiment, slow air movement is used to remove low oxygen air and introduce oxygenated air. The oxygenated air may be ambient air replenished daily through mechanisms including impellers to mechanically agitate the liquid and air atomizers to introduce gas bubbles into the liquid to dissolve oxygen into the liquid.

The method may further include supplementing the culture 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 glycerin; fats and oils such as soybean oil, canola oil, coconut oil, rice bran oil, olive oil, corn oil, sesame oil and/or linseed oil; and so on. These carbon sources can be used independently or in combination of two or more.

In one embodiment, growth factors and micronutrients for microorganisms are included in the medium. This is especially advantageous when growing microorganisms that are not able to produce all the vitamins they need. Inorganic nutrients may also be included in the medium, including trace elements such as iron, zinc, copper, manganese, molybdenum and/or

- 6 039980 cobalt.

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

In some embodiments, the culture method may further include adding additional acids and/or antimicrobials to the liquid medium prior to and/or during the culture process. To protect the culture from infection, antimicrobial agents or antibiotics are used. In addition, it is also possible to add anti-foam agents to prevent the formation and/or accumulation of foam when gas is generated during cultivation.

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

The method and equipment for cultivating microorganisms and obtaining by-products of microorganisms can be performed as batch, quasi-continuous or continuous processes.

Microorganisms can be grown as plankton or as a biofilm. In the case of a biofilm, the vessel may contain a substrate on which the microorganisms can be grown in the 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.

According to one embodiment, the method for cultivating microorganisms is carried out from about 5° to about 100°C, preferably from 15 to 60°C, more preferably from 25 to 50°C. According to a further embodiment, the cultivation can be carried out continuously at a constant temperature. According to another embodiment, the cultivation can be subjected to a change in temperature.

According to one embodiment, the equipment used in the culture method and process is sterile. Culture equipment such as a reactor/vessel may be separate from, but connected to, a sterilization device such as an autoclave. Culture equipment may also have a sterilization unit that sterilizes in place before seeding. The air can be sterilized by methods known in the art. For example, before entering the vessel, ambient air may pass through at least one filter. In other embodiments, the medium may be pasteurized or optionally not heated at all, where the use of low activity, low pH water may be employed to control bacterial growth.

The content of biomass in the nutrient broth may be, for example, from 5 to 180 g/l or more. According to one embodiment, the solids content of the broth is between 10 and 150 g/L.

According to one embodiment, the method for producing a microbial metabolite according to the claimed invention comprises the steps of: 1) mixing hydrophobic and hydrophilic particles to form a matrix-forming solid; 2) contacting said matrix-forming solid with a medium inoculated with the microorganism of interest, thereby creating a microreactor array; and 3) growing said microorganism in said microreactors.

A byproduct of microbial growth produced by the microorganism of interest may be retained in the microorganism or secreted into the liquid medium. According to another embodiment, the method for producing a microbial growth by-product may further comprise the steps of concentrating and purifying the growing by-product of the microorganisms of interest. According to a further embodiment, the liquid medium may contain compounds that stabilize the activity of the microbial growth by-product.

In one embodiment, the entire microorganism culture composition is removed after culture is complete (eg, after, for example, the desired cell density or broth density of said metabolite has been reached). In this batch method, after the first batch is collected, a completely new batch is started.

According to another embodiment, only a portion of the fermentation product is removed at a time. In this embodiment, the viable cell biomass remains in the vessel as an inoculant for the new culture batch. The composition that is removed may be a cell-free broth or contain cells. Thus, a quasi-continuous system is created.

According to one embodiment, the microorganisms are cultured within 100, 50, 25,

- 7 039980

10, 5, 1 or less miles from the location where the microbial based product will be used.

Obtaining products based on microorganisms

One of the microbial products of the claimed invention is simply a 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 necessary, extraction, isolation and/or purification can be easily carried out using standard methods or techniques described herein and/or in the literature.

The microorganisms in a microorganism-based product may be in active or inactive form, in cell form, in spore form and/or in mycelium form. Products based on microorganisms can be used without further stabilization, preservation and storage. Advantageously, direct use of these microbial-based products maintains high viability of the micro-organisms, reduces the possibility of contamination by foreign agents and undesirable micro-organisms, and retains the activity of by-products of microbial growth.

Microorganisms and/or broth (including individual layers or fractions) resulting from the cultivation of microorganisms can be removed from the growth vessel and transferred, for example, by means of a tube for immediate use.

In other embodiments, the composition (microorganisms, broth, or microorganisms and broth) can be placed in containers of a suitable size, considering, for example, the intended use, the intended method of use, the size of the fermentation tank, and any method of transportation from the microorganism growth facility to the place of use. Thus, the containers in which the microbial composition is placed can be, for example, from 1 to 1000 gallons or more. In other embodiments, the containers are 2, 5, 25 gallons or more.

In the context of the present invention, broth includes whole broth or fractions of whole broth.

Once the microbial composition has been collected from the growth vessels, additional components may be added when the collected product is placed in containers and/or piped (or otherwise transported for use). Additives can be, for example, buffers, carriers, other microbial compositions prepared in the same or different plant, viscosity modifiers, preservatives, pH modifiers, microbial growth nutrients, plant growth nutrients, tracers, pesticides, herbicides , animal feed, foodstuffs and other ingredients specifically designed for their intended use.

Preferably, according to the invention, the microbial product may contain a broth in which the microorganism has been grown. The product may, for example, comprise at least 1, 5, 10, 25, 50, 75 or 100% by weight of the broth. The mass amount of biomass in the product may be, for example, from about 0 to 100%, including all percentages in between.

Optionally, the product may be stored prior to use. The storage time is preferably short. Thus, the storage time may be less than 60, 45, 30, 20, 15, 10, 7, 5, 3, 2, 1 day, or 12 hours. In a preferred embodiment, if live cells are present in the product, the product is stored at cold temperature, such as, for example, less than 20°C, 15°C, 10°C or 5°C. On the other hand, the biosurfactant composition can usually be stored for longer periods of time and at ambient temperature.

According to one embodiment, the culture products can be obtained as a spray-dried biomass product. Biomass can be separated using 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 culture medium and spray dried.

Microorganism-based products can be formulated in a variety of ways, including liquid, solid, granular, pulverulent, or sustained release products, by means that will be understood by those skilled in the art prior to the present invention.

Formulations for use in humans or animals can be prepared as a liquid, paste, ointment, suppository, capsule or tablet and used in a manner similar to drugs used in the medical drug industry. Formulations may be encapsulated using components known in the pharmaceutical industry. Encapsulation protects the components from unwanted reactions and helps the ingredients withstand adverse environmental conditions or the object or organism being processed, such as the stomach.

The solid formulations according to the invention may be of various shapes and forms, such as cylinders, sticks, blocks, capsules, tablets, briquettes, pellets, strips, commissures, etc. solid finished

- 8 039980 forms can also be ground, granulated or powdered. The granular or powdered material may be compressed into tablets or used to fill prefabricated gelatin capsules or shells. Semi-solid formulations can be prepared as pastes, waxes, gels or creams.

Solid or semi-solid compositions according to 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 especially important for tablets or capsules used in pesticide formulations. The film coating can protect the processor from direct contact with the active ingredient in the formulations. In addition, a bittering agent such as denatonium benzoate or quassin can also be included in pesticide formulations, coatings, or both.

Compositions of the invention may also be prepared in powder form and filled into prefabricated gelatin capsules.

The concentrations of the ingredients in the formulations and the rate of application of the compositions can vary widely depending on the pest, plant or area being treated, or method of application.

In preferred embodiments, the nematode control composition of the present invention comprises an effective amount of a microorganism-produced biosurfactant.

In one embodiment, the microorganism-produced biosurfactant is a glycolipid such as rhamnolipid, sophorolipids (SLP), trehalose lipid, and/or mannosylerythritol lipid (MEL).

In one embodiment, the nematode control composition of the claimed invention comprises an effective amount of a single microorganism-producing biosurfactant and/or a single microorganism producing biosurfactant. In another embodiment, the nematode control composition may comprise a mixture of microbial biosurfactants and/or a mixture of microorganisms that produce these biosurfactants.

In one embodiment, the nematode control composition comprises SLP. The SLP may be in purified form or in a mixture of fermentation products. The composition preferably contains active ingredients such as SLP at a concentration of 0.01-90 wt.%, preferably 0.150 wt.% and more preferably 0.1-20 wt.%. According to another embodiment, the purified SLP may be in combination with an accepted carrier in which the SLP may be present at concentrations of 0.001-50% (v/v), preferably 0.01-20% (v/v), more preferably 0.02- 5% (o/o).

In one embodiment, the nematode control composition comprises MEL. MEL may be in purified form or in a mixture of fermentation products. The composition preferably contains active ingredients such as MEL at concentrations of 0.01-90% by weight, preferably 0.1-50% by weight and more preferably 0.1-20% by weight. According to another embodiment, the purified MEL may be in combination with an accepted carrier in which SLP may be present at concentrations of 0.0001-50% (v/v), preferably 0.005-20% (v/v), more preferably 0.001-5% (o/o).

According to another embodiment, the nematode control composition comprises a mixture of SLP and MEL. SLP and MEL may be mixed in any ratio, provided that the composition contains the active ingredients, the combination of SLP and MEL, at a concentration of 0.01-90 wt.%, preferably 0.1-50 wt.%, and more preferably 0. 1-20 wt.%. According to another embodiment, the purified SLP and MEL may be in combination with an accepted carrier in which the SLP may be present at concentrations of 0.0001-50% (v/v), preferably 0.005-20% (v/v), more preferably 0.001- 5% (o/o).

In a further 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 species, Wickerhamomyces anomalus or Pseudozymas.

Biosurfactants can be used either alone or in combination with other acceptable active or inactive ingredients. These components may be, for example, an oil component such as cinnamon oil, clove oil, cottonseed oil, garlic oil or rosemary oil; other natural surfactant such as Yucca or Quillaja saponins; or the component may be an aldehyde such as cinnamaldehyde. Other oils that can be used as a pesticide component or adjuvants include: almond oil, camphor oil, canola oil, castor oil, cedarwood 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, rapeseed oil, sunflower oil, sage oils, sesame oil, orange oil, thyme oil, vegetable oil, and wintergreen oil.

Other suitable components may be, for example, nematode-attracting substances, such as soluble and gaseous substances produced by the roots of host plants, or microorganisms present in the rhizosphere. One example of a bait for nematodes according to the

- 9 039980 valerian root (Valeriana officianalis). In particular, solutions containing valerian root extract, as well as any other compound or by-product associated with the roots of the valerian plant, can be used as bait according to the claimed composition and methods.

Other suitable additives that may be present in the formulations of the invention include those commonly used in such formulations. Examples of such additives include adjuvants, surfactants, emulsifying agents, plant nutrients, fillers, plasticizers, lubricants, glidants, dyes, pigments, bittering agents, buffering agents, solubility adjusting agents, pH adjusting agents, preservatives, stabilizers and UV-resistant products. Hardening or curing agents may also be included to harden the finished shapes and make them strong enough to withstand pressure or force in some applications, such as tablets for injection into soil, into the top of a root ball, or into a tree.

According to one embodiment, the composition may further comprise buffering agents, including organic and amino acids, or salts thereof. Suitable buffers include citrate, gluconate, tartrate, malate, acetate, lactate, oxalate, aspartate, malonate, glucoheptonate, pyruvate, galactarate, glucarate, tartronate, glutamate, glycine, lysine, glutamine, methionine, cysteine, arginine, and mixtures thereof. You can also use phosphoric and phosphorous acids or their salts. Synthetic buffers are suitable for use, but it is preferred to use natural buffers such as the organic and amino acids or their salts listed above.

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

In one embodiment, additional components such as sodium bicarbonate or carbonate, sodium sulfate, sodium phosphate, sodium biphosphate may be included in the composition.

According to one embodiment, the compositions may contain one or more chemical compounds with nematicidal activity. These include antibiotic nematocides such as abamectin; carbamate nematicides such as benomyl, carbofuran, carbosulfan and cleotocard; nematicides of the carbamate oxime group such as alanicarb, aldicarb, aldoxycarb, oxamyl; organophosphate nematocides such as diamidophos, fenamiphos, fostyethane, phosphamidon, cadusafos, chlorpyrifos, dichlofenthion, dimethoate, etoprofos, fensulfothion, fostiazate, heterophos, isamidophos, isazophos, methomyl, phorate, phosphocarb, terbufos, thiodicarb, thionazine, triazophos, and imicyafos . 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.

According to one embodiment, the compositions may further comprise an effective amount of at least one insecticide. Suitable insecticides include, but are not limited to, nematocidal neonicotinoid insecticides such as 1-(6-chloro-3-pyridylmethyl)-knitroimidazolidine-2-ylidenneamine (imidacloprid); 3-(6-chloro-3-pyridylmethyl)-1,3-thiazolidin-2ylidene-cyanamide (thiacloprid); 1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine (clothianidin); nitempiran; K1-[(6-chloro-3-pyridyl)methyl]-K2 ^- cyano-K1-methylacetamidine (acetamiprid); 3-(2chloro-1,3-thiazol-5-ylmethyl)-5-methyl-1,3,5-oxadiazinan-4-ylidene-(nitro)amine (thiamethoxam); and 1-methyl2-nitro-3-(tetrahydro-3-furylmethyl)guanidine (dinotefuran).

Application of products based on microorganisms

According to another aspect, the claimed invention includes methods, systems and devices for the use of products based on microorganisms.

According to one embodiment, the claimed invention provides a method of improving plant health and/or increasing crop yield by incorporating a composition disclosed herein into soil, seeds, or parts of plants. According to another embodiment, the claimed invention provides a method for increasing the yield of an agricultural crop or plant, including multiple applications of the composition described herein.

Preferably, the method can effectively control nematodes and related pest diseases while increasing yield while avoiding side effects and additional costs.

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 used to treat seeds. Use for seed treatment is advantageous because application can be easily provided and the amount used for treatment can be reduced, further reducing potential toxicity, if any.

In one embodiment, the biosurfactant-producing microorganisms

- 10 039980 and/or biosurfactants can be added to the soil, to the growing medium, to the plants, to the aquatic environment, or to any area to be treated to prevent pest damage. Microorganisms can grow in situ and produce biosurfactants in situ to control nematodes. In addition, natural biosurfactant-producing organisms applied at the application site will be able to grow and produce the biosurfactant. Therefore, a high concentration of biosurfactant and biosurfactant-producing microorganisms can be easily and continuously obtained at a treatment site (eg, soil).

Substances that enhance microbial growth and production of biosurfactants, to support the growth of biosurfactant-producing organisms, and also to provide the desired biosurfactants in situ can also be added to the composition and/or site of treatment to achieve the object of the invention. These substances include, but are not limited to, carbon, organic substrates such as oil, glycerin, sugar, or other nutrients. For example, to produce a biosurfactant in situ and control nematodes, biosurfactant-producing organisms can grow on a substrate. Natural biosurfactant-producing organisms at the site of application (in soil, water system, plant parts, etc.) will produce biosurfactants when using a carbon substrate. During this process, the biosurfactant produced will destroy and paralyze the pests in the target area. According to the invention, it is not necessary to precontaminate the target site or carbon substrate mixture with biosurfactant-producing organisms.

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

One embodiment includes adding or incorporating sufficient specific biosurfactant into the carbon substrate to initiate the emulsification process and inhibit or reduce the growth of other organisms that compete with the biosurfactant-producing organism and to control nematodes. For example, Pseudomonas syringae and Bacillus subtilis produce a series of lipopeptide biosurfactants called porenes. These lipopeptide porenes include pseudomycin, syringomycin, tabtoxin, phaseolotoxin and surfactin. Some lipopeptides are able to create holes in cell membranes, cells and tissues.

Pseudomycin can be used as a pre-sowing treatment for nematode control in crop production. If it is necessary to stimulate the growth of Bacillus subtilis, a small amount of surfactin biosurfactant is added to the carbon substrate medium to help establish a B. subtilis population and produce more surfactin locally.

In one embodiment, the composition may be applied by spraying, pouring, dipping, as concentrated or dilute liquids, solutions, suspensions, powders, and the like, containing such active agent concentrations as are most appropriate for the particular intended purpose. They can be used ready-made or reconstituted before use. For example, they can be applied by direct injection into trees or into the top of the root ball.

In one embodiment, the composition of the claimed invention may be applied in an amount of from about 0.0001 lb/acre to about 10 lb/acre, from about 0.001 lb/acre to about 5 lb/acre, from about 0.01 lb/acre to about 1 lb/acre, about 0.01 lb/acre to about 0.1 lb/acre, or about 0.01 lb/acre to about 0.05 lb/acre.

In one embodiment, the claimed composition is applied to a plant or crop from about 1 to about 100 days, from about 2 to about 50 days, from about 10 to about 40 days, from about 20 to about 30 days after initial application to the soil or on seeds.

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

The application to the seeds can take place, for example, by coating the seeds or by applying the composition to the soil at the same time as the seeds are sown. This can be automated, for example, by providing a device or irrigation system that applies the microbial composition with and/or near the seed, at or near the time the seed is planted. Thus, the microbial composition can be applied within, for example, 5, 4, 3, 2 or 1 days before or after the sowing time or at the same time as the sowing of the seeds.

According to certain embodiments, the codes provided herein

- 11 039980 positions are applied to the soil surface without mechanical application. Beneficial effects resulting from application to the soil can be activated by rainfall, sprinkler, flood or drip irrigation and then carried, for example, to target pests to reduce their population levels to acceptable thresholds, or to plant roots to affect the microbial content of their roots or to facilitate uptake of the microbial product into the vascular system of the culture or plant to which the microbial product is applied. In an exemplary embodiment, the compositions provided herein can be effectively applied by center pivot or seed furrow spray.

Reference herein to administering a composition to or near a pest or plant, or to the environment of the pest or plant, means that the administration is such that the composition is in sufficient contact with the pest or plant such that the desired result is achieved (e.g., killing the pest, increasing yield, prevention of plant damage, regulation of genes and/or hormones, etc.). Typically, this may be, for example, within 10, 5, 3, 2, or 1 feet or less of a pest, plant, weed, or other desired target.

The microbial product can also be used to promote colonization of the roots and/or rhizosphere as well as the plant vasculature to promote plant vigor and vitality. Nutrient fixing micro-organisms such as rhizohium and/or mycorrhzae, as well as other endogenous (already present in the soil) as well as exogenous, micro-organisms or their by-products that fight pests or diseases or otherwise promote growth can thus be stimulated. crop, hardiness and/or yield. The microbial product can also support the plant vasculature, for example by penetrating and colonizing the vasculature and introducing metabolites and nutrients important for plant health and productivity, or metabolites with pest control properties.

Advantageously, the method does not require complex equipment or high energy consumption. The microorganisms of interest can be cultured on a small or large scale in situ and used, even mixed with their medium. Likewise, metabolites of microorganisms can also be produced in large quantities at the desired location.

Advantageously, microorganism-based products can be produced in remote locations. In one embodiment, microbial-based products can be used for human nutrition and/or disease prevention and/or treatment.

Target pests

According to preferred, but non-limiting embodiments of the invention, the control nematodes are selected from (1) nematodes that are plant pathogenic nematodes, such as, but not limited to, root-knot nematodes (Meloidogyne species) in rice (e.g., M. incognita, M. javanica or M. graminicola), soybeans (eg M. incognita or M. arenaria), cotton (eg M. incognita), potatoes (eg M. chitwoodi or M. hapla), tomatoes (eg , M. chitwoodi), tobacco (eg M. incognita, M. javanica or M. arenaria) and corn (eg M. incognita); cyst nematodes (Heterodera spp.) in rice (eg H. oryzae), soybean (eg H. glycines) and corn (eg H. zeae); cyst nematodes (Globodera spp.) in potatoes (eg G. pallida or G. rostochiensis); kidney nematodes (Rotylenchulus spp.) in cotton (eg R. reniformis); root nematodes (Pratylenchus spp.) on banana (eg P. coffeae or P. goodeyi); burrowing nematodes (Radopholus spp.) on the banana (eg R. similis); and other rice-damaging nematodes such as the rice root nematode (Hirschmaniella spp., eg H. oryzae);

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

(3) nematodes capable of infecting animals such as, but not limited to, dogs (hookworms, such as Ancylostoma caninum or Uncinaria stenocephala, roundworms, such as Toxocara canis or Toxascaris leonina, or whipworms, such as Trichuris vulpis), cats (hookworms , e.g. Ancylostoma tubaeforme, roundworms e.g. Toxocara cati), fish (herring or cod worms e.g. Anisakid or tapeworm e.g. Diphyllobothrium), sheep (filamentous e.g. Haemonchus CHartus) and cattle ( intestinal worms, eg Ostertagia ostertagi, Cooperia oncophora);

(4) nematodes that cause unwanted damage to substrates or materials, such as nematodes that damage food, seeds, wood, paint, plastic, clothing, etc.; examples of such nematodes include, but are not limited to, Meloidogyne species (e.g., M. incognita, M.

- 12 039980 javanica, M. arenaria, M. graminicola, M. chitwoodi or M. hapla); Heterodera spp. (eg H. oryzae, H. glycines, H. zeae or H. schachtii); Globodera spp. (eg G. pallida or G. rostochiensis); Ditylenchus spp. (eg D. dipsaci, D. destructor or D. angustus); Belonolaimus species; Rotylenchulus spp. (eg R. reniformis); Pratylenchus spp. (eg P. coffeae, P. goodeyi or P. zeae); Radopholus spp. (eg R. similis); Hirschmaniella spp. (eg H. oryzae); Aphelenchoides spp. (eg A. besseyi); spp. Criconemoides; species of Longidorus; Helicotylenchus species; Hoplolaimus species; Xiphinema species; Paratrichodorus spp. (eg P. minor); Tylenchorhynchus species;

(5) virus vector nematodes (e.g., Longidorus macrosoma: transmits plum necrotizing ring spot virus, Xiphinema americanum: transmits tobacco ring spot virus, Paratrichadorus teres: transmits pea early browning virus, or Trichodorus similis: transmits tobacco rattle virus).

Specific nematode pests include:

Dracunculus medinensis, roundworms that cause dracunculiasis (a disease caused by the guinea helminth); nematodes Loa loa (African eyeworm), Mansonella streptocerca and Onchocerca volvulus, which cause cutaneous filariasis; Mansonella perstans and Mansonella ozzardi, which cause abdominal filariasis; Trichinella, including T. pseudospiralis (infecting mammals and birds worldwide), T. nativa (infecting polar bears), T. nelsoni (infecting African carnivores and scavengers), and T. britovi (infecting European and western Asian carnivores), which causes Trichinellosis; Angiostrongylus cantonensis (rat lung hookworm), 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 causes gnathostomiasis; and Anisakis simplex, or Pseudoterranova decipiens, which causes anisakiasis.

In specific embodiments, the methods and compositions of the claimed invention are used to control root-knot nematodes (Meloidogyne incognital), stinging nematodes (Belonolaimus longicaudatus), soybean cyst nematodes (Heterodera glycines), wounding nematodes (Pratylenchus sp.), dagger nematode (Xiphinema sp. .) and/or citrus nematodes (Tylenchulus semipenetrans).

target plants

Plants that may benefit from the products and methods of the claimed invention include row crops (e.g., corn, soybeans, sorghum, peanuts, potatoes, etc.), field crops (e.g., alfalfa, wheat, cereals, and 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.), turf crops, ornamental crops (e.g. flowers, grapes, etc.), vegetables (e.g. tomato, carrots, etc.), vine crops (e.g. grapes, strawberries, blueberries, blackberries, etc.), forest crops (eg pine, spruce, eucalyptus, poplar, etc.), pasture crops (any mixture of plants used for grazing animals).

The benefit may be in the form of, for example, increased yield, quality, resistance to diseases and pests, reduced stress (eg from salt, drought, heat, etc.) and improved water use.

Other plants that may benefit from the products and methods of the invention include all plants that belong to the superfamily Viridiplantae, in particular monocots and dicots, including fodder or fodder legumes, ornamental plants, food crops, trees or shrubs selected from the species. Acer, Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp. spp., Agrostis stolonifera, Allium spp. spp., Amarantus spp. spp., Ammophila arenaria, Ananas comosus, Annona spp. Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa spp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa spp. (canola, oilseed rape, oilseed turnip)), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum species, Carex ela ta, 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 spp., 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. spp., Eriobotrya japonica, Eucalyptus spp., 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 batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus, Luzula sylvatica, Lycopersicon species

- 13 039980 (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 spp., Momordica, Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (for example, Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum 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 spp., Sambucus spp., Secale cereale, Ses spp. amum, Sinapis spp, 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. 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 species.

Additional examples of plants of interest include, but are not limited to, corn (Zea mays), Brassica spp. (e.g., B. napus, B. rapa, B. juncea), especially those Brassica spp. sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g. American millet (Pennisetum glaucum), common millet (Panicum miliaceum), Italian millet (Setaria italica), millet palmate (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (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), ban en (Musa species), 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 beet (Beta vulgaris), sugar cane (Saccharum species), oats, barley, vegetables, ornamental plants and conifers.

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 Cucumis genus such as cucumber (C. sativus), cantaloupe (C. cantalupensis) and musk 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), carnations (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima) and chrysanthemum. Conifers that can be used in practical embodiments include, for example, pines such as fox pine (Pinus taeda), Elliot pine (Pinus elliotii), Oregon pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine ( Pinus radiata); tissolist pseudosuga (Pseudotsuga menziesii); western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); sequoia evergreen (Sequoia sempervirens); real spruces such as white fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as the giant thuja (Thuja plicata) and the nootkatensis callitropsis (Chamaecyparis nootkatensis). Plants according to embodiments include agricultural plants (eg, corn, alfalfa, sunflower, cabbage, soybean, cotton, sunflower, peanut, sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Lawn grasses include, but are not limited to, annual bluegrass (Poa annua); ryegrass multiflorum (Lolium multiflorum); oblate bluegrass (Poa compressa); red fescue (Festuca rubra); hairy bent grass (Agrostis tenuis); marsh bent grass (Agrostis palustris); comb wheatgrass (Agropyron desertorum); desert couch grass (Agropyron cristatum); harsh fescue (Festuca longifolia); meadow bluegrass (Poa pratensis); hedgehog team (Dactylis glomerate); perennial ryegrass (Lolium perenne); red fescue (Festuca rubra); bent grass (Agrostis alba); common bluegrass (Poa trivialis); grooved fescue (Festuca ovine); awnless bonfire (Bromus inermis); cane fescue (Festuca arundinacea); timothy (Phleum pretense); bent dog (Agrostis canine); drooping beskilnitsa (Puccinellia distans); Smith's wheatgrass (Agropyron smithii); pig fingered (Cynodon species); Augustine grass (Stenotaphrum secundatum); Zoysia Japanese (Zoysia species); noticeable buckwheat (Paspalum notatum); wonderful axonopus (Axonopus affinis); grass centipede (Eremochloa ophiuroides); Kikuya (Pennisetum clandesinum); seaside paspalum (Paspalum vaginatum); butelua (Bouteloua gracilis); bison grass (Buchloe dactyloids); butelua lateral (Bouteloua curtipendula).

Other plants of interest include Cannabis (eg sativa, indica and ruderalis) and industrial hemp.

Plants of interest include cereal plants, which provide seeds of interest, oil plants, and leguminous plants. Seeds of interest

- 14 039980 includes grain seeds such as corn, wheat, barley, rice, sorghum, rye, millet, etc. Oil plants include cotton, soybean, safflower, sunflower, cabbage, corn, alfalfa, palm, coconut, flax, castor bean, olive, etc. Pulses include beans and peas. Beans include guar, carob, fenugreek, soybeans, common beans, cowpeas, mung beans, lima beans, horticultural beans, lentils, chickpeas, etc.

All plants and plant parts can be treated in accordance with the invention. In this context, plants should be understood as all plants and plant populations, such as desired and undesired wild plants or crop plants (including naturally occurring crop plants). Agricultural plants can be plants that can be obtained by conventional breeding and optimization methods, or by biotechnological and recombinant methods, or combinations of these methods, including transgenic plants and plant varieties.

Parts of plants should be understood as all above and below ground plant parts and organs such as shoot, leaf, flower and root, examples that may be mentioned include leaves, needles, cuttings, stems, flowers, fruiting bodies, fruits and seeds, but also roots, tubers and rhizomes. Plant parts also include plant material and material for vegetative and generative propagation, such as cuttings, tubers, rhizomes, shoots and seeds.

Examples

It should be understood that the embodiments described herein are examples and are for illustrative purposes only and that, therefore, various modifications or changes will be suggested to those skilled in the art, which should be included within the spirit and scope of the present invention.

Example 1 Fermentation of S. bombicola to produce sophorolipid (SLP) in a 110 L dispense reactor.

Uses a portable, airlift, fully enclosed reactor controlled by PLC with water filtration, temperature control unit and built-in air blower. The reactor has a working volume of 90 liters when growing Starmerella bombicola to obtain SLP.

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

The reactor is inoculated with 8 L of liquid culture grown in flasks. The duration of the cultivation cycle for obtaining SLP is 7-8 days, at 25°C and pH 3.5, with sampling carried out twice a day.

The final concentration of SLP is approximately 10% of the working volume, in this case, approximately 9 liters of product contains 300-400 g SLP/l.

Example 2 Fermentation of S. bombicola to produce SLP in a portable 14 L dispense reactor.

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

The nutrient medium contains glucose, yeast extract, urea and vegetable oil. The inoculum may be a 1-2 day old culture of Starmerella bombicola in an amount of approximately 5-10% of the total culture volume.

The duration of cultivation and collection of the finished product lasts 5-14 days. The final production of sophorolipid can reach 1-2 kg per cycle.

Example 3 Fermentation of Wickerhamomyces and/or Pichia yeasts to produce SLP in a 450 liter partition reactor.

A mobile airlift reactor controlled by PLC with water filtration, a temperature control unit and an air blower for sufficient aeration is used. The process can be carried out as a batch culture process. The reactor has a working volume of 400 liters, when growing Wickerhamomyces or Pichia to obtain SLP.

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

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

The final concentration of SLP is approximately 20-25% of the working volume, in this case more than 90 liters of product are formed.

Example 4 Fermentation of Wickerhamomyces and/or yeast Pichia to produce cellular and unicellular protein in a 900 liter dispense reactor.

A portable reactor is used, divided into two tanks served by a central

- 15 039980 airlift to facilitate simultaneous mixing of the two tanks. The reactor has a working volume of 600 liters, when growing Wickerhamomyces and/or Pichia to obtain cells.

In a preferred embodiment, the nutrients for cell production are glucose or baker's sugar, urea, yeast extract, magnesium sulfate and potassium phosphate.

The reactor is inoculated with 2% of the seed culture. Fermentation is continued for 48-72 hours without pH stabilization and at a temperature of 26-32°C.

The final cell concentration will be 100 g wet wt/l. Wet biomass concentration can reach 90 kg per cycle with protein concentration up to 45 kg.

Example 5 Fermentation of Wickerhamomyces and/or yeast Pichia to produce cellular and unicellular protein in a 2000 liter dispense reactor.

A portable reactor is used, divided into two square tanks, with 2 mass transfer loops between them. The reactor has a working volume of 2000 liters, when growing Wickerhamomyces and/or Pichia to obtain cells.

In a preferred embodiment, the nutrients for cell production are glucose or baker's sugar, urea, yeast extract, magnesium sulfate and potassium phosphate.

The reactor is inoculated with 2% of the seed culture. Fermentation is continued for 48-72 hours without pH stabilization and at a temperature of 26-32°C.

The final cell concentration will be 100 g ww/l. Wet biomass concentration can reach up to 200 kg per cycle with protein concentration up to 100 kg.

Example 6 Fermentation of Pseudozyma aphidis to produce mannosyl erythritol lipid (MEL) in a portable 14 L dispense reactor.

It is a steam autoclavable jacketed air-purged glass vessel with a Rashtan impeller. It is equipped with a DO, pH, temperature and foam probe, it has a built-in control station with a color touch interface, built-in pumps, gas flow controllers and foam pH/DO level controllers. The working volume of the reactor is 10 liters.

The composition of the nutrient medium: sodium nitrate, potassium phosphate, magnesium sulfate, yeast extract and vegetable oil. The inoculum may be a 1-2 day old Pseudozyma aphidis culture, in an amount of approximately 5-10% of the total culture volume. Duration of cultivation and sample collection: 915 days. Final receipt of MEL: 800-1000

Example 7 Use of SLP to reduce gall nematode eggs on tomato plants.

Starmerella bombicola was grown as described above. At the end of the culture fermentation, the broth precipitates and most of the SLP separates as a dark brown layer (approx. 500 g/l concentration). This brown layer contains mostly SLPs and some cells. The layer is collected and the pH is adjusted to pH 7.0. This solution is then diluted with water to obtain the required concentrations.

day. Germination of tomato seeds (Moneymaker).

day. Preparation of a mixture of sand/clay turface (1:1) with 1% fertilizer (osmokot), transplanting 2-week-old tomato seedlings a day. Infection with microorganisms by soaking the soil of plants at the age of 4 weeks. For each 10 cm x 10 cm square pot, 80-100 ml of culture was sufficient to wet the soil.

day. Four days after inoculation with microorganisms, RKN eggs were applied to each plant (4000 eggs per pot).

day. Six weeks after infection with eggs, harvest the plant.

After harvesting, the plants were rinsed with tap water to remove sand/clay turface located on the root.

The wet weight of the roots was recorded. After that, the roots were cut into small pieces and placed in a small plastic lunch bag. After adding 200 ml of 5% sodium hypochlorite solution, the bag was incubated and gently shaken for 15 minutes. Then the eggs were counted in 10 µl samples in triplicate under a stereomicroscope. The total number of eggs per root was calculated. As a result, the number of eggs in each gram of root biomass was calculated.

SLP 0.25% also showed activity against root-knot nematode (RNK) with a reduction in eggs of approximately 52%.

Example 8 Comparison of the ability of SLP, MEL and Nimitz® to fight root-knot, sting and wound nematodes.

Nimitz® is the first new chemical nematocide developed over 20 years ago. Nimitz® exhibits irreversible and rapid nematicidal activity immediately after application. Within one hour of contact, the nematodes stop eating and quickly become paralyzed. Within 24-48 hours, the death of pests occurs, and not a temporary nematostatic (stop) activity, which is demonstrated by organophosphate and carbamate nematocides.

- 16 039980

Pseudozyma aphidis was grown as described above. At the end of the fermentation, the entire culture broth was used, since MEL is very difficult to separate without the use of a solvent. The MEL is then diluted with water to the required concentration just prior to use.

Starmerella bombicola was grown as described above. At the end of the culture fermentation, the broth precipitates and most of the SLP separates as a dark brown layer (approx. 500 g/L concentration). This brown layer contains mostly SLPs and some cells. The layer is collected and the pH is adjusted to pH 7.0. This solution is then diluted with water to obtain the required concentrations.

The following bioassay was performed: Cavity glass slides were filled with appropriate amounts of test product. A known population of target nematode larvae is added to the solution containing the nematode. Cavity slides are kept at room temperature. The death of nematodes can be assessed by counting the number of nematodes after 24, 48 and 72 hours. The percentage of death was calculated as: [average number of dead larvae per treatment / total number of larvae per treatment]x100.

A. Gall nematodes.

Root-knot nematode (Meloidogyne incognita) death (i.e. how many nematodes died) and immotility (i.e. how many nematodes were fatally affected) were determined 3 days after treatment with SLP (SD10) or MEL (SD12) at different concentrations and compared with Nimitz® treatment and control without treatment (FIGS. 1 and 2). The percentage kill rate was calculated from the number of dead nematodes/the value of the total number of nematodes before testing. The percentage of non-motile nematodes was calculated from the number of non-motile nematodes/number of live nematodes. Thus, motility was calculated as the proportion of those nematodes that did not die, but were determined to be immobile.

In the test, the most effective treatment was a high level of MEL (0.1% v/v). In terms of death, it was significantly more effective than Nimitz®. He observed a clear dose-dependent response. The results also show that SLP was as effective as Nimitz® on root-knot nematode death and immobility.

B. Stinging nematodes.

Mortality and demobilization rates of stinging nematodes (Belonolaimus longicaudatus) were determined 3 days after treatment with SLP or MEL at different concentrations and compared with Nimitz® treatment and untreated control (FIGS. 1 and 2).

The results show that both SLP and MEL increase the mortality and reduction rate of stinging nematodes in a dose-dependent manner. High levels of MEL (0.1% or 0.2% v/v) were the most effective treatments in the test. They were as effective as Nimitz® in terms of death and more effective than Nimitz® in terms of reduction in mobility.

C. Wounding nematodes.

Mortality and motility reduction rates of wounding nematodes (Pratylenchus sp.) were determined 3 days after treatment with SLP or MEL at various concentrations and compared with Nimitz® treatment and untreated control (FIGS. 1 and 2).

The results show that both SLP and MEL increase the mortality and rate of reduction of motility of injuring nematodes in a dose-dependent manner. In the test, MEL was more effective than SLP and comparable to Nimitz®.

Example 9 Evaluation of different treatments against southern root-knot nematodes in vessel tests in a phytopathological laboratory.

SLP (SD10) and MEL (SD12) treatments applied directly to nematode-infected soil were tested at two rates, alone or together, and compared with Nimitz® and control without treatment to reduce live southern root-knot nematode populations. Counting was performed at three different time points after treatment.

Six repetitions of 7 treatments were carried out in glass jars containing 350 cm ³ 99% sandy soil. Vessels were infected with 70 adult southern root-knot nematodes (Meloidogyne incognita). One application of the drug was carried out immediately after infection with nematodes, when 85 ml of the drug was manually poured into each vessel. Drug mixtures were prepared according to Table 1 (below). Live and dead nematodes were counted on days 3, 14 and 23 after treatment. Also, the table summarized the count of immobile nematodes as a proportion of all living ones.

- 17 039980

Table 1 Preparation of mixtures for processing

No. arr. Processing name Form type Norm Norm unit Quantity of product to measure 1 SD10 L 0.25 % v/v 2.5 ml/cm 2 SD10 L 0.5 % v/v 4.999 ml/cm 3 SD12 L 0, 1 % v/v 0.9999 ml/cm 4 SD12 L 0.2 % v/v 2.0 ml/cm 5 SD10+ SD12 L L 0.25 0, 1 % v/v % v/v 2.5 ml/cm 0.9999 ml/cm 6 Nimitz® L 10 ppm rg 9.999 ml/cm 7 Check without processing

Results.

Vessel testing results for SD10 and SD12 products indicate a positive dose response for activity against southern root-knot nematodes. SD10 and SD12 taken together at a low rate were not as effective as either product alone at a higher rate. Nimitz® provided excellent fighting results.

In FIG. 3 and in table. 2 (below) shows the live nematode count for each treatment. In FIG. 4 and in table. 3 (below) shows the live nematode control (ie nematode kill rate) for each treatment.

Table 2. Enumeration of live nematodes

No. arr. Processing name Norm Norm unit Code etc. Preliminary count 3DA-A 14DA-A 23DA-A 1 SD10 0.25 % v/v A 77.00 A 52.00 A 38.17 A 13.5 A b 2 SD10 0.5 % v/v A 77.00 A 47.67 A 36.67 A 11.50 IN 3 SD12 0.1 % v/v A 77.00 A 46, 33 A 36.00 A 11, 83 V 4 SD12 0.2 % v/v A 77.00 A 48.50 A 33.33 A 8.17 With 5 SD10+ SD12 0.25 oh 1 % v/v % v/v A 77.00 A 46.00 A 36.50 A 11, 83 V 6 Nimitz® 10 ppm rg A 77.00 A 16.00 b 4.17 b 1, 67 D 7 Check without processing 77.00 A 50.50 A 39.83 A 15.33 A

As early as day 3 (3 DA-A) there were significantly higher control results reported in vessels treated with Nimitz® (10 ppm). For each of SD10 and SD12, a significant dose response was observed at 23 DA-A, with samples treated with a higher rate having fewer nematodes than samples with a lower rate. When using the lower rate SD10 and SD12 in combination, nematode mortality was not significantly increased to the levels seen with the higher rate treatments.

- 18 039980

Table 3. Control of live nematodes (%)

No. arr. Processing name Norm Norm unit Code pr. 3DA-A 14DA-A 23DA-A 1 SD10 0.25 % v/v A 1.84 bc 9.09% bc 15.56 cd 2 SD10 0.5 % v/v A 8.29 bc 8.03% bc 25.72 With 3 SD12 0.1 % v/v A 8.84 bc 9.24% bc 22.18 With 4 SD12 0.2 % v/v A 9.39 b 18.64% b 44.88 b 5 SD10+ SD12 0.25 0.1 % v/v % v/v A 10.88 b 11.40% bc 20, 90 With 6 Nimitz® 10 ppm rg A 68.24 A 89.94% A 89.77 A 7 Check without processing 0.00 With 0.00% With 0.00 d

In FIG. 5 and Table 4 (below) show the count of immobile nematodes. In FIG. 6 and Table 5 (below) show the count of immobile live nematodes (%).

Table 4. Enumeration of immobile nematodes

No. arr. Processing name Norm Norm unit Code pr. 3DA-A 14DA-A 23DA-A 1 SD10 0.25 % v/v A 3.33 WITH 4, 67 bc 2.5 Ab 2 SD10 0.5 % v/v A 5, 17 Sun 4, 67 bc 3, 17 A 3 SD12 0, 1 % v/v A 7.33 IN 4, 67 bc 1.50 Sun 4 SD12 0.2 % v/v A 11, 8 3 A 6, 67 A 2.00 AL WITH 5 SD10+ SD12 0.25 0, 1 % v/v % v/v A 100 0 A 6, 33 AL 3, 17 A 6 Nimitz® 10 ppm rg A 4, 67 WITH 2.50 D 0.67 WITH 7 Check without processing 2.83 With 3.33 cd 1, 83 ab c

The number of immobile nematodes was the lowest in samples treated with Nimitz®. At 3 DAA there was a higher proportion of non-motile nematodes at elevated rates of either SD10 or SD12, confirming that early pesticide activity did not result in immediate mortality. At 14 DA-A, the proportion of live nematodes assessed as immobile was not statistically different among treated SD vessels.

- 19 039980

Table 5. Count of immobile live nematodes (%)

No. Name Norm Unity Code 3DA-A 14DA-A 23DA-A arr. processing ca norms etc. 1 SD10 0.25 % v/v A 6, 39 E 12.86 b 18.80 A 2 SD10 0.5 % v/v A 11.1 8 De 12.55 b 28, 61 A 3 SD12 0, 1 % v/v A 15, 7 8 CD 13.51 b 13, 16 A 4 SD12 0.2 % v/v A 25.0 3 Ab 20, 85 b 22.59 A 5 SD10+ SD12 0.25 0, 1 % v/v % v/v A 22.0 0 Sun 17.89 b 27.85 A 6 Nimitz® 10 ppm rg A 30.5 5 A 43, 06 A 36, 67 A 7 Check without processing 5, 91 e 8.54 b 13, 01 A

Example 10 Use of MEL as a nematocide in a cucumber plant microplot system.

An outdoor microplot located in Thonotosassa, Florida, USA was used to study MEL nematode treatment for cucumber plants compared to Nimitz® and no treatment controls. Six different treatment groups were studied, each consisting of 8 plants. For groups using SD12 treatment (0.2% ai/v), the drug was applied to the soil once, twice or three times. Both the nematode count and the rate of gall formation were determined at harvest. Plant power, yield, and plant weight were also measured.

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

Nematode counts and gall rates were performed for all groups 31 days after final SD12 treatment in the triple treatment group. Plant vigor (0-10 index/scale), yield (lb) and weight (lb) were measured the next day.

Results.

Normal response was observed by gall formation (FIG. 7 and Table 6 below) and by nematode count after treatment (FIG. 8 and Table 7 below). SD12 are made to commercial standards. There was no significant difference between the SD12 treatments and the Nimitz® treatment, but significant differences were observed between the SD12 treatments and the no-treatment control. Table 8 below shows the power, yield and weight of plants at harvest.

Table 6. Rating of gall formation (1-10 index/scale)

No. arr. Processing name Norm Unit norms Code pr. Rating gall formation 1 without processing 2.9 A 2 Nimitz® 5 Pt/a A eleven IN 3 SD12*1 0.2 % ai/o IN 1.1 IN 4 SD12*2 0.2 % ai/o sun 0.7 IN 5 SD12*3 0.2 % ai/o BCD 0.3 IN

LSD (P=0.05)=0.88; SD=0.86; CV=69.39; X2 Bartlett=11.754; P (X2 Bartlett)=0.019*; repeat F=3.712; repeat Prob(F)=0.0061; processing F=11.133; processing prob(F)=0.0001

- 20 039980

Table 7. Number of nematodes

No. arr. Processing name Norm Norm unit Code pr. Quantity nematodes 1 without processing 59 A 2 Nimitz® 5 Pt/a A 27 IN 3 SD12*1 0.2 % ai/o IN 20 IN 4 SD12*2 0.2 % ai/o sun 18 IN 5 SD12*3 0.2 % ai/o BCD 9 IN

LSD (P=0.05)=17.33; SD=16.3; CV=63.88; X2 Bartlett=17, 263; P(X2 Bartlett)=0.02*; repeat F=2.774; repeat Prob(F)=0.0261; treatment F=10.44; processing prob(F)=0.0001

Table 8. Plant power (0-10), yield (f.) and weight (f.). Measurements when harvesting plants

No. arr. Processing name Norm Norm unit Code pr. Power Harvest Weight 1 without processing 6, 2 IN 2.18 8 A 1, 13 1 A 2 Nimitz® 5 Pt/a A 8.0 A 3, 06 3 A 1.55 0 A 3 SD12*1 0.2 ai/o IN 8, 1 A 2.43 8 A 1.28 8 A 4 SD12*2 0.2 ai/o sun 8.4 A 2, 63 8 A 1.26 3 A 5 SD12*3 0.2 ai/o BCD 8.2 A 3.28 1 A 1.42 5 A

Example 11 Nematode Bait Performance Evaluations.

Percentage numbers and infestations of southern root-knot nematodes were obtained in four sealed chambers of 11.6 in x 7.6 in containing lacustrine fine sand soil with added bait material. To obtain the bait, a pre-prepared, purchased valerian root extract was mixed with water, vegetable glycerin and 20% grain alcohol.

Each plot was infected with nematodes in a zone with a diameter of 2 cm. In a zone of 3 cm (h) x 1 cm (w), 10 ml of nematode bait was added, 2 cm from the zone of infection (Fig. 9).

Percentage counts and nematode infestations were obtained at three locations, 3 days post-treatment and 8 days post-treatment. The three sites tested were the center of the infection zone, the bait zone, and the untreated zone.

Results.

The results are shown in FIG. 10 and in Table 9 (below).

Migration towards the bait area or no treatment differed significantly from the challenge area as a proportion of the total population. In the case of sampling after 24 and 48 hours, the number of nematodes in the bait zone than in the zones without chamber treatment was more than 14%; however, the central zone, where nematode infestation occurred, contained the most nematodes.

- 21 039980

Table 9

Quantity Infection % No. arr. Processing area Norm Unit norms 3DA-A 8DA-A 3DA-A 8DA-A 1 Center 63.50 A 22.50 A 62.17 A 76, 52 A 2 Treatment -Bait 10 ml/group 27.50 b 6.00 b 28.67 b 18.66 b 3 Without processing 8.75 b 1.75 b 9.16 With 4.82 With

All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated herein by reference in their entirety, including all figures and tables, to the extent that they do not conflict with the express provisions of this specification.

The description in this document of any aspect or embodiment of the invention, using such terms as comprising, having, including, or including with reference to an element or elements, is intended to provide support for a similar aspect or embodiment of the invention, which consists of, consists essentially of or essentially contains that particular element or elements, unless otherwise indicated or clearly inconsistent with the context.

Claims (11)

CLAIM 1. A method for controlling plant pests with nematodes, wherein said method includes obtaining a nematocidal composition based on microorganisms by cultivating microorganisms under such conditions that the microorganism produces a biosurfactant based on mannosyl erythritol lipid (MEL), wherein said nematocidal composition contains microorganisms and/or nutrient broth, in which the microorganism is cultivated, wherein said broth contains MEL, and wherein said method includes mixing the nematicidal composition with water and applying the nematicidal composition to nematodes, soil, and/or to treat seeds. 2. The method of claim 1 wherein the microorganism is Pseudozyma aphidis. 3. The method of claim 1, wherein the method further comprises treating the nutrient broth to separate the MEL-based biosurfactant from the cell mass, followed by mixing the biosurfactant with water and applying the biosurfactant to nematodes, soil, and/or seed dressing. 4. The method of claim 1, wherein the nutrient broth is applied to nematodes, soil, and/or seed dressing without first separating the MEL-based biosurfactant from the cell mass. 5. The method of claim 1, wherein the nematodes are selected from the following: root-knot nematode (Meloidogyne incognital), stinging nematode (Belonolaimus longicaudatus), soybean cyst nematode (Heterodera glycines), wounding nematode (Pratylenchus sp.), burrowing nematode (Xiphinema sp. .) and citrus nematode (Tylenchulus semipenetrans). 6. The method of claim 1 wherein the plants are selected from tomato, soybean, corn, citrus and turf grasses. 7. The method according to claim 1, which further includes the use of valerian root extract. 8. A method of controlling plant pests with nematodes, wherein said method comprises applying a composition consisting of a mannosyl erythritol lipid (MEL) biosurfactant mixed with water to nematodes, soil, and/or to treat seeds. 9. The method of claim 8 further comprising applying the SLP to the nematodes, soil and/or seeds. 10. The method of claim 9, further comprising applying the valerian root extract. 11. The method according to claim 9, used to control nematodes selected from the following: root-knot nematode (Meloidogyne incognital), stinging nematode (Belonolaimus longicaudatus), soybean cyst nematode (Heterodera glycines), wounding nematode (Pratylenchus sp.), burrowing nematode (Xiphinema sp.) and citrus nematode (Tylenchulus semipenetrans).