Oil dispersion

Abstract

The present invention relates to a process for preparing an oil dispersion comprising microbial cells, characterized by the steps of: a) Mixing a hectorite and an oil to prepare a gel, wherein said mixing is performed at a shear force of from 0.1 to 20 kW / m3, and, b) Mixing a composition comprising microbial cells with the gel prepared in step a). The invention also relates to an oil dispersion obtainable by the process according to the invention and a method of using the oil dispersion in agriculture.

Description

[0001] OIL DISPERSION

[0002] The present invention relates to a process for preparing an oil dispersion comprising microbial cells. The invention also relates to an oil dispersion obtainable by a process according to the present invention, and a method of using the oil dispersion comprising microbial cells in agriculture, such as in treating plant against phytopathogenic microorganisms, reducing abiotic stress, or improving plant health or yield.

[0003] BACKGROUND

[0004] Oil dispersions are well known types of formulations for active ingredients that are used as biocontrol agents in agriculture. However, not every type of oil dispersion is suitable for every active ingredient that is used in agriculture, depending on the stability of the active ingredient, phase separation of the oil dispersion and envirnomental factors, such as temperature, humidity / moisture etc during storage and use.

[0005] There are suggestions in the art for the development of oil dispersions for microorganisms. However, it appears to be very challenging to develop a stable oil dispersion containing living microbial cells.

[0006] W02020 / 100093 discloses an oil dispersion composition comprising algae, a water immiscible solvent, a surfactant and an agrochemical excipient, wherein the particle size of the composition is in the range of 0.1 to 50 microns. The oil dispersion is made by mixing a water immiscible solvent, the algae and a surfactant under high shear to obtain on oil dispersion with the desired particle size.

[0007] US2011 / 0195946 discloses an oil dispersion comprising a) a biocide, b) a polymeric surfactant, c) an anti-settling agent selected from fumed silica, calcium carbonate, modified castor oils, modified cellulose, bentonite, hectorite, or chemically modified derivatives thereof, and d) a carrier liquid. The biocide that is used in the process disclosed in US2011 / 0195946 is trafopyril, zinc, pyrithione or tolyltluanid. The biocide and the anti-settling agent are mixed under high shear mixing.

[0008] A disadavantage of the processes disclosed in W02020 / 100093 and US 2011 / 0195946 is that high shear is used when mixing the algae or the biocide, which can lead to disrupton of living cells.

[0009] There is a need for improved processes for preparing an oil dispersion comprising microbial cells, wherein the oil dispersion remains stable and the microbial cells sufficiently active.

[0010] SUMMARY

[0011] The present invention relates to a process for preparing an oil dispersion comprising microbial cells, characterized by the steps of: a) Mixing a hectorite and an oil to prepare a gel, wherein said mixing is performed at a shear force of from 0.1 to 20 kW / m3, and, b) Mixing a composition comprising microbial cells with the gel prepared in step a).

[0012] In a second aspect the invention relates to an oil dispersion comprising microbial cells, wherein the oil dispersion is obtainable by a process according to the present invention.

[0013] In a third aspect the present invention relates to a method for controlling or preventing infestation of a plant, plant propagation material and / or harvested food crops by a phytopathogenic microorganism or an insect pest, or for improving agronomic performance, characterized by treating the plant, plant propagation material, the locus thereof, and / or harvested food crops with an oil dispersion according to the present invention.

[0014] DETAILED DESCRIPTION

[0015] The present invention relates to a process for preparing an oil dispersion comprising microbial cells, characterized by the steps of a) Mixing a hectorite and an oil to prepare a gel, wherein said mixing is performed at a shear force of from 0.1 to 20 kW / m3, and, b) Mixing a composition comprising microbial cells with the gel prepared in step a).

[0016] Surprisingly, it was found that the process for preparing an oil dispersion according to the present invention resulted in an oil dispersion comprising microbial cells which was stable for a period of at least 10 days, preferably at least 20 days, preferably at least 30 days at a temperature of 25 °C. Preferably the oil dispersion is stable for a period of at least 2 months, preferably at least 3, 4, 5 or at least 6 months at a temperature of 25°C, preferably for a period of at least 9 months at a temperature of 25 °C, preferably for a period of at least 1 year at 25°C, preferably for a period of at least 2 years at 25°C. A stable oil dispersion is defined herein as an oil dispersion which has a phase separation of less than 10%, preferably less than 9, 8, 7, 6, 5, 4, 3, 2, or 1 %, preferably an oil dispersion has no separation for a period as indicated above. Suitably, a stable oil dispersion has a phase separation is between 0 to 10%. Phase separation is measured by the height of a supernatant layer divided by the total height of the oil dispersion in a storage vessel times 100%. A storage vessel may for instance be a bottle. It was surprisingly found that the microbial cells remained living microbial cells after preparation of the oil dispersion, and after a period as indicated above.

[0017] Advantageously, the oil dispersion prepared according to a process according to the present invention has a G’ value of more than 5 Pa, such as more than 10 Pa. The G’ value is below 500 Pa, such as below 250 Pa. Andvantageously, the oil dispersions have a G’ value of 5 to 500 Pa, such as a G’ value of 10 to 250 Pa.

[0018] Advantageously, the oil dispersion has a critical strain of 0.05 to 2%, such as 0.1 to 1.8%, such as 0.15 to 1.5%.

[0019] Advantageously the oil dispersion has a water activity (aw) of 0.1 to 0.7, suitably a water activity of 0.15 to 0.65, suitably a water activity of 0.2 to 0.6. These low water activities were found advantageous for the viability of the microbial cells in the oil dispersion.

[0020] As used herein an oil dispersion, also indicated as an oil dispersion formulation, or an oil based suspension concentrate (OD), is defined as a stable suspension of an active ingredient(s) in an oil. The active ingredient in a oil dispersion according to the present invention comprises a microbial cell. The oil can vary from paraffinic to aromatic solvent types and vegetable oil or methylated seed oils. Typically, an oil is also indicated as a water immiscible solvent.

[0021] The oil dispersion comprising microbial cells disclosed herein may further comprise derivatives or metabolites produced by the microbial cells. Metabolites produced by microbial cells may have pesticidal activity. The oil dispersion comprising microbial cells, comprises a microbial cell count of 1*104to 1*1012cfu

[0022] 1 ml, such as a microbial cell count of 1*105to 1 *1011cfu I ml, such as a microbial cell count of 1*106to 1*101° cfu I ml, such as a microbial cell count of 1*107to 5*109cfu I ml, such as a microbial cell count of 1 *107to 1 *1010cfu I ml, such as a microbial cell count of 5*107to 5*109cfu I ml.

[0023] The process for preparing an oil dispersion comprises mixing a hectorite and an oil at a shear force of 0.1 to 20 kW / m3. Preferably the hectorite and the oil are mixed at a shear force of 0.2 to 15 kW / m3, preferably of 0.5 to 10 kW / m3, preferably of 0.8 to 5 kW / m3to a prepare a gel.

[0024] In one embodiment, step a) of mixing a hectorite and an oil in a process according to the present invention further comprises adding an activator. Adding an activator is preferably performed after mixing the hectorite and the oil, preferably the activator is added to a mixture of the hectorite and the oil. It was found that adding an activator may result in improved gel formation and gel stability. Preferably, adding an activator to step a) of the process according to the present invention comprises mixing an activator with the hectorite and the oil at a shear force of from 0.1 to 20 kW / m3, preferably at a shear force of 0.2 to 15 kW / m3, preferably of from 0.5 to 10 kW / m3, preferably of from 0.8 to 5 kW / m3. Any suitable activator may be added to step a) of a process according to the present invention. An activater can be a polar molecule, for instance a quaternary ammonium salt, an alkali metal salt such as sodium salt or water. Preferably, the activator comprises propylene carbonate.

[0025] A gel as disclosed herein is also indicated as an anti-settling agent is used interchangebly.

[0026] In one embodiment the gel disclosed herein comprises 1 to 30 w / w% of the hectorite, preferably

[0027] 2 to 25 w / w% of the hectorite, preferably 4 to 20 w / w% of the hectorite, preferably 5 to 15 w / w% of the hectorite. A hectorite as used herein is also known as an organoclay.

[0028] In one embodiment, the hectorite comprises a natural hectorite or a synthetic hectorite. Preferably, the hectorite comprises a synthetic hectorite, preferably the hectorite comprises AhFWeSi (Bentone® 27) and / or HLiMgNaOnSi4 (quaternium 18-hectorite, Bentone ® 38).

[0029] In one embodiment, the gel comprises and 70 to 99 w / w% of the oil, preferably from 75 to 98 w / w% of the oil, preferably from 80 to 97 w / w% of the oil, preferably from 82 to 95 w / w% of the oil.

[0030] In one embodiment, the oil that is mixed with the hectorite comprises a mineral oil and / or a vegetable oil. Preferably the oil comprises a vegetable oil, such as soya oil, rapeseed oil, castor oil, palm oil, olive oil, sunflower oil, linseed oil and I or methylated oils thereof, such as methylated soya oil, methylated rapeseed oil, methylated castor oil, methylated palm oil, methylated olive oil, methylated sunflower oil, methylated linseed oil or mixtures thereof. Preferably the oil comprises rape seed oil, soya oil, and I or linseed oil, and I or methylated rape seed oil, methylated soya oil, and I or methylated linseed oil.

[0031] Preferably, the oil dispersion prepared in and I or obtainable by a process according to the present invention comprises 1 to 60 w / w% of the gel prepared in step a), preferably 5 to 50 w / w% of the gel, preferably 8 to 45 w / w% of the gel, preferably 10 to 40 w / w% of the gel.

[0032] In one embodiment, the process for preparing an oil dispersion comprising microbial cells, comprises mixing a composition comprising microbial cells with the gel of step a). Mixing a composition comprising microbial cells with the gel may be perfomed in any suitable way, for instance by stirring a composition of microbial cells and the gel at a speed of 500 to 5000 rpm, for instance a speed of 800 to 4000 rpm, for instance a speed of 1000 to 3000 rpm, for instance a speed of 1200 to 2000 rpm. Mixing a composition comprising microbial cells with the gel is perfomed at a temperature of 1 to 60 °C, for instance a temperature of 2 to 50° C for instance a temperature of 5 to 45° C, for instance a temperature of 10 to 40 °C.

[0033] In one embodiment, a process according to the present invention further comprises adding a carrier, a polymeric surfactant, a dispersant and / or an emulsifier to the gel. Adding a carrier, a polymeric surfactant, a dispersant and I or an emulsifier may be performed prior, during and I or after mixing the composition comprising microbial cells. Preferably, adding a carrier, a polymeric surfactant, a dipsersant and I or an emulsifier to the gel of step a) is performed prior to mixing a composition comprising microbial cells with the gel.

[0034] A step of adding a carrier, a polymeric surfactant, a dispersant and I or an emulsifier to the gel of step a) comprises mixing the carrier, the polymeric surfactant, the dipsersant and I or the emulsifier with the gel. Said mixing may be performed in a suitable way known to a person skilled in the art, for instance bulk mixing. Said mixing may be performed by stirring the gel with a carrier, a polymeric surfactant, a dispersant and I or an emulsifier at a speed and temperature as indicated herein above.

[0035] The carrier as used herein comprises a water immiscible solvent such as an oil. Preferably the carrier comprises a mineral and I or vegetable oil. Preferably, the carrier comprises a vegetable oil, such as soya oil, rapeseed oil, castor oil, palm oil, olive oil, sunflower oil, linseed oil and I or methylated oils thereof, such as methylated soya oil, methylated rapeseed oil, methylated castor oil, methylated palm oil, methylated olive oil, methylated sunflower oil, methylated linseed oil or mixtures thereof. Preferably, the carrier comprises rape seed oil, soya oil, linseed oil, and I or methylated rape seed oil, methylated soya oil, and I or methylated linseed oil. Suitably, the carrier is added in step b) in a process according to the present invention in an amount 1 to 50 wt / wt%, such as 2 to 40 wt / wt%, such as 5 to 35 wt / wt% of the oil dispersion.

[0036] Advangeously, the oil dispersion comprises a carrier in an amount of 1 to 50 wt / wt%, such as 2 to 40 wt / wt%, such as 5 to 35 wt / wt%

[0037] A polymeric surfactant as used herein comprises ionic and I oror non-ionic surfactants, such as a polysorbate product (eg Tween® 20, polyoxyethylene polyoxypropylene sorbitan monolaurate (Tween® L-05-15), or Tween® 80), or a nonionic polymeric surfactant for instance BREAK-THRU® S201. Suitably, a polymeric surfactant is added in step b) in a process according to the present invention in an amount of 1 to 30 wt / wt%, such as 5 to 20 wt / wt% of the oil dispersion. Suitably, the oil dispersion comprises a polymeric surfactant in an amount of 1 to 30 wt / wt%, such as 5 to 20 wt / wt%.

[0038] Suitable dispersants as used herein include, but are not limited to, surfactants and wetting agents. In general, the dispersant(s) will have low toxicity for the microorganism(s). A suitable dispersant may comprise ionic and anionic surfactants, for instance a pyrrolidone polymer (eg. A C16 alkylated polyvinylpyrrolidone, Agrimer AL-22). Suitably a dispersant is added to step b) in a process according to the present invention in an amount of 1 to 30 wt / wt%, such 5 to 20 wt / wt% of the oil dispersion. Suitably, the oil dispersion comprise a dispersant in an amount of 1 to 30 wt / wt%, such 5 to 20 wt / wt%.

[0039] Suitable emulsifiers comprise alcohol ethoxylates / alcoxylates, such as C16 / 18 ethoxylates, or C16 / C18 alcoxylate, or block co-polymers or mixtures thereof. An emulsifier may comprise oleyl alcohol polyglycol ether (Emulsogen M). Suitably, an emulsifier is added in step b) in a process according to the present invention in an amount in amount of from 1 to 30 w / w%, such 5 to 20 wt / wt% of the oil dispersion .Suitably, the oil dispersion comprises an emulsifier in amount of from 1 to 30 w / w%, such 5 to 20 wt / wt%.

[0040] Suitably, the oil dispersion prepared and / or obtainable by a process according to the present invention comprises a carrier, a polymeric surfactant, a dispersant and I or an emulsifier.

[0041] In one embodiment the oil dispersion prepared by a process according to the present invention comprises 5 to 90% dry w / w% of the composition comprising microbial cells, preferably 10 to 80 dry w / w % of the composition comprising microbial cells. Preferably, the oil dispersion comprises 20 to 75 dry w / w % of a composition comprising microbial cells, preferably 25 to 70 dry w / w % of a composition comprising microbial cells, preferably 30 to 65 w / w % of a composition comprising microbial cells.

[0042] In one embodiment, the composition comprising microbial cells comprises living microbial cells and I or spores. The composition comprising microbial cells may be any suitable composition comprising microbial cells. The composition may be a fermentation broth, for instance a wet, or whole or purified fermentation broth. Preferably, the composition comprises a spray-dried or freeze-dried fermentation broth comprising microbial cells. The composition comprising microbial cells may further comprise metabolites that have been produced by the microbial cells. Accordingly, a composition comprising microbial cells may be a composition comprising microbial cells and metabolites produced by the microbial cells. As used herein metaboliets may have pesticidal activity.

[0043] A fermentation broth comprising microbial cells is typically obtained by cultivating a microbial cell in a suitable fermentation medium and under suitable fermentation conditions. Cultivating a microbial cell usually comprises producing metabolites. Suitable fermentation conditions such as temperature and pH for cultivating microbial cells depend on the microbial strain used and are known to a person skilled in art. A cultivation medium comprises a suitable carbon and I or nitrogen source. A suitable carbon source may be molasses, such as beet or cane molasses, polysaccharides, flour, starch, sugar, and I or glucose. A suitable nitrogen source may be casein hydrolysate, tryptone, ammonium sulphate, ammonia, yeast extract, peptone and I or urea. A process for producing or fermentation broth comprising a microbial strain may be performed in a batch, fed-batch or continuous culture. A process for cultivating microbial cells optionally comprises a step of recovering the microbial cells.

[0044] Preferably, a process for preparing a fermentation broth comprising microbial cells comprises a step of spray drying or freeze drying the fermentation broth comprising microbial cells. Spray drying or freeze drying a fermentation broth are known technologies to a person skilled in the art. Spray drying is a method of forming a dry powder from a liquid or slurry by rapidly drying with a hot gas. Freeze drying is a low temperature dehydration process that involves freezing a product and lowering pressure thereby removing the ice by sublimation.

[0045] In one embodiment, the composition comprising microbial cells comprises particles of microbial cells. The particles comprising microbial cells preferably comprise particles of spray-dried microbial cells or freeze-dried microbial cells. Preferably, the particles of microbial cells have a particle size of 1 to 500 pm, such as a particle size of 2 to 400 pm, such as a particle size of 5 to 300 pm, such as a particle size of 10 to 250 pm, such as a particle size of 20 to 200 pm, such as a particle size of 25 to 150 pm, such as a particle size of 30 to 100 pm.

[0046] In one embodiment, the composition comprising microbial cells, comprises a microbial cell count of 1 * 103to 1 * 1013cfu / ml, such as a microbial cell count of 1 * 104to 1 * 1012cfu / ml, such as a microbial cell count of 1 * 105to 1 * 1011cfu I ml, such as a microbial cell count of 1 * 106to 5 * 1 O10cfu I ml, such as a microbial cell count of 1 * 107to 1 * 1 O10cfu I ml, such as a microbial cell count of 5 * 107to 5 * 109cfu / ml.

[0047] In one embodiment the microbial cells disclosed herein comprise fungi, bacteria and I or algae, their derivatives, or mixtures thereof.

[0048] Suitable fungi include but are not limited to Aspergillus sp. such as A. flavus, A. niger, Isara sp., such as I. fumosorosea or I. cicadae, Metarhizium sp. such as Metarhizium anisoplia, Talaromyces flavus, Aureobasidium sp. such as A. pullulans, Pseudozyma aphidis, Trichoderma sp, for instance T. atroviride, T. asperelloides T. harzianum, T. harmatum, T. asperellum, T. polysporum, T. virens, T. gamsii, their derivatives or mixtures thereof.

[0049] Suitable bacteria include but are not limited to Azotobacter sp., such as A. salinestris, Bacillus sp. such as B. amyloliquefaciens, B. cereus, B. mycoides, B. velezensis, B. subtilis, B. thuringiensis, B. pumilus, Curtobacterium sp. for instance C. salicis, Paenibacillus sp. for instance P. polymyxa, P. epiphyticus, Pseudomonas sp. such as P. proradix, P. chlororophis, P. fluorescens. P. aeruginosa, or P. siliginis preferably the bacteria comprise Streptomyces sp., for instance S. rimosus, S. chrestomyceticus, S. griesoviridis, S. galbus. S. lydicus, S. lavendulae, S. nojiriensis, S. xanthopheus and I or S. paramomycinus, preferably Streptomyces chrestomyceticus deposited at the Westerdijk Institute with accession number CBS149411 , their derivatives or mixtures thereof.

[0050] Suitable algae disclosed herein are Spirulina sp., Arthrospira sp., Chlorella sp., Anabaena sp., Sargassum sp., Scenedmus sp., Aphanixomenon sp., Dunaliella sp., Phymaioliihion sp., Lithoihamnium sp., Ascophyllum sp., Entcromorpha sp., Tctrasclmis sp., Prymnesium sp., Chltunydomonas sp., Euglcna sp., Caulctpa sp., Padina sp., Urophora sp., Chondria sp., Caulerpa sp., Lyngby sp., Prasiola sp., Gymnopilus sp., Melanothamnus sp., Turbeneria sp., Masiigocladopsis sp., Hydroclathrus sp., Padina sp., Cysioscira sp., Laminaria sp., Fucus sp., Diva sp. or their derivatives, species or mixtures thereof.

[0051] In a second aspect the present invention relates to an oil dispersion comprising microbial cells, wherein the oil dispersion is obtainable by a process according to the present invention. Surprisingly, it was found that the oil dispersion remained stable for a period of at least 10 days, preferably at least 20 days, preferably at least 30 days, preferably for a persion fo at least 2, 3, 4, 5, 6 months at a temperature of 25°C, preferably at least 9 months at a temperature of 25 °C, preferably for a period of at least 1 year at 25°C, preferably for a period of at least 2 years at 25°C. Advantageously, the microbial cells remained living microbial cells in the oil dispersion for a prolonged period of time.

[0052] The features disclosed above related to an oil dispersion prepared by a process according to the present invention are applicable to the oil dispersion obtainable by the present invention.

[0053] Advantageously, the oil dispersion obtainable by a process according to the present invention has a G’ value of more than 5 Pa, such as more than 10 Pa. The G’ value is below 500 Pa, such as below 250 Pa. Andvantageously, the oil dispersions have a G’ value of 5 to 500 Pa, such as a G’ value of 10 to 250 Pa.

[0054] Advantegeously, the oil dispersion has a critical strain of 0.05 to 2, such as 0.1 to 1 .8, such as

[0055] 0.15 to 1.5. In a third aspect the present invention relates to a method for controlling or preventing infestation of a plant, plant propagation material and / or harvested food crops by a phytopathogenic microorganism, or for improving agronomic performance of plants, characterized by treating the plant, plant propagation material and / or harvested food crops with an oil dispersion according to the present invention.

[0056] Controlling or preventing means reducing infestation by phytopathogenic microorganisms or insect pests to such a level that an improvement is demonstrated.

[0057] Phytopathogenic microoganisms that may be affected by an oil dispersion comprising microbial cells as according to the present invention are fungi, for instance fungi belonging to the genus of Zymoseptoria, Puccinia, Mycorsphearella, Pyricularia, Rhizoctoonia, Blumeria, Alternaria, Colletotrichum, Ramularia, Parastagonospora, Rhynchosporium, Oculimacula, Fusarium, Gaeumannomyces, Botrytis, or Sclerotinia, preferably a fungus belonging tot he species Zymoseptoria tritici, Puccinia recondita, Puccinia striiformis, Mycorsphearella fijiensis, Mycorsphearella arachidis Pyricularia oryzae, Rhizoctonia solani, Blumeria graminis f.sp. tritici, Alternaria solani, Colletotrichum lagenarium, Ramularia collo-cygni, Parastagonospora nodorum, Rhynchosporium secalis, Oculimacula yallandae, Fusarium avenaceum, F. graminearum, F. culmorum, F. virguliforme, F. subglutinans, F. pseudograminearum, F. verticillioides, F. fujikuroi, F subglutinans, F. oxysporum, for instance, F. oxysporum f. sp. cubense, F. oxysporum f. sp. melonis, F. oxysporum f. sp.vasinfectum, F. oxysporum f. sp. lycopesici, Gaeumannomyces graminis Botrytis cinerea, or Sclerotinia sclerotiorum, or bacteria, such as a bacterium belonging tot he genus Xanthomonas, preferably Xanthomonas oryzae pv. oryzae or oomycetes, such as an oomycete belonging to Aphanomyces, preferably Aphanomyces cochlioides.

[0058] The method according to the present invention does not include a method for treatment of the human or animal body by surgery or therapy and diagnostic methods practised on the human or animal body.

[0059] The term “plant” refers to all physical parts of a plant, including seeds, seedlings, saplings, roots, tubers, stems, stalks, foliage, and fruits. Germinated plants and young plants which are to be transplanted after germination or after emergence from the soil, may also be mentioned. These young plants can be protected before transplantation by a total or partial treatment by immersion.

[0060] The term “plant propagation material” is understood to denote generative parts of the plant, such as seeds, which can be used for the multiplication of the latter, and vegetative material, such as cuttings or tubers, (for example potatoes), roots, fruits, bulbs, rhizomes or parts of plants. The plant propagation material can be treated with the oil dispersion according to the invention before the material is sown or planted. Alternatively, the plant propagation material may be treated with the oil dispersion of the invention during sowing or planting. .

[0061] Suitably, the plant according to the disclosure may be selected from the group consisting of: cereals, such as wheat, barley, rye, oats, rice, maize or sorghum; beet, such as sugar or fodder beet; fruit, for example pomaceous fruit, stone fruit or soft fruit, such as apples, pears, plums, peaches, almonds, cherries or berries, for example strawberries, raspberries or blackberries; leguminous crops, such as beans, lentils, peas or soya; oil crops, such as oilseed rape, mustard, poppies, olives, sunflowers, coconut, castor, cocoa or ground nuts; cucurbits, such as pumpkins, cucumbers or melons; fibre plants, such as cotton, flax, hemp or jute; citrus fruit, such as oranges, lemons, grapefruit or tangerines; vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes or bell peppers; Lauraceae, such as avocado, Cinnamonium or camphor; and also tobacco, nuts, coffee, eggplants, sugarcane, tea, pepper, grapevines, hops, the plantain family and latex plants.

[0062] Suitably, the plant may be an ornamental and / or vegetable crop, including flowers, shrubs, broad-leaved trees and evergreens.

[0063] Suitably, the plant may be selected from the group consisting of: grains, fruits and tree nuts, vegetables, field crops, oil seed crops, forage crops, fiber crops, forest plants, horticulture crops, floriculture, greenhouse and nursery plants, propagative materials, culinary herbs and spices, and medicinal herbs.

[0064] Suitably, the plant may be selected from the group consisting of: apples, almonds, bananas, cherries, citrus, grapes, grapevines, peaches, nectarines, blueberries, caneberries, raspberries, strawberries, tomatoes, potatoes, cucurbits, cucumbers, eggplants, lettuce, beans, brassicas, peas, oilseed rapes, soybeans, sugar beets, sunflowers, rice, peanuts, coffee, ornamental plants, and turfgrass.

[0065] The term “locus” as used herein means fields in or on which plants are growing, or where seeds of cultivated plants are sown, or where seed will be placed into the soil. It includes soil, seeds, and seedlings, as well as established vegetation.

[0066] Improving agronomic performance of a plant includes improving plant health, plant nutrition, such as nutrient use efficiency or nutrient uptake, or plant yield, or improving abiotic stress resistance or tolerance, such as resistance against low or high temperature, deficient or excessive water, high salinity, heavy metals, and ultraviolet radiation.

[0067] EXAMPLES

[0068] MATERIALS AND METHODS

[0069] Streptomyces chestomyceticus.

[0070] Streptomyces sp. Saigon413, a S. chrestomyceticus, which was isolated in Vietnam before 1961 , was deposited at the Westerdijk institute under accession number CBS149411 as disclosed in in WO2024 / 074637, Example 1 .

[0071] Large scale fermentations

[0072] For large scale production, standard procedures and media were applied for cultivating Streptomyces chrestomyceticus to high cell density using fed-batch fermentation. After harvesting, the broth was spray dried or freeze dried according to methods known to a person skilled in the art.

[0073] The final product (TGAI) after spray- or freeze drying had a cell count of 1*106to 1*1013CFU / g dry mass. TGAI: technical grade active ingredient. The particle size of theTGAI was from 10 to 100 pm.

[0074] Other microbial products

[0075] Conidia spores from the fungus Metarhizium anisoplia were prepared by rice solid state fermentation in plastic bags according to the method disclosed in Loera-Corral et al, Production of Conidia by the Fungus Metarhizium anisopliae using Solid State Fermentation, p. 61-69, Chapter 6, In: Travis R. Glare and Maria E. Moran-Diez (eds.), Microbial-Based Biopesticides: Methods and protocols, Methods in Molecular Biology vol. 1477, DOI 10.1007 / 978-1 -4939-6367-6_6, © Springer Science+Business Media New York 2016.

[0076] Taegro®, a product from Syngenta Crop protection, comprises Bacillus amyloliquefaciens strain FZB24 (13% w / w, minimum of 1x1013cfu / kg).

[0077] Tiros® is a product from Unium bioscience ltd and comprises Curtobaceterium salicis and Pseudomonas siliginis.

[0078] Nutribio N®, is a product from Ceres Biotics and comprises Azotobacter salinestris - CECT9690. CoStar WG®, can be obtained from Certis Belchim, and comprises Bacillus thuringiensis subsp. kurstaki.

[0079] Formulation technology

[0080] If not indicated otherwise in the examples, general formulation technology was used as disclosed in Croda Crop Care, the Nouryon formulator toolbox and in: Formulation of Microbial Biopesticides: Beneficial microorganisms, nematodes and seed treatments (412 p., 6 December 2012) eds. Burges H.D., Springer, ISBN 978-94-011-4926-6. A person skilled in the art understands how to consult this documentation for polymeric surfactants, disperants, emulsifiers and the like.

[0081] Emulsogen M, which contains oleyl alcohol polyglycol ether, can be obtained from Clariant international, Muttenz, Switzerland.

[0082] Suitable polymeric surfactants which can be used include sorbic acid containg products like Tween® L05-15, Tween®20, or Tween®80, or the non-ionic trisiloxan containing additive BreakThru® S301. Methylated rapeseed oil is commercially available and can be obtained from BASF.

[0083] Linseed oil (CAS No. 8001-26-1) can be obtained from Sigma-Aldrich.

[0084] Soya oil refined was obtained from OLVEA vegetable oils.

[0085] Measurement of rheological properties

[0086] The gel strengths of the antisettling agent (gel) and the oil dispersion formulations (eg. OD400 or OD 350) was measured through offline rheology measurements, using an Anton Paar MCR 502 modular compact rheometer. The method employed for the rheological measurements normally uses a Bob-and- cup configuration CC27 at 25°C.The viscoelasticity properties of the formulation can be described by the complex dynamic modulus G (Macosko C.W. 1994 Rheology: Principles, Measurements and applications, 576 pages, ISBN, 978-0-471 -18575-8):

[0087] G=G’+ / G”; where G’ is the storage modulus and G” is the loss modulus, 12= -1 .

[0088] Critical strain

[0089] Critical strain is a physical parameter obtained during amplitude sweep oscillatory tests. When the storage modulus starts to drop (usually >5% in LVE), the strain (deformation) is defined as critical strain (%) for the system.

[0090] Storage stability

[0091] The formulations were stored in HDPE plastic bottles (or Glass) in a cabinet at constant temperature

[0092] 25 °C. The degree of settling was assessed on a regular basis by measuring the formation of a clear supernatant layer at the top of the sample. This was expressed as a percentage of the total height of the liquid, and the values given after 30 days of storage are noted in the table below.

[0093] Water activity (aw)

[0094] An amount of 5 g of formulations prepared in the Examples was put in a sample pot. The sample pot was placed into the seal chamber in the water activity meter from AQUALAB, Benchtop. When the temperature was settled at 25°C, the vapour pressure (aw)was measured from the sample.

[0095] Measurement of microbial cell counts

[0096] To assess the viability of the microbe within a formulation orTGAI, the colony forming unit (CFU) method was used. The CFU protocol was carried out as follows: an initial dilution of a formulation or TGAI was performed in a sterile diluent (dH2O or 0.01 % Tween(R)20 in dFLO), the suspension was homogenised through vortexing, using glass beads for additional agitation. The initial dilution was then serially diluted with a consecutive series of 1 :10 dilutions in sterile de-ionised dFLO. The dilutions were plated onto Luria Broth (LB) or Trypticase Soya (TSA) agar (Oxoid) plates (100 pL per plate). The plates were incubated at 25°C for 2-4 days and monitored daily. Colonies were counted within a statistically relevant range. The protocol was performed using three independent formulation / TGAI sample dilutions. The final CFU per mL (formulation) I g (TGAI) was calculated.

[0097] EXAMPLE 1 : Preparation of an oil dispersion based on methylated soya oil comprising S. chrestomyceticus

[0098] An oil dispersion formulation (OD) was prepared in a two-step process

[0099] The first step entailed the formation of the anti-settling system:

[0100] A modified hectorite clay, ALFWSi (Bentone® 27, Elementis), was mixed with methylated rape seed oil under high shear mixing (Silverson mixer) at 1 Kw / M3. Upon achieving a high level of dispersion of the hectorite clay a propylene carbonate was added and high shear mixing was continued. A stable antisettling system (gel) was formed. Table 1 shows the composition in w / w% of the gel (anti-settling agent).

[0101] In the second step an oil dispersion comprising microbial cells was prepared:

[0102] The anti-settling agent was mixed with a carrier, a polymeric surfactant and an emulsifier as indicated in Table 2. Subsequently, the TGAI of S. chrestomyceticus prepared as descibed above was added to produce the final oil dispersion. The composition of the oil dispersion formulation is shown in Table2. The rheological properties, storage stability and microbial cell counts of the oil dispersion are presented in Table 14 and 15. Table 1. Anti settling agent of Example 1

[0103] Table 2. Composition of the oil dispersion formulation Example 1

[0104] EXAMPLE 2: Preparation of an oil dispersion based on methylated soya oil comprising fungal spores of Metarhizium anisoplia

[0105] An oil dispersion formulation (OD) was prepared in a two-step process. In a first step an antisettling system was prepared: A modified hectorite organoclay (HLiMgNaO11 Si4, molecular weight 343.58, Bentone® 38, Elementis) was mixed with methylated rape seed oil under high shear mixing at 7 PA / (Kw / M3). Upon achieving a high level of dispersion of the organic clay, propylene carbonate, the chemical activator, was introduced. A stable anti-settling system (gel) was formed. Table 3 shows the composition in w / w% of the antisettling agent. Subsequently, the anti-settling system was combined with a carrier, a dispersant, and an emulsifier and subsequently fungal spores of Metarhizium anisoplia prepared as described above were added to produce the final oil dispersion formulation. Table 4 shows the composition of the oil dispersion comprising the fungal spores of this Example. The rheological properties and storage stability of the oil dispersion are presented in Table 14. Table 3. Anti settling agent Example 2

[0106] Table 4. Composition of the oil dispersion formulation of Example 2

[0107] EXAMPLE 3: Preparation of an oil dispersion based on linseed oil comprising S. chrestomyceticus

[0108] An oil dispersion formulation (OD) was prepared in a two-step process. In a first step an antisettling system was prepared:

[0109] A modified hectorite clay, AhFLOSi (Bentone® 27, Elementis), was mixed with linseed oil under high shear mixing at at 7 PA / (Kw / M3). Upon achieving a high level of dispersion of the organic clay, propylene carbonate as chemical activator was introduced. A stable anti-settling system (gel) was formed. Table 5 shows the composition in w / w% of the anti-settling agent.

[0110] Subsequently, the anti-settling system was mixed with a carrier, a dispersant, and emulsifier and a polymeric surfactant. Polyether modified polysiloxan (from Evonik) was used as a dispersant. Finally, the TGAI of S. chrestomyceticus prepared as descibed above was added to produce the final oil dispersion. Table 6 shows the composition of the oil dispersion comprising microbial cells of this Example. The rheological properties and storage stability of the oil dispersion are presented in Table 14. Table 5. Anti-settling agent Example 3

[0111] Table 6. Composition of the oil dispersion formulation of Example 3 EXAMPLE 4: Preparation of an oil dispersion based on methylated rapeseed oil comprising Bacillus amyloliquefaciens

[0112] A stable anti-settling system (gel) was prepared as described in Example 1 , Table 1 .

[0113] Subsequently, an oil dispersion comprising microbial cells was prepared:

[0114] The anti-settling agent was mixed with a carrier, a polymeric surfactant and emulsifiers as shown in Table 7. Subsequently, Taegro® (B. amyloliquefaciens FZB24) was added to produce the final oil dispersion. The composition of the oil dispersion formulation is shown in Table?. The rheological properties and storage stability of the oil dispersion are presented in Table 14.

[0115] Table 7. Composition of the oil dispersion formulation Example 4

[0116] EXAMPLE 5: Preparation of an oil dispersion based on methylated rapeseed oil comprising Curtobacterium salicis and Pseudomonas siliginis strains

[0117] A stable anti-settling system (gel) was prepared as described in Example 1 , Table 1 .

[0118] In the second step an oil dispersion comprising microbial cells was prepared:

[0119] The anti-settling agent was mixed with a carrier, a polymeric surfactant as indicate din Table 8 and the emulsifiers Emulsogen M and Atlas G-1086. Atlas G-1086 is an ethoxylated sorbitol hexaester which is derived from oleic acid. Subsequently, Tiros® comprising Curtobaceterium salicis and Pseudomonas siliginis was added to produce the final oil dispersion. The composition of the oil dispersion formulation is shown in Table8. The rheological properties and storage stability of the oil dispersion are presented in Table 14.

[0120] Table 8. Composition of the oil dispersion formulation of Example 5

[0121] Example 6: Preparation of an oil dispersion based on soybean oil comprising S. chrestomyceticus

[0122] An oil dispersion formulation (OD) was prepared in a two-step process. The first step entailed the formation of the anti-settling system:

[0123] A modified hectorite clay, AhFWSi (Bentone® 27, Elementis), was mixed with soybean oil under high- shear mixing (Silverson mixer) at 1 Kw / M3. Upon achieving a high level of dispersion of the hectorite clay, a propylene carbonate was added and high shear mixing was continued. A stable antisettling system (gel) was formed. Table 19 shows the composition in w / w% of the gel (anti-settling agent).

[0124] In the second step an oil dispersion comprising microbial cells was prepared:

[0125] The anti-settling agent was mixed with a carrier, a polymeric surfactant and an emulsifier as shown in Table 10. Subsequently, the TGAI of S. chrestomyceticus prepared as described above was added to produce the final oil dispersion. The composition of the oil dispersion formulation is shown in Tablel O.

[0126] The rheological properties and storage stability of the oil dispersion are presented in Table 14. Table 9. Anti settling agent of Example 6

[0127] Table 10. Composition of the oil dispersion formulation Example 6

[0128] Table 10A. Composition of an alternative oil dispersion formulation Example 6

[0129] EXAMPLE 7: Preparation of an oil dispersion based on methylated rapeseed oil comprising Azotobacter salinestris

[0130] An oil dispersion formulation (OD) was prepared in a two-step process A stable anti-settling system (gel) was prepared as described in Example 1 , Table 1 .

[0131] In the second step an oil dispersion comprising microbial cells was prepared:

[0132] The anti-settling agent was combined with a carrier, a polymeric surfactant and emulsifier as indicated in Table 11. Subsequently, Nutribio N® comprising Azotobacter salinestris- CECT9690 was added to produce the final oil dispersion. The composition of the oil dispersion formulation is shown in Tablel 1 .

[0133] The rheological properties and storage stability of the oil dispersion are presented in Table 14.

[0134] Table 11. Composition of the oil dispersion formulation of Example 7

[0135] EXAMPLE 8: Preparation of an oil dispersion based on methylated rapeseed oil comprising Bacillus thuringiensis An oil dispersion formulation (OD) was prepared in a two-step process

[0136] A stable anti-settling system (gel) was prepared as described in Example 1 , Table 1 .

[0137] In the second step an oil dispersion comprising microbial cells was prepared:

[0138] The anti-settling agent was combined with a carrier, a polymeric surfactant and emulsifier as indicated in Table 12. Subsequently, Bacillus thuringiensis CoStar WG® was added to produce the final oils dispersion. The composition of the oil dispersion formulation is shown in Tablel 2. COMPARATIVE EXAMPLE: Preparation of an oil dispersion based on methylated rapeseed oil comprising S. chrestomyceticus

[0139] In a first step an antisettling system was prepared:

[0140] A polymeric rheological modifer, the polyester block co-polymer Atlox Rheostrux™ 100 (Croda), was mixed with methylated rapeseed oil under high shear mixing at at 7 P / V (Kw / M3) and water bath (80 °C) for 0.5 hours.

[0141] A dispersant, an emulsifier, a polymeric surfactant, and subsequently the TGAI of S. chrestomyceticus prepared as descibed above were combined with the rheological modifier to produce the final oil dispersion formulation. Table 13 shows the composition of the oil dispersion comprising microbial cells of this Example. The rheological properties and storage stability of the oil dispersion are presented in Table 14.

[0142] Table 13. Composition of the oil dispersion formulation of comparative Example

[0143] Rheological properties of the oil dispersions prepared in Examples 1 to 9

[0144] The rheological properties and storage stability of the oil dispersions prepared in Examples 1 to 9 was assessed according to the methods disclosed above under Materials and Methods and summarized Tables 14 and 15. Table 14. Rheological properties of the oil dispersions prepared in Examples 1 to 9

[0145] Microbial cell count The microbial cell count (cfu) of the oil dispersion prepared in Example 1 was assessed as described under Materials and Methods. The results are shown in Table 15. G’ value and the stability of the oil dispersion prepared in Example 1 did not change during storage of up to 12 months (data not shown).

[0146] Table 15. Microbial count of S. chrestomyceticus cells of the oil dispersion prepared in Example 1

[0147] CONCLUSION

[0148] The results in the Examples show that oil dispersions prepared and obtainable by a process according to the present invention are stable and have a value of G’ above 5.

[0149] In addition, microbial cells such as those from S. chrestomyceticus remain viable in the oil dispersion for a period of at least 12 months.

Claims

CLAIMS1 . A process for preparing an oil dispersion comprising microbial cells, characterized by the steps of a) Mixing a hectorite and an oil to prepare a gel, wherein said mixing is performed at a shear force of from 0.1 to 20 kW / m3, and, b) Mixing a composition comprising microbial cells with the gel prepared in step a).

2. The process according to claim 1 , wherein step a) further comprises mixing an activator, preferably wherein the activator comprises propylene carbonate.

3. The process according to any one of the claims 1 to 2, wherein step b) further comprises adding a carrier, a polymeric surfactant, a dispersant and I or an emulsifier, preferably prior to mixing the composition comprising microbial cells.

4. The process according to any one of the claims 1 to 3, wherein the gel comprises 1 to 30 w / w% of the hectorite and 70 to 99 w / w% of the oil.

5. The process according to any one of the claims 1 to 4, wherein the hectorite comprises a synthetic hectorite, preferably wherein the synthetic hectorite comprises AhFWeSi and I or HLiMgNaOnSi4.

6. The process according to any one of the claims 1 to 5, wherein the oil is a vegetable oil, preferably rape seed oil, soya oil, linseed oil, or methylated rape seed oil, methylated soya oil, methylated linseed oil.

7. The process according to any one of the claims 1 to 6, wherein the oil dispersion comprises 1 to 60 w / w% of the gel prepared in step a).

8. The process according to any one of the claims 1 to 7, wherein the oil dispersion comprises 5 to 90 (dry) w / w % of the composition comprising microbial cells.

9. The process according to any one of the claims 1 to 8, wherein the composition comprising microbial cells comprises particles comprising microbial cells, wherein the particles have a particle size of 1 to 500 pm.

10. The process according to any one of the claims 1 to 9, wherein the oil dispersion has a water activity (aw) of 0.1 to 0.6.

11. The process according to any one of the claims 1 to 10, wherein the oil dispersion comprises a microbial cell count of 1 * 104to 1 * 1012cfu / ml.

12. The process according to any one of the claims 1 to 11 , wherein the microbial cells comprise fungi, bacteria and I or algae.

13. The process according to claim 12, wherein the bacteria comprise Streptomyces sp., preferably Streptomyces rimosus, Streptomyces chrestomyceticus, Streptomyces. griesoviridis, Streptomyces. galbus. Streptomyces lydicus, Streptomyces. lavendulae, Streptomyces nojiriensis, S. xanthopheus Streptomyces nojiriensis, and / or Streptomyces paramomycinus, preferably Streptomyces chrestomyceticus deposited at the Westerdijk institute with accession number CBS149411 .

14. An oil dispersion comprising microbial cells, wherein the oil dispersion is obtainable by a process according to any one of the claims 1 to 13.

15. A method for controlling or preventing infestation of a plant, plant propagation material and / or harvested food crops by a phytopathogenic microorganism or an insect pest, or for improving agronomic performance of a plant, characterized by treating the plant, plant propagation material or the locus thereof, and / or harvested food crops with an oil dispersion to claim 14.

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