Enhanced microbial and biorational control of nematode pests of plants

Abstract

This disclosure describes a biological system of plant growth promotion, pest resistance and disease resistance by application of strains of T. viride, Trichoderma harzianum K2, Bacillus amyloliquifaciens AS2, Bacillus amyloliquifaciens AS3, or a combination thereof. These strains can be used in conventional or organic agriculture for the promotion nematode resistance when applied topically as a foliar spray or as a seed coating. The highly active strains also are expected to increase plant productivity and improve quality of fruits, vegetables, flowers or other plant products.

Claims

1. A composition to enhance activity of herbicides against plant pests or plant diseases comprising: an emulsion containing water and a vegetable oil in a ratio of from about 1:1 to 0.5:4, 0.5 to 1.2% weight/volume of lecithin, 1 to 3% by weight based on the total weight of the emulsion of sodium stearate surfactant to form a stable emulsion, and an herbicide composition that includes at least one microbial metabolite that increases the resistance of plants to plant pests or plant diseases to create an herbicide microbial metabolite mixture; and wherein the stable emulsion can be diluted more than 100-fold in water without phase separation.

2. The composition of claim 1, wherein the vegetable oil is soy, corn, canola or safflower.

3. The composition of claim 1, wherein the lecithin is from soy or egg.

4. The composition of claim 1, wherein the at least one microbial metabolite is derived from Trichoderma spp.

5. The composition of claim 1, wherein the at least one microbial metabolite includes 6-pentyl pyrone, harzianic acid, hydtra 1, harzinolide, 1 octene-3-ol or mixtures thereof.

6. The composition of claim 1, wherein the at least one microbial metabolite is derived from Bacillus spp.

7. The composition of claim 1, wherein the at least one microbial metabolite includes sufactin, iturin, fengycin, polyketides or other bacterial metabolites.

8. A method for enhancing activity of herbicides comprising the steps of: a.) preparing a stable emulsion consisting of a vegetable oil, water, 0.5% to 1.2% weight/volume of lecithin, and 1 to 3% by weight based on the total weight of the emulsion of a surfactant, wherein the ratio of water to vegetable oil is from about 1:1 to 0.5:4 in the emulsion, wherein the emulsion can be diluted more than 100 fold in water without phase separation; b.) adding to the stable emulsion an herbicide composition to form an herbicide emulsion mixture, wherein the herbicide composition includes at least one microbial metabolite that increases the resistance of plants to plant pests or plant diseases to create an herbiciden microbial metabolite mixture; and wherein the plant pest or plant disease control properties of herbicide are enhanced.

9. The method according to claim 8, comprising the further step of contacting a plant or plant seed with the herbicide emulsion mixture or a dilution thereof in additional water, wherein said contacting is carried out by broadcast application, liquid or dry in-furrow application, spray application, irrigation, injection, dusting, pelleting, or coating of the plant or the plant seed or a planting medium.

10. The method of claim 8, wherein the emulsion contains 1 to 3% by weight sodium stearate as the surfactant.

11. The method of claim 8, wherein the vegetable oil is soybean, canola, sunflower, peanut, or safflower.

12. The method of claim 8, wherein the lecithin is vegetable lecithin or egg lecithin.

13. The method of claim 8, wherein the plant pest or the plant disease control comprises plant pests or plant disease controls selected from the group consisting of nematodes, insects, arthropods, animals, bacterial species, viral species, fungal species, yeast species, prions, plant species, spores, systemic foliar disease control, and any combination thereof.

14. The method of claim 8, wherein the plant disease or plant pest controlled includes fungal plant pathogens, Oomcetes, insects, mites or nematodes.

15. The method of claim 8, wherein the plant disease controlled for is caused by nematodes Heterodera, Melodigyne, Pratylemchus, or Longidorus.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a picture of greenhouse tested models of FMD. FIG. 1A shows plants of one variety of corn treated with nothing (chemical seed treatment only), SabrEx, a mixture of K2, K4 and As2 and K5As2. FIG. 1B shows plants of same variety with the chemical seed treatment only (same plant as at the one at the left of FIG. 1A and with a formulation containing a low level of a metabolite of Trichoderma).

(2) FIG. 2 is a graph showing data from nematode control experiments.

(3) FIG. 3A is a picture showing enhanced root growth of corn grown in a PVC pipe.

(4) FIG. 3B is a picture showing enhanced root growth of corn grown in the field.

(5) FIG. 4A is a table showing seed treatments that were applied to soybeans. FIG. 4B is a graph showing the effects of treatment of soybean yields. FIG. 4C is a graph showing plants with root systems at different stages of development. FIG. 4D is a graph showing the effect of seed treatments on nematode soil egg counts.

(6) FIG. 5A is a graph showing stand establishment and yield of field-planted corn that were treated for nematode control. FIG. 5B is a graph showing plants with visible root symptoms at different stages of development. FIG. 5C is a graph showing overall nematode numbers by type.

(7) FIG. 6 is a graph showing data from experiments showing control of aphids using foliar treatments.

(8) FIG. 7 shows graphs from experiments demonstrating the efficacy of pesticide-Trichoderma combinations, where wheat root growth (shoot growth and stand establishment followed a similar pattern following seed treatments).

(9) FIG. 8 shows a model of a hydrophobin molecule, where the hydrophobic portion is shown in green.

(10) FIG. 9 shows a pesticide (bright red) overlain with a wax barrier (palm oil) that melts at 95 F (36 C). This then was overlain with the Trichoderma spore suspension in soybean oil.

(11) FIG. 10 shows the survival of Trichoderma spores in pesticide S in a palm oil encapsulation system.

(12) FIG. 11 shows the appearance of Aqua Tex oil and wax based formulations of Trichoderma (left) and Bradyrhizobium (right).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

(13) At the outset, it should be clearly understood that like reference numerals are intended to identify the same structural elements, portions or surfaces consistently throughout the several drawing figures, as such elements, portions or surfaces may be further described or explained by the entire written specification, of which this detailed description is an integral part. Unless otherwise indicated, the drawings are intended to be read together with the specification, and are to be considered a portion of the entire written description of this invention.

(14) Overview.

(15) Applicants have investigated the uses of many new microbial agents including fungi in the genus Trichoderma and bacteria in the genus Bacillus. Some of the uses that have been identified are for plant growth promotion and yield enhancement, while other uses are for pest control. Many strains and formulations have both types of functions.

(16) All of these developments are made possible through the concepts Applicants have developed in Smart Selection™ and Focused Molecular Diversity™. Smart Selection requires (a) the inclusion of endophytic strains of microorganisms that provide season long benefits to plants, (b) a knowledge of the mechanisms of action, especially of the ability of the strains to induce systemic alteration of gene expression/ability to cause benefits throughout plants, and (c) efficient screening methods that provide good applicability to the field. Focused Molecular Diversity is the concept that we can introduce specific microorganisms (usually endophytic) to replace or enhance the microbial community that ordinarily would exist with organisms that we add and that provide plant benefits that otherwise would not occur. It is augmented by the ability to create formulations that allow the organisms (or metabolites) selected through Smart Selection or Focused Molecular Diversity to function and persist in agricultural practices.

(17) This results in plants that have (a) enhanced resistance to disease, (b) markedly enhanced resistance to abiotic stresses such as drought, salt, flooding and others, (c) bigger and deeper roots, (d) increased abilities to utilize nitrogen and other fertilizers, and (e) enhanced seed germination (Harman, Cadle-Davidson, et al., 2015, Mastouri, Bjorkman, et al., 2010, Shoresh, Mastouri, et al., 2010). The coordinated up-regulation of entire pathways are critically essential. Among the most important changes is an increase photosynthetic efficiency; all of the other changes are energy-dependent and without an increase in photosynthate (energy) none of the other changes can occur. The concepts underlying the induction of stress resistance in plants are unique.

(18) The capabilities of Applicant's seed treatments with FMD gave plants as shown in FIG. 1. In FIG. 1A, plants of one variety of corn treated with nothing (chemical seed treatment only), SabrEx, a mixture of K2, K4 and As2 and K5As2 is shown, while FIG. 1B shows plants of same variety with the chemical seed treatment only (same plant as at the one at the left of FIG. 1A and with a formulation containing a low level of a metabolite of Trichoderma. The metabolite was applied only to the seeds at less than 1 μl/seed, but caused season-long effects, by epigenetic effects. As predicted from greenhouse testing, both above ground plant parts and roots were larger from the microbial or metabolite seed treatments, yield of grain and silage was increased by more than 10% and C sequestration (a measure of photosynthetic efficiency) and N uptake (a measure of N use efficiency (NUE) were increased. All values were significant from these replicated plots and the results were replicated over five separate varieties.

(19) These results are therefore the result of the integration of systems biological approaches that begin with identification of superior strains in the laboratory, continue through predictive greenhouse evaluations, and that are a component of testable and predictive models that have largely been shown to be correct.

(20) Systems and Observations Reaching Commercial Stages.

(21) Many of Applicant's strains have multiple functionality and, as an example of this versatility, Applicants present data on one such strain, with reference to others that are appropriate for the purpose. Information on the stage of development of these strains and formulations relative to them are provided for each application.

(22) Nematode Control.

(23) Experiments were contracted with Nemalab, Durbanville, SA, as follows and shown at FIG. 2 herein:

(24) Tomatoes were grown for a 6-8 weeks and then the stems were split and the resulting plant had two separate root systems but a single shoot.

(25) Each of the two roots were separately placed in potting soil/separate pot.

(26) One pot was treated with a microbial strain plus nematodes and the other side contained only nematodes. Data on nematode control was taken 45 days later.

(27) The purpose of this system was to measure direct control of nematodes by contact with the biocontrol agents versus control via systemic resistance conferred by the contact of the biocontrol agents plus nematodes. Results of nematode control is reported with the two root systems as treated pot vs systemic pot.

(28) This experiment is another validation of the model based FMD and shown above. The only way that the roots without the biological treatment can be protected from nematodes is via systemic induction of resistance, this time for control of nematodes. There were ten other microbials and microbial combinations evaluated, and only two others gave results comparable to those shown herein.

(29) Experiments were conducted on tomato with Meloidgyne javacina (plant pathogenic nematodes) on tomato.

(30) There were six replicates per treatment.

(31) The experiments were designed to indicate whether control was of an induced systemic nature or direct effects of the strains on the nematodes. Data shown therefore is for the systemic effect (i.e., nematodes applied but Trichoderma not applied to that root side) versus direct effects (i.e., both nematodes and the agent applied to the split root in question). Data is shown in the accompanying graph in FIG. 2. In every case, the implied comparison of the water (nematode only) control and the treated roots or systemic roots is statistically significant. The data suggest that the biological agent reduced all parameters measured, and did so on roots where the agent was applied directly with the nematodes and on the other root, where only nematodes were applied.

(32) This data indicates a high degree of systemic resistance that can be transferred to plant portions where the biocontrol organism is not added. The galling index was reduced and it must be noted that the reduction of galling is not linear, so the decrease is probably underestimated. Egg numbers were reduced very greatly, to almost zero, even though the numbers of female nematodes were at less reduced numbers. This suggests that, even though female nematodes were present, they were incapable of reproducing. Other strains and strain combinations were about as effective as K5, although some were much less effective.

(33) In the above tomato plant experiments, nematode growth and reproduction occurred in root portions not exposed to the agents, thus demonstrating that this system was operational in tomato roots tested. This experiment is not determinative if, in addition, there was also direct effects of the agents where the agent and nematodes were present together. Further experiments have now shown such effects.

(34) In this disclosure, Applicants provide the following:

(35) Enhanced season-long root development in corn (expanding upon the information in the co-terminally filed U.S. provisional patent application No. 62/479,080 entitled “Coating materials for seeds and particulate materials, including fertilizers, to enhance plant growth and productivity.”)

(36) High levels of efficacy of nematode control on corn and soybeans, as well as increasing the range of efficacy to the following nematodes on corn: root lesion nematodes=Pratylenchus spp.; spiral nematode=Helicotylenchus, but it is also sometimes applied to other genera in the family Hoplolaimidae including Rotylenchus, Aorolaimus, Scutellonema, and Peltamigratus; Dagger nematode=Xiphinema; these nematodes transmit viruses; Lance nematode=Hoplolaimus; these insects are damaging to field crops, but even more so on turf, and on soybeans the soybean cyst nematode=Heterodera glycines.

(37) Demonstration that control on different plants occurs in the field as well as in the greenhouse. Applicants also include definitive field results with corn and soybeans. FIGS. 3A and 3B show pictures of enhanced root growth in corn in both PVC pipes (FIG. 3A) and in the field (FIG. 3B).

(38) FIGS. 4A-D show results involving the control of nematodes for soybeans in field trials in Wisconsin in the summer of 2016. FIG. 4A is a chart showing the specific seed treatments that were applied. FIG. 4B indicates the yields generated with each treatment. FIG. 4C is a chart showing plants with root symptoms at different stages of development. FIG. 4D is a chart showing the effect of these treatments on soil egg counts.

(39) FIGS. 5A-C show results involving the control of nematodes for corn in field trials in Wisconsin in the summer of 2016. Seeds were separately treated with Omega (1-octene-3-ol), SabrEx, K5, and a control (chemical seed treatment only). FIG. 5A is a chart showing stand establishment and yield. FIG. 5B is a chart showing plants with root symptoms at different stages of development. FIG. 5C is a chart showing nematode numbers by type.

(40) Control of Aphids Using Foliar Treatments.

(41) Applicant's systems that are composed of selected strains and development of formulations have proven effective in the control of aphids, as shown in FIG. 6, and similar treatments also are effective on white flies. The graph in FIG. 6 provides results in greenhouse cage experiments on cabbage. In each case, results were from counts on individual plants considered as replicates, but all the plants were contained in the same cage. This was necessary to avoid transfer of the winged forms of the aphids between treatments. Consequently, the starting point in aphid numbers varied between the treatments, so the change over time is the important parameter.

(42) Three of a number of treatments are shown in FIG. 6. The control was on plants treated with aphids only. All of the other treatments contained Applicant's proprietary adjuvant organic pesticide base (“OPB”). This material is suitable for organic certification and contains an OMRI listed surfactant as well as other materials. It forms highly stable emulsions for spray application; typically Applicants add 3-6 ml of the concentrated OPB per liter of water used for applications. By itself, OPB has insecticidal properties. The two microbial strains shown in the example are K5 (added in the OPB-based formulation) and Bacillus amyloliquifaciens strain TR4, also added with OPB.

(43) In the experiment having results shown in FIG. 6, spray applications were made on day 1 and day 7, and numbers of live aphids were counted daily. In the data shown, there was no effect in aphid numbers in the control (water-only) spray, but with OPB and OPB+K5, numbers were immediately and significantly reduced and remained at a fairly low levels throughout the experiment. However, numbers with OPB+TR4 were significantly lower. In Applicant's experiments, OPB is necessary as adjuvant both for its insecticidal activity and because it allows wetting and potentiation of the microbial agents. In this case FMD is an effective tool, but the most effective strain TR4 is different from those identified in root studies.

(44) Enhanced and Improved Formulations of Biological Agents as Seed Treatments.

(45) Microbial agents have greatly untapped potential to improve agricultural productivity and profitability. However, they have some potential issues that can reduce their acceptability in agricultural practices. Two of the more important (and sometimes overlooked) factors follow. First, for agricultural practices, especially for seed treatments, but other applications as well, currently have chemical pesticides as components. These frequently are toxic to microbial agents, either through direct effects of the active ingredients or through toxicity of the adjuvants in chemical formulations such as surfactants and the like. Applicants have shown repeatedly that one can add biologicals to seeds already treated with a chemical with good efficacy, even just after the chemical is applied. This can be accomplished with a separate tank on the seed treater for the biological, but this has proven inconvenient, which limits sales of our products. To overcome this issue, you need to be able to mix biological agents directly with the tank mix with no separate tank. Beyond this, some potential users would like to be able to make a direct ‘in can’ system in which the disclosed biologicals are fully compatible with the concentrated pesticide for extended periods of time.

(46) Second, seed treatment and other agricultural application equipment frequently contain elements that impose high shear forces upon microbial cells. These may easily disrupt the cells, a fact that many or most biological agent manufacturers ignore. Nevertheless, recent advances by the Applicants in microbial formulation have overcome many of these objections. For evaluation of new systems, Applicants worked with a commercial pesticide mixture that contains three fungicides and an insecticide. It also contains a colorant, and high levels of surfactants and emulsifiers. Each of these components can be lethal to Trichoderma spores; if spores themselves with no added protection are added to this mix, they are killed instantly. Applicants considered that the only possible solution to provide compatibility is to create systems in which the spores are physically separated from the pesticide. Initial steps were on a macro scale, but the desired outcome required a physical mixture of the pesticide and the biological agent, at least at the tank mix level.

(47) Shelf Life of New Formulations in Pesticide.

(48) Applicants have been successful in creating new formulations that are compatible with the chemical pesticide. Novel encapsulation/formulation systems allow the disclosed Trichoderma strains to retain full viability three weeks after mixing. To reiterate, without the formulation advance, the biological agents are killed immediately upon contact. Three weeks allows full chemical compatibility of our formulations with chemical pesticides at least in tank mixes. Three weeks does not give a full in-can solution, but the advances made suggest that such a goal is within reach. The new system also allows seeds to be treated with the Hege spinning disc system, demonstrating the compatibility of the new formulation with high shear forces. Compatibility was demonstrated by direct planting of seeds and by dilution plantings after seed washings.

(49) Efficacy of Pesticide-Trichoderma Combinations.

(50) Data is given for root weight; shoot weight and stand establishment follow the exact same pattern. Data is from greenhouse tests with four replicates per treatment and shown in FIG. 7. The results of the planting in infested soil verify the results reported above for protection of seeds by K5; these experiments were conducted with K2, but as mentioned earlier, all of the strains have excellent abilities to protect seeds against soil-borne pathogens. The results with K2 (represented by T if the graph in FIG. 7) are, in fact superior to the complex mixture within the chemical pesticide preparation (designated by P; P+T is the combination treatment). This is attributed to the fact that the pesticides protect the seeds only, but the systemic protection afforded by K2 not only protects the seed, but provides longer term protection to the entire plant.

(51) Advanced Microbial Agent Encapsulation Systems for Plant Agriculture.

(52) In accord with the foregoing discussion and details regarding formulations and modality of application, the Applicants appreciate that microbial agents are proving to have substantial utility and capabilities in plant agriculture. However, they must be produced at high levels and with good quality, and they must be formulated both to allow them to be used in chemically-intensive commercial agriculture and to provide adjuvants that enhance the activity of the microbes in plant agriculture. With the exception of organic agricultural production, almost all commercial production is reliant upon chemical fungicides. If biological agents are to reach their full potential and diversity of uses, living microorganisms need to be formulated to allow them to survive and flourish in this chemical environment.

(53) Among the items addressed by the present technology are (i) formulations that permit living organisms to be formulated and survive in harsh chemical environments. This included survival in concentrated chemical formulations; usually to aid in shipping and handling chemical pesticide products are concentrated with the expectation that they will be diluted with water before application; (ii) in addition, many delivery systems for agricultural delivery includes devices designed to apply high shear and gravitational forces to the products. This is an advantage for chemicals where a high degree of particle dispersement and singulation is desirable but it may be lethal to microbial cells where cellular integrity is necessary. Such devices include seed treatment systems where spinning discs or high shear pumping systems are employed; and (iii) in addition, an issue for both biological and chemical agricultural products is high levels of dusting and removal of product from seed surfaces by the use of vacuum planters. This is a particular issue when seeds are treated with some chemical insecticides; the talc dusts from the planter due to the vacuum and the insecticide laced powder it deposited in the environment and it has been implicated as a major factor in honey bee decline.

(54) The present technology also entails a product, GraphEx (see U.S. Pat. No. 9,102,893), that contains talc. Honey bees are not an issue, but loss of product during planting has been a very serious problem. If the product is removed from seeds during the planting process, then the benefits of the microbes are lessened. This problem has contributed to less-than-optimal performance of the product in field situations. To this point, most formulations development of biologicals have focused on aqueous or dry hydrophilic materials. This is reasonable since the organisms themselves are grown in aqueous or at least environments that are quite moist. However, substitution of nonaqueous formulations have advantages in many circumstances. These are defined by the examples that follow.

Examples

(55) Preparation of Oil-Based Trichoderma Preparations. Trichoderma spores frequently contain proteins on their surface designated hydrophobins. These proteins are amphiphilic, in that they contain both a polar (water soluble) end and a nonpolar end (lipid soluble). The properties of these materials are very useful to the present disclosure. See FIG. 8 showing a model of a hydrophobin molecule, where the hydrophobic portion is shown highlighted at the bottom portion of the model (Linder, 2009). Most notably, they assemble on the air/water or oil/water surfaces. As a consequence, if dry spores of Trichoderma are mixed with water, they will be suspended in water and will not mix with oil, if an oil layer is applied to the surface. Remarkably, if the opposite process is done, in which spores are suspended in oil, then the resulting spore suspension will not migrate into a water layer if water is overlain on the oil. Thus, stable suspensions in either water or oil are readily obtainable simply by mixing dry spores with either water or oil in the absence of the other. This is a very useful property in preparing formulations of these highly useful microbial agents. In addition, these proteins also are important for the outstanding biological activity of these fungi in plant agriculture. Selected strains of the fungi are highly active both in reducing diseases and other pests and to increase plant growth and yield when properly formulated into agriculturally beneficial formulations (e.g., see U.S. Pat. Nos. 8,716,001, 8,877,480 and 8,877,481).

(56) Applicant sells an aqueous-based product, SabrEx LQ, that has proven highly useful especially for treating seeds by commercial seed treaters and seed companies. It is very active, only about 6 ml are required to treat sufficient corn seeds to treat about 1 hectare of farm land. However, it may be even more useful to prepare a similar material but with that is oil based. Preparation is extremely easy, dried spores are added to soybean or other oil, but preferably a plant-based triglyceride. Such oils are readily susceptible to degradation by Trichoderma lipases and other enzymes, and therefore become a carbon source for the microorganism. Later examples demonstrate wax-based encapsulation will show, and the ability of the microbe to escape an encapsulating material is critical for rapid growth of the organism on the planted seed.

(57) Such oil-based formulations may be advantageous over, for example, SabrEx LQ, which is a suspension of spores in water with a preservative. Advantages include, since the preparations are made with dry spores that have smaller volumes than the fully hydrated ones used in the aqueous-based product, higher potency (colony forming units; cfus) are possible. Second, the shelf life of SabrEx LQ is only about 90 days at room temperature, probably in large part because the high level of hydration allows significant levels of microbial respiration. However, the oil-based formulations are much drier, and so no respiration should be possible. The water activity of the aqueous material is about 0.92 and that of the oil-based formulation about 0.10. However, for oil-based bacterial preparations, different steps must be used since the bacteria lack the hydrophobins on the outer cell or spore wall. These organisms are suspendable in aqueous solutions but not readily in oils. If, however, concentrated bacterial suspensions are added to soybean oil containing about 0.2% lecithin, then a stable emulsion can be obtained and this can be used for further formulations.

(58) Formulation of Chemical Pesticide-Stable Trichoderma Preparations.

(59) Large agbio corporations are dominated by chemically-based paradigms. If biological companies are achieve maximum success, they have to adapt to the business and technology models imposed by the chemically-dominated markets and market drivers. The material in question is a typical pesticide that contains three different fungicides, an insecticide and formulations that contain high levels of surfactants, emulsifiers and coloring agents. Hereafter this material is termed pesticide S. If any of the disclosed dry or aqueous-based Trichoderma products are added to pesticide S, they were killed instantly. It was considered that the only possible solution was to prevent direct contact of the strains with the toxic solution.

(60) The first attempt was to prepare a physical separation as shown in FIG. 9, and that is termed the parfait solution. In this system, the pesticide (bright red) is added, and this is overlain with a wax barrier (palm oil) that melts at 95 F (36 C). This then was overlain with the Trichoderma spore suspension in soybean oil described in herein. It is important to note that the relative amounts of Trichoderma spore suspension and pesticide S are correct for application to seeds. The pesticide solution is concentrated and is typically diluted 15× for application to seeds, but even so, the volume of the Trichoderma solution is less. This is because the Trichoderma suspension can be highly concentrated since more dry spores can be added to the oil suspension than if aqueous suspensions are used, and because only a very small amount of Trichoderma is used for field seed treatments because the agent colonizes plant roots and may increase up to 10,000-fold within the first few days of seed germination. Thus, the amount added to seeds is only the starter inoculum and most of the growth of the organism is on roots and on developing plants.

(61) This parfait solution conferred stability to the Trichoderma spores overlaying the barrier layer. Applicants considered that this might be a viable solution. However, Applicants further considered the addition of the biological agent directly to the pesticide solution. There were two next steps.

(62) The first was demonstration that it is the pesticide base, probably the surfactants, that were immediately toxic. This was shown by microscopic observation that as soon as spores came into contact with the solution, the spores were immediately destroyed, and effect too rapid to be due to the chemical fungicides present in the mix.

(63) The second step was a consideration of the nature of the oil-spore interaction. As indicated above, the hydrophobins on the spore surface interact with the oil, and from the literature and observations, forms a shell around the spore. Thus, there was a reasonable expectation that, if the oil-based Trichoderma spore suspensions were mixed with the aqueous pesticide S, there would be formed a “pseuo-encapsulation” of the oil base around the spores that would prevent its contact with the pesticide. This did indeed occur. This step was performed with soybean oil, which at room temperature is a liquid, so the system consists of the spore surrounded by a shell of the liquid oil that is in turn surrounded by the aqueous pesticide S.

(64) A next step was to produce a “micro-parfait” mixture. To accomplish this, rather than using soybean oil as the suspending oil, liquid palm oil or wax at the 36 C melting point was added, and immediately this was mixed rapidly into Pesticide S. This process resulted in a suspension of the wax-encapsulation with small (approximately 5-20 μm) particles with the wax surrounding the Trichoderma spores.

(65) This still was not an ideal solution, since the wax probably has too low a melting point to meet all temperature conditions to which the preparation may be subjected. Other waxes, such as bee's wax, cadelilla wax and carnauba wax have higher melting points, up to 86 C. The method described herein to prepare useful microbial products is generally not possible with these high melting point waxes since the microbial agents will be killed by the heat of the molten wax solutions. However, there is a relatively simple method and apparatus to overcome this difficulty.

(66) Buchi manufactures a device that produces encapsulated products, including microorganisms, with a nozzle system. In the preferred embodiment, the hot wax is extruded in the outer shell of concentric nozzles and the material to be encapsulated is extruded in the center nozzle. This gives a liquid encapsulated by a solid wax layer, and this is immediately cooled by expulsion into cold water or alcohol. This encapsulation system avoids exposure of the microbes to the hot wax, or if it is exposed at all, it is for only a second. This system will provide encapsulation of microbes for a wide variety of applications. Even without this system, the palm oil/wax method gave good results. FIG. 10 gives an example.

(67) FIG. 10 indicates that the cfu levels actually increased in the mix. However, this probably is not correct. It is important to note that the original encapsulation contained many particles that contain multiple Trichoderma spores. Thus, over time it is anticipated that the surfactants and other materials in pesticide S probably resulted in disaggregation of some of the larger particles, which led to the apparent increase and overestimation. Nonetheless, given that the spores if exposed to pesticide S are immediately killed, the result is excellent.

(68) The biological activity of the new formulations with or without pesticide S is very good. FIG. 7 provides data on growth of wheat treated with pesticide S (P), S plus the new formulations of Trichoderma, the new formulations of Trichoderma alone or the untreated control when the treated seeds were planted in soil infested with the soil pathogens Pythium or Rhizoctonia. As is evident, the treatment with the new formulation of Trichoderma was superior to the pesticide used alone. This data is also remarkable because the seeds were treated using a spinning disc (Hege) device, which illustrates that the encapsulation system also eliminates damage to the spores by the spinning disc.

(69) Development of Wax-Based Formulations of Microorganisms to Provide Highly Potent Products and to Avoid Seed Dusting and Blow-Off.

(70) As noted at the outset, seed dusting with dry products, especially talc is a problem. Not only can the dusts pose environmental issues, but active ingredients contained within the talc formulations may be removed from the seeds, thereby creating issues with efficacy. Waxes have been shown to adhere more firmly to seeds than other materials such as talc (e.g., US Patent Application 20150013221). As noted, Applicants have dust applications of various products that contain talc, and a non-clumping formulation that adheres well to seeds is needed.

(71) This disclosure provides such a method. It comprises: (i) making the soybean oil based suspensions of Trichoderma spores or bacteria as described herein (with or without lecithin as needed for the organism). Other oils can be used with equal efficacy, but for maximum efficacy these may need to be vegetable oils that can be hydrolyzed by the microbe, thus providing a nontoxic food base for the microbes; and (ii) mixing the suspension with a waxy substance with appropriate properties for seed treatments.

(72) Applicants used Aqua-Tex 350 from Micropowders Inc. The ratio of wax to oil is preferably 5:1 or greater; the concept is that the oil in preparation will be dissolved into the wax. This expectation was realized and a free-flowing powder with excellent seed coating properties was obtained. The use of the oil suspensions provides starting materials with very high potency, up to 1011 cfu for bacteria and 1010 cfu/g for fungi. Many materials would be effective as the waxy component. This includes bee's wax, cadelilla wax and carnauba wax, synthetic waxes such as Aqua-Tex 350. There are a wide variety from which to choose. The powder so obtained can be further diluted or mixed to provide final products with different properties as desired. FIG. 11 shows an example of the products obtained.

(73) The present disclosure is not to be limited in terms of the particular embodiments described in this application. Many modifications and variations can be made without departing from its scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

(74) As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 conduits refers to groups having 1, 2, or 3 conduits. Similarly, a group having 1-5 conduits refers to groups having 1, 2, 3, 4, or 5 conduits, and so forth.

(75) While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope being indicated by the following claims.

(76) All references cited herein are incorporated by reference herein in their entireties and for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually incorporated by reference in its entirety for all purposes.