阅读说明：本技术 稳定的杀真菌组合物 (Stable fungicidal compositions ) 是由 A·格尔兹 C·基梅尔舒尔 李燕 C·S·泰勒 C·R·威尔逊 K·芬塞思 于 2019-05-14 设计创作，主要内容包括：本发明涉及一种稳定的杀真菌组合物,其包含：a)产生杀镰孢菌素的类芽孢杆菌属种菌株；以及b)稳定剂,其选自胺、季铵化合物、磷酸盐或柠檬酸盐、具有2至10个碳原子的短链多元醇、脲及其组合。本发明还提供了农业上可接受的稳定的水性制剂,其包含产生杀镰孢菌素的类芽孢杆菌属种菌株和稳定剂,以及使用所述杀真菌组合物和水性制剂防治植物病害的方法。(The present invention relates to a stable fungicidal composition comprising: a) a fusaricidin producing paenibacillus species strain; and b) a stabilizer selected from the group consisting of amines, quaternary ammonium compounds, phosphates or citrates, short chain polyols having 2 to 10 carbon atoms, urea and combinations thereof. The present invention also provides agriculturally acceptable stable aqueous formulations comprising a fusaricidin-producing strain of bacillus sp.)

1. A stable fungicidal composition comprising:

a) a fusaricidin producing paenibacillus species strain; and

b) a stabilizer selected from the group consisting of amines, quaternary ammonium compounds, phosphates or citrates, short chain polyols having 2 to 10 carbon atoms, urea, and combinations thereof.

2. The fungicidal composition of claim 1, wherein the fusaricidin producing paenibacillus species strain is: paenibacillus agaricus, Paenibacillus agarophilus, Bacillus alginolyticus, Paenibacillus fonticola, Paenibacillus amyloliquefaciens, Paenibacillus anaerobicus, Paenibacillus antarctica, Paenibacillus assamica, Paenibacillus azoreducens, Paenibacillus azotobacterium, Paenibacillus Baselina, Paenibacillus beidella, Paenibacillus brazilian, P.brassicae, Paenibacillus camphensis, Paenibacillus jin, Paenibacillus chitinolyticus, Paenibacillus chondroitin, P.cineris, Paenibacillus kushii, Paenibacillus coagulans, Paenibacillus endosphaenii, Paenibacillus trellissimus, Paenibacillus cereus aiensis, Paenibacillus angsiensis, Paenibacillus cerevisiiphenii, Bacillus depolymeriticus, Paenibacillus cerevisiiphenii, Paenibacillus gasseri, Paenibacillus cereus, Paenibacillus cerevisionius, Paenibacillus cerevisionii, Paeni, Paenibacillus subtilis, Paenibacillus cereus, Paenibacillus korea, Paenibacillus mucilaginosus, Paenibacillus lactis, Paenibacillus larvae, Paenibacillus lautus, P.lentimorbobus, Paenibacillus macerans, Paenibacillus markularensis, Paenibacillus massiliensis, Paenibacillus menensis, Paenibacillus benthicus, Paenibacillus naportus, Paenibacillus nematophilus, novel species epiphytic species of Paenibacillus cereus, Paenibacillus odoriferus, Paenibacillus cereus, Paenibacillus pulus pitelii, P.enophonis, Paenibacillus phyllus, Paenibacillus polymyxa subspecies, Paenibacillus japonicus, Paenibacillus cereus, paenibacillus staurospora, Paenibacillus taizhou, Paenibacillus terrae, Paenibacillus thiaminolyticus, Paenibacillus tsumadiensis, P.tylopili, Paenibacillus thuringiensis, Paenibacillus robenii, Paenibacillus vortexes, Paenibacillus vulnificus, Paenibacillus cereus or Paenibacillus xylanolyticus.

3. The fungicidal composition of claim 1, wherein the fusaricidin producing paenibacillus species strain is: paenibacillus sp strain NRRL B-50972, Paenibacillus sp strain NRRL B-67129, Paenibacillus sp strain NRRL B-67304, Paenibacillus sp strain NRRL B-67306, Paenibacillus sp strain NRRL B-67615 or fungicidal mutant strains thereof.

4. The fungicidal composition of claim 3, wherein the genomic sequence of the fungicidal mutant strain has greater than about 90% sequence identity to the respective strain.

5. The fungicidal composition according to any one of claims 1 to 4, wherein the stabilizer is urea.

6. The fungicidal composition of claim 5, wherein the urea is carbamide (CO (NH)₂)₂) Or substituted ureas of the formula (I)

(I)

Wherein

R₁、R₂And R₃Independently is-H, -OH, C₁-C₆Alkyl or-R₅OH；

R₄is-R₅OH; and

R₅is C₁-C₆An alkyl group.

7. The fungicidal composition according to claim 5 or 6, further comprising a phosphate or citrate.

8. The fungicidal composition according to claim 7, wherein the phosphate or citrate is selected from the group consisting of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen citrate, dipotassium hydrogen citrate, and combinations thereof.

9. The fungicidal composition according to claim 7 or 8, wherein the urea and phosphate or citrate are present in the fungicidal composition in a combined concentration of from about 0.1% to about 3%.

10. The fungicidal composition according to any one of claims 1 to 4, wherein the stabilizer is an amine.

11. The fungicidal composition according to claim 10, wherein the amine is guanidine hydrochloride or triethanolamine.

12. The fungicidal composition according to any one of claims 1 to 4, wherein the stabilizer is a quaternary ammonium compound.

13. The fungicidal composition of claim 12, wherein the quaternary ammonium compound is a betaine hydrochloride.

14. The fungicidal composition according to any one of claims 1 to 4, wherein the stabilizer is a phosphate or citrate.

15. The fungicidal composition of claim 14, wherein the salt is potassium phosphate (dibasic) or potassium citrate (tribasic).

16. The fungicidal composition according to any one of claims 1 to 4, wherein the stabilizer is a short-chain polyol having 2 to 10 carbon atoms.

17. The fungicidal composition of claim 16, wherein the short chain polyol having 2 to 10 carbon atoms is selected from the group consisting of diethylene glycol, triethylene glycol, propylene glycol, 1, 3-butanediol, 1, 4-butanediol (tetramethylene glycol), 1, 5-pentanediol (pentaethylene glycol), 1, 6-hexanediol (hexamethylene glycol), ethylene glycol, 1, 3-propanediol, 1, 4-butanediol, neopentyl glycol, 3-methyl-1, 5-pentanediol, p-xylene glycol, 1, 4-di- (. beta. -hydroxyethoxy) benzene, 1, 3-di- (. beta. -hydroxyethoxy) benzene, cyclohexane 1, 4-dimethanol, octane-1, 8-diol, decane-1, 10-diol, and combinations thereof.

18. The fungicidal composition of claim 17, wherein the short chain polyol having 2 to 10 carbon atoms is propylene glycol.

19. The fungicidal composition according to any preceding claim, comprising a biologically pure culture of a fusaricidin-producing strain of a bacillus sp.

20. The fungicidal composition according to any preceding claim, wherein the weight ratio of the fusaricidin-producing bacillus sp.

21. The fungicidal composition according to any preceding claim, wherein the fungicidal composition is formulated as one selected from the group consisting of: liquid compositions, emulsified concentrates, suspoemulsions, suspension concentrates and aqueous solutions.

22. The fungicidal composition according to any preceding claim, further comprising an agriculturally acceptable polar water-miscible organic solvent.

23. An agriculturally acceptable stable aqueous formulation comprising:

a) a fusaricidin producing paenibacillus species strain in an amount of 3% w/w to 90% w/w;

b) a stabilizer selected from the group consisting of amines, quaternary ammonium compounds, phosphates or citrates, short chain polyols having 2 to 10 carbon atoms, urea and combinations thereof in an amount of 0.1% w/w to 20% w/w;

c) water; and

d) optionally, a polar water-miscible organic solvent in an amount of 2% w/w to 60% w/w.

24. The aqueous formulation of claim 23, wherein the fusaricidin-producing paenibacillus strain is paenibacillus strain NRRL B-50972, paenibacillus strain NRRL B-67129, paenibacillus strain NRRL B-67304, paenibacillus strain NRRL B-67306, paenibacillus strain NRRL B-67615, or a fungicidal mutant strain thereof.

25. The aqueous formulation of claim 23 or 24, wherein the stabilizer is urea.

26. The aqueous formulation of claim 25, wherein said urea is carbamide (CO (NH)₂)₂) Or substituted ureas of formula (I)

(I)

Wherein

R₁、R₂And R₃Independently is-H, -OH, C₁-C₆Alkyl or-R₅OH；

R₄is-R₅OH; and

R₅is C₁-C₆An alkyl group.

27. An aqueous formulation according to claim 25 or 26, further comprising a phosphate or citrate.

28. The aqueous formulation according to claim 27, wherein the phosphate or citrate is selected from the group consisting of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen citrate, dipotassium hydrogen citrate, and combinations thereof.

29. The aqueous formulation according to claim 27 or 28, wherein the urea and phosphate or citrate are present in the aqueous formulation at a combined concentration of about 0.1% to about 3%.

30. The aqueous formulation of claim 23 or 24, wherein the stabilizer is an amine.

31. The aqueous formulation of claim 30, wherein the amine is guanidine hydrochloride or triethanolamine.

32. The aqueous formulation of claim 23 or 24, wherein the stabilizing agent is a quaternary ammonium compound.

33. The aqueous formulation of claim 32, wherein said quaternary ammonium compound is a betaine hydrochloride.

34. The aqueous formulation of claim 23 or 24, wherein the stabilizer is a phosphate or citrate.

35. The aqueous formulation of claim 34, wherein the salt is potassium phosphate (dibasic) or potassium citrate (tribasic).

36. The aqueous formulation of claim 23 or 24, wherein the stabilizer is a short chain polyol having 2 to 10 carbon atoms.

37. The aqueous formulation of claim 36, wherein the short chain polyol having 2 to 10 carbon atoms is selected from the group consisting of diethylene glycol, triethylene glycol, propylene glycol, 1, 3-butanediol, 1, 4-butanediol (tetramethylene glycol), 1, 5-pentanediol (pentaethylene glycol), 1, 6-hexanediol (hexamethylene glycol), ethylene glycol, 1, 3-propanediol, 1, 4-butanediol, neopentyl glycol, 3-methyl-1, 5-pentanediol, p-xylene glycol, 1, 4-di- (. beta. -hydroxyethoxy) benzene, 1, 3-di- (. beta. -hydroxyethoxy) benzene, cyclohexane 1, 4-dimethanol, octane-1, 8-diol, decane-1, 10-diol, and combinations thereof.

38. The aqueous formulation of claim 37, wherein the short chain polyol having 2 to 10 carbon atoms is propylene glycol.

39. A method of treating plants to control diseases, wherein the method comprises applying an effective amount of a fungicidal composition according to any one of claims 1 to 22 or an aqueous formulation according to any one of claims 23 to 38 to the plants, plant parts and/or the locus of the plants.

40. The method of claim 39, wherein the method comprises applying the fungicidal composition or the aqueous formulation to the plant parts of the foliage.

41. The method of claim 39 or 40, wherein the plant disease is caused by a fungal pathogen selected from the group consisting of Alternaria alternata, Alternaria solani, Botrytis cinerea, Chaetomium anthracis, Fusarium culmorum, Septoria nodorum, Mycosphaerella graminicola, Phytophthora crypthecicola, Phytophthora infestans, Peronospora cucumis, Pythium ultimum, Magnaporthe oryzae, Rhizoctonia solani var nigricans, Avena nigrospora sativa, Ruscus aculeatus, and Puccinia tritici.

Technical Field

The present invention relates to a bacterial strain preparation and a method for controlling plant diseases by using the same. In particular, the present invention relates to stable fungicidal compositions and formulations of Paenibacillus sp strains that produce fusaricidin.

Background

Fungicides have numerous uses, including use in crop protection; as food, feed and cosmetic preservatives; and as therapeutic agents for human and veterinary applications. Crop reduction, food-borne diseases, and fungal infections of humans and animals are problematic in both developed and developing countries.

Synthetic pesticides or fungicides are generally non-specific and therefore can act on organisms other than the target organism, including other naturally occurring beneficial organisms. Due to their chemical nature, they may also be toxic and non-biodegradable. Consumers worldwide are becoming increasingly aware of potential environmental and health issues related to chemical residue, particularly in food products. This has led to increasing consumer pressure to reduce the use of, or at least reduce the number of, chemical (i.e. synthetic) pesticides. Thus, there is a need to manage food chain requirements while still allowing effective pest control.

Another problem that arises with the use of synthetic insecticides or fungicides is that repeated and individual application of the insecticide or fungicide often results in the selection of an anti-pathogenic microorganism. Typically, such strains are also cross-resistant to other active ingredients with the same mode of action. After this, effective control of pathogens using the active compounds will no longer be possible. However, active ingredients with new mechanisms of action are difficult and expensive to develop.

The risk of resistance development in pathogen populations and concerns about the environment and human health have prompted interest in identifying alternatives to synthetic pesticides and fungicides for managing plant diseases. The use of biological control agents is an alternative.

Paenibacillus-based biocontrol agents can provide a valuable tool to control the spectrum of plant diseases while reducing the risk of pathogen resistance. Fusaricidin and other antifungal compounds are produced during fermentation of a paenibacillus species strain. However, these antifungal compounds are susceptible to degradation during storage and transportation of paenibacillus-based biocontrol agents. The degradation of these antifungal compounds is promoted by the temperature increase that is common during the growing season. There is a need to identify stable compositions and formulations of paenibacillus-based biocontrol agents that will retain their activity for controlling plant diseases for long periods of time at elevated temperatures.

Disclosure of Invention

The present invention is based on the identification of stabilizers that inhibit the degradation of antifungal compounds produced by strains of Paenibacillus species. These stabilizers retain the antifungal activity of the formulated paenibacillus species strain and reduce the decomposition of fusaricidin and fusaricidin-like compounds.

In certain embodiments, the present invention relates to a stable fungicidal composition comprising: a) a fusaricidin producing paenibacillus species strain; and b) a stabilizer selected from the group consisting of amines, quaternary ammonium compounds, phosphates or citrates, short chain polyols having 2 to 10 carbon atoms, urea and combinations thereof.

In other embodiments, the present invention relates to a stable fungicidal composition comprising: a) a fusaricidin producing paenibacillus species strain; and b) a stabilizer selected from the group consisting of amines, quaternary ammonium compounds, phosphates or citrates, short-chain polyols having 2 to 10 carbon atoms, urea, alkanolamines, amides, and combinations thereof.

In some embodiments, the present invention relates to a stable fungicidal composition comprising: a) a fusaricidin producing paenibacillus species strain; and b) stabilizers selected from the group consisting of carbamide (CO (NH)₂)₂) Guanidine hydrochloride, triethanolamine, betaine hydrochloride, potassium phosphate (dibasic), potassium citrate (tribasic), and combinations thereof.

In certain aspects, the compositions or formulations comprise a biologically pure culture of a fusaricidin-producing strain of a bacillus sp.

In one embodiment, the fusaricidin producing strain of a bacillus species is: bacillus agaricus (p.agarexendens), paenibacillus agaricus (p.agaroidevans), paenibacillus alginolyticus (p.algolyticus), paenibacillus curiosa (p.algalitere), paenibacillus alveolius (p.alveii), paenibacillus amyloliquefaciens (p.amyloliquefaciens), paenibacillus anaerobically growing (p.anaerobacter), paenibacillus antarctica (p.antarcticas), paenibacillus assamica (p.assamensis), paenibacillus azoreducidum (p.azoreducans), paenibacillus azotoformans (p.azotofibracteris), paenibacillus barnacinia (p.barcinonensis), paenibacillus beifengensis (p.borealis), paenibacillus brasiliensis (p.brasiliensis), paenibacillus brasiliensis (p.chondriospecies), paenibacillus brasiliensis (p.echinococcus), paenibacillus reticulatus sp.sp.sp.chondriosus sp.chondriosus, paenibacillus purpureus (p.sp.sp.chondriosus), paenibacillus crispatus (p.chondriosus), paenibacillus sp.chondriosus sp.sp.chondriosus sp.sp., Bacillus firmus (p.ehimensis), paenibacillus erygii (p.elsigii), paenibacillus mellea (p.favispora), paenibacillus degelatinatus (p.glucanolyticus), paenibacillus depolymeriticus (p.glucanolyticus), paenibacillus gomerdae (p.gordonae), paenibacillus graminis (p.grandis), paenibacillus granulosus (p.granivorans), p.hodogayensis, paenibacillus illinois (p.illinoinensis), paenibacillus gamsii (p.jamesonensis), paenibacillus shensukuchenensis (p.kobensis), paenibacillus cerevisiae (p.koleovani), paenibacillus koreanus (p.koremensis), paenibacillus mucilaginosus (p.kuchenensis), paenibacillus lactis (p.lactis), paenibacillus maculatus sp.malanibacillus sp.sp.sp.sp.sp.sp., Bacillus naphylogenens (p.naphylenovorans), paenibacillus nematophilus (p.nematophilus), paenibacillus epiphyticus (p.nov. spec. epiphyticus), paenibacillus odoriferus (p.odorifer), paenibacillus foddensis (p.papuli), paenibacillus perrei (p.peoria), p.phoenis, paenibacillus phyllosporans (p.phyllosporane), paenibacillus polymyxa (p.polymyxa), paenibacillus polymyxa (p.polymyxa sp.polymyxa), paenibacillus polymyxa subsp (p.polymyxa sp.plantarum), paenibacillus japonicus (p.pophyllus), paenibacillus japonicus (p.po), paenibacillus pusilvadensis (p.pusilvadensis), paenibacillus rhizophilus, paenibacillus sphaeroides (p.purpureus), paenibacillus sp.vorax (p.sp.sp), paenibacillus sp., Paenibacillus vulnificus (p.vulneris), paenibacillus went (p.wynnii), or paenibacillus xylanolyticus (p.xylanilyticus).

In another embodiment, the fusaricidin producing strain of a bacillus species is: paenibacillus polymyxa, Paenibacillus polymyxa subspecies, Paenibacillus epiphytic new species, Paenibacillus terrae, Paenibacillus macerans or Paenibacillus alvei. In yet another embodiment, the fusaricidin-producing strain of a Paenibacillus species is a Paenibacillus terreus.

The fungicidal composition may be in any formulation form, in particular a liquid composition, such as an emulsified concentrate, a suspoemulsion, a suspension concentrate or a solution, such as an aqueous solution.

In some aspects, the weight ratio of the fusaricidin producing strain of Bacillus species to the stabilizer is from about 500: 1 to about 1: 500, from about 400: 1 to about 1: 400, from about 300: 1 to about 1: 300, from about 200: 1 to about 1: 200, from about 100: 1 to about 1: 100, from about 75: 1 to about 1: 75, or from about 50: 1 to about 1: 50. In one embodiment, the weight ratio of the fusaricidin producing strain of Bacillus species to stabilizer is from about 200: 1 to about 1: 200. In another embodiment, the weight ratio of the fusaricidin producing strain of Bacillus sp and the stabilizing agent is from about 50: 1 to about 1: 50.

In certain aspects, the fungicidal compositions further comprise a polar water-miscible organic solvent. The polar water-miscible organic solvent may be any one and/or combination of the following organic solvents: 1, 3-butanediol, 2-pyrrolidone, acetone, acetonitrile, aliphatic alcohols, alkyl esters of aliphatic carboxylic acids, cyclohexanone, diethylene and triethylene glycols, diacetone alcohol, dialkyl ketones, diethylene glycol dimethyl ether, Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethanol, ethyl acetate, formamide, furfuryl alcohol, gamma-butyrolactone, glycerol, glycofurol (glycofurol), glycol ethers, ethylene glycol, 1, 6-hexanediol (hexane glycol), isopropanol, methyl ethyl ketone, N-methylpyrrolidone, 1, 5-pentanediol (pentaethylene glycol), phosphate esters, polyethylene glycols, polyglycols, polyhydroxylated alkanes, propanol, propylene carbonate, propylene glycol, pyrrolidine, sulfolane, tetrahydrofuran, 1, 4-butanediol (tetramethylene glycol), Thiodiglycol, triethylene glycol.

In certain aspects, the invention relates to a composition wherein the fusaricidin producing paenibacillus species strain is paenibacillus species strain NRRL B-50972, paenibacillus species strain NRRL B-67129, paenibacillus species strain NRRL B-67304, paenibacillus species strain NRRL B-67306, paenibacillus species strain NRRL B-67615, or a fungicidal mutant strain thereof. The composition may comprise fermentation products of Paenibacillus sp strain NRRL B-50972, Paenibacillus sp strain NRRL B-67129, Paenibacillus sp strain NRRL B-67304, Paenibacillus sp strain NRRL B-67306, Paenibacillus sp strain NRRL B-67615, or fungicidal mutant strains thereof.

In some embodiments, the genomic sequence of the fungicidal mutant strain has greater than about 90% sequence identity to paenibacillus species strain NRRL B-50972, paenibacillus species strain NRRL B-67129, paenibacillus species strain NRRL B-67304, paenibacillus species strain NRRL B-67306, or paenibacillus species strain NRRL B-67615. In other embodiments, the fungicidal mutant strain has comparable or better fungicidal activity and/or levels of fusaricidin than paenibacillus species NRRL B-50972.

In one aspect, the present invention relates to an agriculturally acceptable stable aqueous formulation comprising: (a) a fusaricidin producing paenibacillus species strain in an amount of 3% w/w to 90% w/w; (b) a stabilizer selected from the group consisting of amines, quaternary ammonium compounds, phosphates or citrates, short chain polyols having 2 to 10 carbon atoms, urea and combinations thereof in an amount of 0.1% to 10% w/w; (c) water; and (d) optionally, a polar water-miscible organic solvent in an amount of 2% w/w to 60% w/w.

In another aspect, the invention relates to agriculturally acceptable stabilizedAn aqueous formulation comprising: (a) a fusaricidin producing paenibacillus species strain in an amount of 3% w/w to 90% w/w; (b) a stabilizer selected from the group consisting of carbamide (CO (NH)₂)₂) Guanidine hydrochloride, triethanolamine, betaine hydrochloride, potassium phosphate (dibasic), potassium citrate (tribasic), and combinations thereof in an amount of 0.1% w/w to 10% w/w; (c) water; and (d) optionally, a polar water-miscible organic solvent in an amount of 2% w/w to 60% w/w.

The amount of fusaricidin producing paenibacillus species strain applied is preferably from about 3% w/w to about 90% w/w of the total formulation. More preferably, the fusaricidin producing paenibacillus species strain is present in the following amounts: from about 4% to 80% w/w, more preferably from 5% w/w to about 75% w/w, and in some embodiments, from about 10% w/w to about 70% w/w or even from about 20% w/w to about 70% w/w.

The amount of stabilizer applied is preferably from about 0.1% w/w to about 20% w/w of the total formulation. More preferably, the stabilizer is present in the following amounts: from about 0.5% to 10% w/w, more preferably from 0.5% w/w to about 5% w/w, and in some embodiments, from about 0.5% w/w to about 2% w/w.

The amount of polar water-miscible organic solvent applied is optionally from about 2% w/w to about 60% w/w of the total formulation. Alternatively, the polar water-miscible organic solvent is optionally present in the following amounts: from about 5% to 50% w/w, more preferably from 10% w/w to about 40% w/w, and in some embodiments, from about 20% w/w to about 40% w/w.

In certain aspects, the present invention relates to a stable dry formulation comprising: (a) a fusaricidin producing paenibacillus species strain; and (b) a stabilizer selected from the group consisting of amines, quaternary ammonium compounds, phosphates or citrates, short chain polyols having 2 to 10 carbon atoms, urea and combinations thereof.

In other aspects, the present invention relates to a stable dry formulation comprising: (a) a fusaricidin producing paenibacillus species strain; and (b) a stabilizer selected from the group consisting of amines, quaternary ammonium compounds, phosphates or citrates, short chain polyols having 2 to 10 carbon atoms, urea, alkanolamines, amides, and combinations thereof.

The dry formulation may be any of wettable powder, soluble powder, dust (dust), granule (granule), tablet, water-soluble and water-dispersible granule, water-soluble and water-dispersible tablet, and water-soluble and water-dispersible powder.

In one embodiment, the present invention relates to an agriculturally acceptable stable formulation comprising: (a) a fusaricidin producing paenibacillus species strain in an amount of 3% w/w to 90% w/w; (b) a stabilizer selected from the group consisting of amines, quaternary ammonium compounds, phosphates or citrates, short chain polyols having 2 to 10 carbon atoms, urea and combinations thereof in an amount of 0.1% to 10% w/w; and (c) other inert agents.

In yet another embodiment, the present invention relates to a method of treating a plant to control a disease, wherein the method comprises applying an effective amount of a composition or formulation disclosed herein to the plant, plant part, and/or locus of the plant. In certain aspects, the compositions comprise a fermentation product of paenibacillus sp.nrrl B-50972, paenibacillus sp.nrrl B-67129, paenibacillus sp.nrrl B-67304, paenibacillus sp.nrrl B-67306, paenibacillus sp.nrrl B-67615, or a fungicidal mutant strain thereof. In other aspects, the method comprises applying the composition to plant parts of the foliage. In still other aspects, at about 1 × 10 per hectare¹⁰To about 1X 10¹²The composition is applied to Paenibacillus sp strain NRRL B-50972, Paenibacillus sp strain NRRL B-67129, Paenibacillus sp strain NRRL B-67304, Paenibacillus sp strain NRRL B-67306, Paenibacillus sp strain NRRL B-67615 or a fungicidal mutant strain thereof of a Colony Forming Unit (CFU). In one embodiment, the composition is applied at from about 0.5kg to about 5kg of fermentation solids per hectare.

In some aspects, the plant disease is caused by a fungus. In other aspects, the plant disease is mildew or rust. In one embodiment, the mildew is powdery mildew or downy mildew. In another embodiment, the rust is selected from wheat leaf rust, barley leaf rust, rye leaf rust, brown leaf rust, crown rust and stem rust.

In some embodiments, the fungus is selected from the group consisting of Alternaria alternata (Alternaria alternata), Alternaria solani (Alternaria solani), Botrytis cinerea (Botrytis cinerea), Colletotrichum (Colletotrichum lagenarium), Fusarium culmorum (Fusarium culmorum), septoria nodorum (phaosporania nodorum), mycosphaerella graminicola (zymospora tritici), Phytophthora crypthecellum (Phytophthora crypthecogenum), Phytophthora infestans (Phytophthora infestans), Pythium ultimum (Pythium), pyricularia oryzae (Magnaporthe oryzae), rhizoctonia solani (solanacearum), triticum aestivum var nigricans (triticale var. niger), trichomonas oryzae (trichomonas oryzae), triticum purpurea (wheat trichomonas sp., and triticum purpurea (wheat).

The invention also relates to the use of the disclosed compositions and formulations for controlling phytopathogenic organisms in useful plants. In certain aspects, the plant pathogenic organism is selected from the group consisting of alternaria alternata, alternaria solani, botrytis cinerea, colletotrichum, fusarium culmorum, septoria nodorum, mycosphaerella graminicola, phytophthora crypthecogenum, phytophthora infestans, pythium ultimum, pyricularia oryzae, rhizoctonia solani, ustilago avenae variety, puccinia verruculosa and puccinia tritici. In other aspects, the plant pathogenic organism is selected from the group consisting of Xanthomonas campestris (Xanthomonas campestris), Pseudomonas syringae (Pseudomonas syringae), and Erwinia carotovora (Erwinia carotovora).

In other aspects, useful plants are selected from the group consisting of apple, banana, citrus, kiwi, melon, peach, pear, pineapple, pome fruit, pomegranate, cabbage, cauliflower, cucumber, cucurbit, tomato, potato, wheat, rice, and soybean.

Drawings

FIG. 1 depicts the relative fusaricidin levels in liquid formulations of Bacteroides strain NRNR B-50972 Broth Concentrate (BC) without any stabilizer or in admixture with urea, guanidine hydrochloride, triethanolamine or ethyl glycerate (glycoethyl oxylate) before and after storage for 2 weeks at 54 ℃.

FIG. 2 depicts the relative fusaricidin levels in liquid formulations of Bacteroides strain NRRL B-50972BC without any stabilizer or in admixture with urea, guanidine hydrochloride, triethanolamine or ethyl glycerinooxalate before and after storage for 2, 4 and 8 weeks at 40 ℃.

FIG. 3 depicts the relative fusaricidin levels in liquid formulations of Bacteroides strain NRRL B-50972BC without any stabilizer or in admixture with urea, betaine hydrochloride, or dipotassium hydrogen phosphate before and after storage for 2 weeks at 54 ℃. Error bars represent 95% confidence intervals for the values shown.

FIG. 4 depicts the relative fusaricidin levels in liquid formulations of Bacteroides strain NRRL B-50972BC without any stabilizer or in admixture with urea, betaine hydrochloride, or dipotassium hydrogen phosphate before and after 8 weeks of storage at 40 ℃. Error bars represent 95% confidence intervals for the values shown.

FIG. 5 depicts the relative fusaricidin levels in liquid formulations of Paenibacillus sp strain NRRL B-50972BC diluted 50: 50 with water or propylene glycol before and after storage for 2 weeks at 54 ℃.

FIG. 6 depicts the relative fusaricidin levels in liquid formulations of Paenibacillus sp strain NRRL B-50972BC diluted 50: 50 with water or propylene glycol before and after storage for 2, 4 or 8 weeks at 40 ℃.

FIG. 7 depicts photographs of samples of Paenibacillus strain NRRL B-50972BC ("BC-urea") without urea and of a Paenibacillus strain NRRL B-50972BC ("BC + urea") with urea after storage for 2 weeks at 54 ℃.

FIG. 8 depicts the relative fusaricidin levels in liquid formulations of Bacteroides strain NRRL B-50972BC without any stabilizer or mixed with urea before and after storage at 23 ℃ for 2, 4, 8, 26, 52 and 104 weeks.

Figure 9 depicts the relative fusaricidin levels in liquid formulations of paenibacillus species strain NRRL B-50972BC without any stabilizer or mixed with urea and paenibacillus species strain NRRL B-67304 BC and paenibacillus species strain NRRL B-67306 BC each mixed with urea before and after storage at 40 ℃ for 4 and 8 weeks.

Figure 10 depicts the relative fusaricidin levels in liquid formulations of paenibacillus species strain NRRL B-50972BC without any stabilizer or mixed with urea and paenibacillus species strain NRRL B-67304 BC and paenibacillus species strain NRRL B-67306 BC each mixed with urea before and after 2, 4, 8, 26 and 52 weeks of storage at 23 ℃.

FIG. 11 depicts the relative fusaricidin levels in liquid formulations of Paenibacillus sp strains NRRL B-67304, NRRL B-67306, and NRRL B-67615 during storage at 40 ℃ for 8 weeks. The liquid formulations were urea-free whole broth ("WB-urea") and urea-containing broth concentrate ("BC + urea").

FIG. 12 depicts the relative fusaricidin levels in liquid formulations of Paenibacillus sp strains NRRL B-67304, NRRL B-67306, and NRRL B-67615 during storage at 4 ℃ or 23 ℃ for 52 weeks. The liquid formulations were urea-free whole broth ("WB-urea") and urea-containing broth concentrate ("BC + urea").

Detailed Description

It has been found that certain stabilizers maintain the antifungal activity of paenibacillus species strains such that they can be stored and transported at elevated temperatures and/or over extended periods of time. The Paenibacillus strain comprises Paenibacillus strain NRRL B-50972, Paenibacillus strain NRRL B-67129, Paenibacillus strain NRRL B-67304, Paenibacillus strain NRRL B-67306, Paenibacillus strain NRRL B-67615 and antifungal mutants derived from the same.

Paenibacillus species strain NRRL B-50972 and Paenibacillus species strain NRRL B-67129 have previously been identified as producers of a unique group of fusaricidin and fusaricidin-like compounds with broad spectrum antifungal activity (WO 2016/154297). As described in U.S. patent application No. 62/671,067, Paenibacillus sp.NRRL B-50972 is related to Paenibacillus sp.NRRL B-67304 and Paenibacillus sp.NRRL B-67306. Fusarium-like compounds include Paeniserine and Paeniprolixin described in WO 2016/154297. Additional fusaricidin-like compounds include the fusaricidin class compounds described in WO 2016/020371 (e.g., compounds 1A, 1B, 2A, 2B, 4A, 4B, 5A and 5B).

As used herein, the verb "to comprise" and its conjugations are used in this specification and claims in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that one and only one of the elements is present. Thus, the indefinite article "a" or "an" generally means "at least one".

In certain embodiments, the present invention relates to a stable fungicidal composition comprising: a) fusaricidin and/or fusaricidin-like compounds; and b) a stabilizer selected from the group consisting of amines, quaternary ammonium compounds, phosphates or citrates, short chain polyols having 2 to 10 carbon atoms, urea and combinations thereof.

In other embodiments, the present invention relates to a stable fungicidal composition comprising: a) fusaricidin and/or fusaricidin-like compounds; and b) a stabilizer selected from the group consisting of amines, quaternary ammonium compounds, phosphates or citrates, short-chain polyols having 2 to 10 carbon atoms, urea, alkanolamines, amides, and combinations thereof.

In some embodiments, the present invention relates to a stable fungicidal composition comprising: a) fusaricidin and/or fusaricidin-like compounds; and b) stabilizers selected from the group consisting of carbamide (CO (NH)₂)₂) Guanidine hydrochloride, triethanolamine, betaine hydrochloride, potassium phosphate (dibasic), potassium citrate (tribasic), and combinations thereof.

In certain embodiments, the stable composition comprises a cell-free preparation of a fermentation broth of a paenibacillus species strain. In one aspect, the cell-free preparation comprises a fusaricidin and/or a fusaricidin-like compound.

It should be understood that all numerical ranges recited herein are intended to include all sub-ranges subsumed therein. For example, a range of 1 to 10 is intended to include all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, i.e., having a minimum value equal to or greater than 1 and a maximum value of equal to or less than 10.

The term "alkyl" as used herein, unless otherwise defined, refers to a straight, branched, or cyclic saturated group derived from an alkane by removal of a hydrogen atom. Representative straight chain alkyl groups include-methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and n-heptyl. Representative branched alkyl groups include-isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, -neopentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1-dimethylpropyl, and 1, 2-dimethylpropyl. Representative cyclic alkyl groups include cyclohexyl, cyclopentyl, and cyclopropyl.

In one embodiment, the stabilizer is "urea". As used herein, the term "urea" refers to a compound having a functional group consisting of a carbonyl group attached to two amine residues, wherein each amine residue is independently a primary, secondary, or tertiary amine. The simplest urea is carbamide (CO (NH)₂)₂) Wherein both amine residues are primary amines.

In certain aspects, the urea is a substituted urea of formula (I)

Wherein R is₁，R₂And R₃Independently is-H, -OH, C₁-C₆Alkyl or-R₅OH；R₄is-R₅OH; and R₅Is C₁-C₆An alkyl group.

Illustrative classes of substituted ureas are hydroxymethyl urea, hydroxyethyl urea, hydroxypropyl urea; bis (hydroxymethyl) urea; bis (hydroxyethyl) urea; bis (hydroxypropyl) urea; n, N' -dimethylol urea; n, N' -dihydroxyethyl urea; n, N' -dihydroxypropyl urea; n, N' -trishydroxyethyl urea; tetrakis (hydroxymethyl) urea; tetra (hydroxyethyl) urea; tetra (hydroxypropyl) urea; N-methyl-N' -hydroxyethyl urea; N-ethyl-N' -hydroxyethyl urea; N-hydroxypropyl-N '-hydroxyethyl urea and N, N' -dimethyl-N-hydroxyethyl urea. In the case where the term hydroxypropyl appears, the meaning is common to 3-hydroxy-n-propyl, 2-hydroxy-n-propyl, 3-hydroxy-isopropyl or 2-hydroxy-isopropyl groups.

In some embodiments, the stabilizer is carbamide (CO (NH)₂)₂) Or a substituted urea. In other embodiments, the stabilizer is carbamide (CO (NH)₂)₂)。

In certain aspects, the stabilizer in the disclosed fungicidal compositions or formulations comprises urea and a phosphate or citrate salt. In one aspect, the phosphate or citrate is selected from the group consisting of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen citrate, dipotassium hydrogen citrate, and combinations thereof.

As used herein, the term "combined concentration" refers to the sum of the concentrations of the individual components mentioned in a composition or formulation. In some embodiments, urea and phosphate or citrate are present in the fungicidal composition or formulation at a combined concentration of from about 2% to about 3%. In other embodiments, urea and phosphate or citrate are present in the fungicidal composition or formulation at a combined concentration of from about 0.1% to about 3%, from about 0.5% to about 3%, from about 1% to about 3%, or from about 1.5% to about 3%. In yet other embodiments, urea and phosphate or citrate are present in the fungicidal composition or formulation at a combined concentration of from about 0.1% to about 2%, from about 0.25% to about 2%, from about 0.5% to about 2%, or from about 1% to about 2%. Non-limiting examples of such mixtures of urea and phosphate or citrate are given in table 1.

Certain advantages arise from the combination of urea and phosphate or citrate in the composition or formulation. Multiple stabilizers increase the stability of fusaricidin and fusaricidin-like compounds, and in the event that one stabilizer fails under adverse conditions, another stabilizer may remain active and exert a protective effect. In addition, the salts of the polybasic acids (i.e., phosphate and citrate) provide a pH buffering effect to the composition or formulation, thereby further stabilizing the system.

In some aspects, the stabilizer is an alkanolamine. As used herein, an "alkanolamine" is a compound that contains both hydroxyl (-OH) and amino (i.e., primary, secondary, or tertiary amino) functional groups on the alkane backbone.

In one aspect, the alkanolamine is an aminoalcohol. In another aspect, the aminoalcohol is ethanolamine, diethanolamine, triethanolamine, isopropanolamine, Diisopropanolamine (DIPA), Triisopropanolamine (TIPA), methanolamine, aminomethylpropanol, heptaminol, dimethylethanolamine, or N-methylethanolamine. In yet another aspect, the aminoalcohol is ethanolamine, diethanolamine, triethanolamine, isopropanolamine, Diisopropanolamine (DIPA), Triisopropanolamine (TIPA), or methanolamine. In some embodiments, the aminoalcohol is ethanolamine, diethanolamine, or triethanolamine. In certain embodiments, the aminoalcohol is triethanolamine.

In yet other embodiments, the stabilizer is an amide. In some embodiments, the amide is an organic amide, sulfonamide, or phosphoramide.

In one aspect, the organic amide is a carboxamide. In some embodiments, the carboxamide is acetamide or benzamide.

In some embodiments, the disclosed fungicidal compositions or formulations further comprise a protease inhibitor. In certain aspects, the protease inhibitor is an aspartic protease inhibitor, a cysteine protease inhibitor, a metalloprotease inhibitor, a serine protease inhibitor, a threonine protease inhibitor, or a trypsin inhibitor. In one aspect, the trypsin inhibitor is a Kunitz soybean trypsin inhibitor, also known as a Kunitz STI protease inhibitor. In other aspects, the protease inhibitor is a suicide inhibitor, a transition state inhibitor, a protein protease inhibitor, or a chelator. In one aspect, the protein protease inhibitor is a serine protease inhibitor (serpin).

Unless otherwise specifically stated, the microorganisms and specific strains described herein are isolated from nature and grown under artificial conditions, such as shake flask culture or by expanding the manufacturing process (e.g., in a bioreactor), for example to maximize production of bioactive metabolites. Growth under such conditions results in "acclimatization" of the strain. Typically, such "domesticated" strains are distinguished from their naturally occurring counterparts in that the "domesticated" strains are cultured as homogeneous populations (homogenetic outputations) that are not subjected to the selection pressure existing in the natural environment, but are subjected to artificial selection pressure.

The microorganism or culture of the invention or isolate thereof may be described as being in "isolated" or "biologically pure" form. These terms are intended to mean that the microorganism has been separated from the environment or one or more components, cells, or other constituents, which may be associated with the microorganism if in nature or under other conditions. The term "isolated" or "biologically pure" should not be used to indicate the degree to which a microorganism is purified. However, in one embodiment, the isolate or culture of the microorganism contains predominantly a microorganism of the invention.

Culturing paenibacillus species strain NRRL B-50972 produced a spontaneous variant strain with stable colony morphology, referred to herein as paenibacillus species strain NRRL B-67129. Paenibacillus sp strain NRRL B-67129 is closely related to Paenibacillus sp strain NRRL B-50972, except that Paenibacillus sp strain NRRL B-67129 expresses Spo0A with a single amino acid substitution compared to Spo0A expressed by Paenibacillus sp strain NRRL B-50972.

In one embodiment, mutant strains of Paenibacillus sp strain NRRL B-50972, Paenibacillus sp strain NRRL B-67129, Paenibacillus sp strain NRRL B-67304, Paenibacillus sp strain NRRL B-67306 or Paenibacillus sp strain NRRLB-67615 are provided. The term "mutant" refers to a genetic variant derived from a paenibacillus species strain. In one embodiment, the mutant has one or more or all of the identified (functional) characteristics of a paenibacillus species strain. In particular cases, the mutant or its fermentation product controls (as an identified functional characteristic) fungi and/or bacteria at least as well as the paenibacillus species parent strain. Such mutants may be genetic variants having a genomic sequence with greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% sequence identity to a parent strain of the paenibacillus species. The mutants can be obtained by: treating cells of a paenibacillus species strain with chemicals or radiation, or by selecting spontaneous mutants (e.g., phage-resistant or antibiotic-resistant mutants) from a population of cells of a paenibacillus species strain, by genomic shuffling (as described below), or by other means well known to those skilled in the art.

Genomic rearrangement between paenibacillus species strains can be promoted by using a method called protoplast fusion. The method begins with the formation of protoplasts from vegetative bacterial cells. Peptidoglycan cell walls are typically removed using lysozyme and osmotic stabilizers, which results in the formation of protoplasts. This process was accompanied by appearance of spherical cells as seen under light microscopy. The addition of PEG and polyethylene glycol then induces fusion between protoplasts, bringing genetic components of two or more cells into contact, thereby facilitating recombination and genomic rearrangement. The fused cells were then redistributed and recovered on solid growth media. During recovery, protoplasts reconstitute the peptidoglycan cell wall and convert back to bacterial shape. See Schaeffer, et al, (1976) PNAS USA, vol.73, 6: 2151-2155).

Paenibacillus sp strain NRRL B-50972, Paenibacillus sp strain NRRL B-67129, Paenibacillus sp strain NRRL B-67304, Paenibacillus sp strain NRRL B-67306, Paenibacillus sp strain NRRL B-67615 and mutants thereof have activity against a variety of plant pathogens. In one aspect, the strain has antifungal, anti-oomycete and/or antibacterial activity against fungi such as cucumber anthracnose, cucumber powdery mildew, wheat leaf rust, barley powdery mildew, Alternaria (Alternaria) and Botrytis (Botrytis); oomycetes such as tomato late blight, cucumber downy mildew and canola downy mildew; bacteria such as Pseudomonas (Pseudomonas), Xanthomonas (Xanthomonas) and Erwinia (Erwinia).

The invention also includes a method of controlling plant disease by applying an aqueous formulation or fungicidal composition comprising a fusaricidin-producing bacillus-like strain and a stabilizer selected from the group consisting of urea, guanidine hydrochloride, triethanolamine, betaine hydrochloride, potassium phosphate (dibasic), potassium citrate (tribasic), and combinations thereof to a plant or plant part (e.g., leaf, stem, flower, fruit, root, or seed) or by applying to a locus where the plant or plant part is growing (e.g., soil).

In the method according to the invention, the compositions and formulations can be applied to any plant or any part of any plant growing in any type of medium used for growing plants (such as soil, vermiculite, shredded paper board and water) or to aerial growing plants or plant parts (such as orchid or platycerium). The composition or formulation may be applied, for example, by spraying (nebulizing), atomizing (atomizing), evaporating (vaporizing), scattering (scattering), dusting (dusting), watering (watering), spraying (watering), sprinkling (sprinkling), pouring (sprinkling) or fumigating (fumigating). As already indicated above, application can be carried out at any desired location where the plants of interest are located, such as agricultural environments, horticulture, forests, plantations, orchards, nurseries, organically grown crops, turf and urban environments.

The compositions of the present invention may be obtained by culturing a fusaricidin-producing strain of a paenibacillus species according to methods well known in the art, including by using culture media and other methods described in the examples below. Conventional large-scale microbial culture methods include submerged fermentation, solid state fermentation, or liquid surface culture. Towards the end of fermentation, the cells begin to shift from the growth phase to the sporulation phase as nutrients are consumed, so the final fermentation products are mainly spores, metabolites and residual fermentation medium. Sporulation is part of the natural life cycle of paenibacillus and is usually initiated by the response of the cell to nutrient limitation. The fermentations were set to obtain high levels of colony forming units and to promote sporulation. The bacterial cells, spores and metabolites in the fermentation-produced medium can be used directly or concentrated by conventional industrial methods (e.g., centrifugation, tangential flow filtration, depth filtration and evaporation).

The compositions of the present invention comprise a fermentation product. In some embodiments, the concentrated fermentation broth is washed (e.g., by a diafiltration process) to remove residual fermentation broth and metabolites. As used herein, the term "broth concentrate" refers to a whole broth (fermentation broth) that has been concentrated by conventional industrial processes, as described above, but remains in liquid form. As used herein, the term "fermentation solid" refers to solid material that remains after the fermentation broth is dried. As used herein, the term "fermentation product" refers to a whole broth, a broth concentrate, and/or a fermentation solid. The compositions of the present invention comprise a fermentation product.

Cell-free preparations of fermentation broths of the strains of the invention can be obtained by any method known in the art, such as extraction, centrifugation and/or filtration of the fermentation broths. One skilled in the art will appreciate that, depending on the technique used to remove the cells (e.g., the speed of centrifugation), so-called cell-free preparations may not be cell-free, but rather largely cell-free or substantially cell-free. The resulting cell-free preparation can be formulated with components that facilitate its application to plants or plant growth media. The above-described method of concentrating the fermentation broth and the drying technique are also applicable to cell-free preparations.

In one embodiment, the fermentation product comprises at least about 1X 10⁴Colony Forming Units (CFU) microorganisms (e.g., Paenibacillus sp. strain NRRL B-50972 or a fungicidally mutated strain thereof) per mL of fermentation broth. In another embodiment, the fermentation product comprises at least about 1X 10⁵Colony Forming Units (CFU) microorganisms (e.g., Paenibacillus sp. strain NRRL B-50972 strain or a fungicidally mutated strain thereof) per mL of fermentation broth. In another embodiment, the fermentation product comprises at least about 1X 10⁶CFU microorganism (e.g., Paenibacillus sp. strain NRRL B-50972 or a fungicidal mutant strain thereof)/mL fermentation broth. In yet another embodiment, the fermentation product comprises at least about 1X 10⁷CFU microorganism (e.g., Paenibacillus sp. strain NRRL B-50972 or a fungicidal mutant strain thereof)/mL fermentation broth.In another embodiment, the fermentation product comprises at least about 1X 10⁸CFU microorganism (e.g., Paenibacillus sp. strain NRRL B-50972 or a fungicidal mutant strain thereof)/mL fermentation broth. In another embodiment, the fermentation product comprises at least about 1X 10⁹CFU/mL fermentation broth of a microorganism (e.g., Paenibacillus sp. strain NRRL B-50972 or a fungicidal mutant strain thereof). In another embodiment, the fermentation product comprises at least about 1X 10¹⁰CFU microorganism (e.g., Paenibacillus sp. strain NRRL B-50972 or a fungicidal mutant strain thereof)/mL fermentation broth. In another embodiment, the fermentation product comprises at least about 1X 10¹¹CFU microorganism (e.g., Paenibacillus sp. strain NRRL B-50972 or a fungicidal mutant strain thereof)/mL fermentation broth.

The compositions of the invention can be used as such or, depending on their specific physical and/or chemical properties, in the form of their formulations or the use forms prepared therefrom, for example aerosols, capsule suspensions, cold atomized concentrates, hot atomized concentrates, capsule granules, microgranules (fine granules), flowable concentrates for seed treatment, ready-to-use solutions, powders, emulsified concentrates, oil-in-water emulsions, water-in-oil emulsions, macrogranules, microgranules, oil-dispersed powders, oil-miscible flowable concentrates, oil-miscible liquids, gases (under pressure), gas-generating products, foaming agents, pastes, pesticide-coated seeds, suspension concentrates, oil dispersions, suspoemulsion concentrates, soluble concentrates, suspensions, wettable powders, soluble powders, powders and granules (dus and granules), water-soluble and water-dispersible granules or tablets, Water-soluble and water-dispersible powders (powder) for seed treatment, natural and synthetic substances impregnated with active ingredient, and microcapsules in polymeric substances and coating materials for seeds, and ULV cold-and hot-fogging formulations.

In some embodiments, the compositions of the present invention are liquid formulations. Non-limiting examples of liquid formulations include suspension concentrates and oil dispersions.

The compositions of the present invention may comprise inert agents added to compositions comprising cells, cell-free agents or metabolites to improve efficacy, stability and availability and/or facilitate processing, packaging and end-use applications. Such inert agents and ingredients may include carriers, stabilizers, nutrients or physical property modifiers, which may be added alone or in combination. In some embodiments, the carrier may include liquid materials, such as water, oils, and other organic or inorganic solvents, as well as solid materials, such as minerals, polymers, or polymer composites obtained by biological methods or chemical synthesis. In some embodiments, the carrier is a binder or adhesive (adhesive) that promotes adhesion of the composition to a plant part, such as a seed or root. See, for example, Taylor, A.G. et al, "Concepts and Technologies of Selected Seed Treatments", Annu.Rev.Phytopathol.28: 321-339(1990). Stabilizers may include anti-caking agents, antioxidants, drying agents, protective agents or preservatives. Nutrients may include sources of carbon, nitrogen and phosphorus such as sugars, polysaccharides, oils, proteins, amino acids, fatty acids and phosphates. The physical property modifier may include a filler, wetting agent, thickener, pH modifier, rheology modifier, dispersion, adjuvant, surfactant, antifreeze, or colorant. In some embodiments, the composition comprising cells produced by fermentation, cell-free preparations, or metabolites may be used directly with or without water as a diluent, without any other preparation. In some embodiments, the inert formulation is added after concentrating the fermentation broth and during and/or after drying.

All plants and plant parts can be treated according to the invention. In the context of the present invention, plants are understood to mean all plants and plant populations, such as desired and undesired wild plants or crop plants (including naturally occurring crop plants). Crop plants may be plants which are obtained by conventional breeding and optimization methods or by biotechnological and recombinant methods, or by combinations of these methods, including transgenic plants and including plant varieties which are or are not protected by plant breeders' rights. Plant parts are to be understood as meaning all parts and organs of plants above and below the ground, such as seedlings (shoots), leaves, flowers and roots, examples which may be mentioned being leaves, needles, stalks, stems, flowers, fruit bodies, fruits and seeds, and also roots, tubers and rhizomes. Plant parts also include crop material and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, grafts (slips) and seeds.

As already mentioned above, all plants and parts thereof can be treated according to the invention. In a preferred embodiment, plant species and plant varieties and parts thereof, which are grown wild or obtained by traditional biological breeding methods, such as crossing or protoplast fusion, are treated. In a further preferred embodiment, transgenic plants and plant varieties (genetically modified organisms) obtained by recombinant methods, if appropriate in combination with conventional methods, and parts thereof are treated. The terms "part", "part of a plant" and "plant part" have been explained above. It is particularly preferred according to the invention to treat plants of the plant variety which are in each case commercially available or in use. Plant variety is understood to mean plants which have been cultivated by traditional breeding, by mutagenesis or by recombinant DNA techniques and which have new traits. Plant species may be expressed in the form of varieties, subspecies (race), biotypes and genotypes.

The treatment of plants and plant parts with the compositions according to the invention can be carried out directly or by working the environment, habitat or storage space with conventional treatment methods, for example by dipping (watering), spraying, atomizing (atomizing), misting (vaporizing), atomizing, fogging (scattering), scattering (frothing), painting (painting on), scattering (spraying), injecting (injecting), drenching (drenching), drip irrigation (rickle irrigation), and also in the case of propagation material, in particular in the case of seeds, by dry seed treatment methods, wet seed treatment methods, slurry treatment methods, by forming a shell, by coating with one or more coatings, etc. Furthermore, it is also possible to apply the active substance by the ultra-low volume method, or to inject the active substance preparation or the active substance itself into the soil.

A preferred direct treatment of plants is the application treatment to the leaves, i.e.the application of the composition according to the invention to the leaves, with a treatment frequency and application rate which can be matched to the infection pressure of the pathogen in question.

In the case of systemically active compounds, the compositions according to the invention reach the plants via the root system. In this case, the plant treatment is carried out by allowing the compounds according to the invention to act on the plant environment. This can be done, for example, by: drenched, mixed in soil or in a nutrient solution, i.e. poured in liquid form on the site where the plants are located (e.g. soil or hydroponic culture systems), or applied through soil, i.e. the composition according to the invention is incorporated in solid form (e.g. in the form of granules) on the site where the plants are located. In the case of rice cultures, this can also be achieved by adding the compositions according to the invention in solid use form (for example in the form of granules) to (meter) flooded paddy fields.

Preferred plants are those useful plants, ornamentals, lawns, trees commonly used as ornamentals in public and homes, and forestry trees. Forestry trees include trees used to produce wood, cellulose, paper, and products produced from tree parts.

The term "useful plants" as used in the present context refers to crop plants which are used as plants for obtaining food, feed, fuel or for industrial purposes.

Useful plants that may be treated and/or improved with the compositions and methods of the present invention include, for example, the following types of plants: turf, vines, cereals, such as wheat, barley, rye, oats, rice, maize and millet/sorghum; beets, such as sugar beets and fodder beets; fruits such as pomes, stone fruits and berries, e.g. apples, pears, plums, peaches, almonds, cherries and berries, e.g. strawberries, raspberries, blackberries; leguminous plants, such as beans, lentils, peas and soybeans; oil crops, such as rape, mustard, poppy, olive, sunflower, coconut, castor oil plants, cocoa and peanut; cucurbits, such as squash (pumpkin/squash), cucumber, and melon; fiber plants, such as cotton, flax, hemp and jute; citrus fruits such as oranges, lemons, grapefruits and mandarins; vegetables, such as spinach, lettuce, asparagus, cabbage species, carrots, onions, tomatoes, potatoes and sweet peppers; lauraceae (Lauraceae), such as avocado, Cinnamomum (Cinnamomum), camphor or other plants, such as tobacco, nuts, coffee, eggplant, sugarcane, tea, capsicum, grapevine, hops (hop), bananas, latex plants, and ornamental plants, such as flowers, shrubs, deciduous trees, and coniferous trees. This list is non-limiting.

The following plants are considered as target crops for which the compositions and methods of the present invention are particularly useful: cotton, eggplant, lawn, pome fruit, stone fruit, berry, corn, wheat, barley, cucumber, tobacco, vine, rice, cereal, pear, bean, soybean, rape, tomato, sweet pepper, melon, cabbage, potato, and apple.

The present invention is also applicable to any turf grass, including cool-season turf grass and warm-season turf grass. Examples of cool season turfgrass are: poa annua (Poa spp.) such as Poa kentuckahi (Poa pratensis L.), Poa canescens (Poa trivialis L.), Poa canadensis (Poa compansa L.), Poa annua (Poa annua L.), Poa alpina (Poa glauca Gaudin)), Poa linnei (Poa nemoralis L.) and Poa aleuca (Poa bulbosa L.); bentgrass (Agrostis spp.) such as creeping bentgrass (creeping bentgrass), thin bentgrass (weak bentgrass Sibth.), villous bentgrass (Agrostis marina L.), mixed bentgrass in south germany (bentgrass species including thin bentgrass, villous bentgrass and creeping bentgrass) and small bran grass (small bran grass L.);

fescue (Festuca spp.), such as fescue (Festuca rubra L. spp. rubra), fescue (stoca (Festuca rubra L.),), fescue (Festuca stoca (Festuca rubra L.), fescue (Festuca rubra Gaud.),), fescue (Festuca ovina L.), Festuca arundinacea (Festuca longifolia), fescue (Festuca capilata Lam.), Festuca arundinacea (fescuju capilata Lam.), Festuca arundinacea (fescue aru arundinacea Schreb.), and oxtail (Festuca elanogen L.);

ryegrass (Lolium spp.) such as annual ryegrass (Lolium multiflorum Lam.), perennial ryegrass (Lolium perenne L.) and italian ryegrass (Lolium multiflorum Lam.);

and wheatgrass (Agropyron spp.), such as, for example, edgeworthia (Agropyron cristatum (L.) gartn.), Agropyron arenaria (Agropyron desusertum (Fisch.) Schult.), and echeveria glauca (Agropyron smithii Rydb.).

Examples of other cold season turfgrass are jungle grass (Ammophila breviligilata Fern.), awnless brome (Bromus inermis Leys.), cattail (catail), such as timothy grass (Phleum pratensis L.), and cattail grass (Phleum sublibrium L.), Dactylis glomerata (Dactylis glomerata L.), Dactylis glomerata (Puccinellia distans (L.) Parl.), and setaria viridis (Cyrtotrachelus crispus L.).

Examples of warm-season turfgrass are: bermuda grass (Cynodon spp. L.C.Rich), Japanese lawngrass (Zoysia spp. Willd.), blumea lanceolaris (Stenothera segetum (Stenophora segetum Walt Kuntze)), eremophila pseudomollis (Eremochloa ophioides Munro Hack.), carpet grass (Axonopus Affinisis Chase), and Pasteur camptotrichum (Pasteum nodatum F.), pennisetum alopecuroides (Pennisetum clindestinum hochst. ex chiav.), Pennisetum alopecuroides (Buchloe dactyloids (Nutt.) Engelm.), glan mae (bruteloua gracilis (h.b.k.)), seashore Paspalum (Paspalum vaginatum Swartz)) and stringy grasses (peuteloua curtipentula (michx.tor.)) are generally preferred for use according to the invention.

The compositions according to the invention have effective microbicidal activity and can be used for controlling undesirable microorganisms, such as fungi and bacteria, in crop protection and in the protection of materials.

The invention also relates to a method for controlling unwanted microorganisms, characterized in that the composition according to the invention is applied to phytopathogenic fungi, phytopathogenic bacteria and/or their habitat.

Fungicides can be used for controlling phytopathogenic fungi in crop protection. Fungicides are characterized by a pronounced efficacy against a broad spectrum of phytopathogenic Fungi, including soil-borne pathogens, in particular members of the classes Plasmodiophoromycetes, Oomycetes, Chytridiomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes.

The fungicides can be used in crop protection for controlling Pseudomonadaceae (pseudomonas adaceae), Rhizobiaceae (Rhizobiaceae), Enterobacteriaceae (Enterobacteriaceae), Corynebacteriaceae (Corynebacteriaceae) and Streptomycetaceae (Streptomycetaceae).

Non-limiting examples of pathogens of fungal diseases that can be treated according to the present invention include:

diseases caused by powdery mildew pathogens, e.g., powdery mildew species (Blumeia) such as Blumeia graminis (Blumeia graminis); sphaerotheca species (Podosphaera), such as, for example, apple powdery mildew (Podosphaera leucotricha); sphaerotheca species, such as, for example, cucumber powdery mildew (Sphaerotheca fuliginea); devil's claw (Uncinula) species, such as, for example, grape powdery mildew (Uncinula necator);

diseases caused by rust pathogens, such as, for example, the genus Gymnosporangium (Gymnosporangium) species, such as, for example, Puccinia pyrifolia (Gymnosporangium sabinae); camelina rust (Hemileia) species, such as coffee rust (Hemileia vastatrix); phakopsora species (Phakopsora), such as Phakopsora pachyrhizi (Phakopsora pachyrhizi) and Phakopsora meibomiae (Phakopsora meibomiae); puccinia species, such as Puccinia recondite (Puccinia recondite), Puccinia tritici (p.triticina), Puccinia graminis (p.graminis), or Puccinia striiformis (p.striiformis); species of the genus, Puccinia (Uromyces), such as, for example, Puccinia verrucosa (Uromyces apendiculus);

diseases caused by oomycete pathogens, such as, for example, white rust (Albugo) species, such as white rust (Albugo Candida); species of the genus Bremia (Bremia), such as Bremia lactucae (Bremia lactucae); conidiophora clinopora species, such as Peronospora pisi (Peronospora pisi) or Peronospora brassicae (p.brassicae); phytophthora species (Phytophthora), such as late blight of potato (Phytophthora infestans); plasmopara species, such as Plasmopara viticola (Plasmopara viticola); pseudoperonospora species (Pseudoperonospora), such as Pseudoperonospora humuli (Pseudoperonospora humuli) or Pseudoperonospora cubensis; pythium species, such as Pythium ultimum;

leaf spot and leaf wilting caused by pathogens such as: alternaria species (Alternaria), such as Alternaria solani (Alternaria solani); cercospora species (Cercospora), such as Cercospora saccharina (Cercospora betacola); cladosporium species, such as, for example, Cladosporium cucumerinum; species of the genus Sporotrichum (Cochliobolus), such as, for example, Cochliobolus graminis (Cochliobolus sativus) (conidia form: Deuteromyces (Drechslera), synonym: Helminthosporium (Helminthosporium)), Cochliobolus oryzae (Cochliobolus miyabenus); colletotrichum species, such as Colletotrichum leguminosum (Colletotrichum lindelminthium); members of the genus cyclonigrospora (Cycloconium), such as the species alternaria oleae (Cycloconium oleginum); diaporthe species, such as citrus brown rot (Diaporthe citri); elsinoe species, such as Elsinoe fawcettii; species of the species Pediobolus (Gloeosporium), such as the peach tree rot pathogen (Gloeosporium laetiicolor); pleurotus species (Glomeella), such as anthrax (Glomeella cingulate); species of the species globisporus (Guignardia), such as globisporus viticola (Guignardia bidwelli); leptosphaeria species (Leptosphaeria), such as Leptosphaeria maculans (Leptosphaeria maculans), Leptosphaeria nodorum (Leptosphaeria nodorum); species of the genus Sphaerotheca (Magnaporthe), such as Magnaporthe grisea; species of the genus discorea (Marssonia), such as apple discoidea (Marssonia coronaria); microdochium species, such as, for example, Rhizoctonia cerealis (Microdochium nivale); mycosphaerella species (Mycosphaerella), such as Mycosphaerella graminicola (Mycosphaerella graminicola), Mycosphaerella arachidicola (m.arachidiocola), and Mycosphaerella fijiensis (m.fijiensis); mycosphaerella species (phaespharia), such as fusarium graminearum (phaespharia nodorum); pyrenophora species, such as Pyrenophora teres (Pyrenophora teres), Pyrenophora tritici-repentis (Pyrenophora tritici repentis); species of the genus Podospora (Ramularia), such as, for example, Podospora postrema (Ramularia collo-cygni), Podospora leucoderma (Ramularia areola); rhinochloropsis species (Rhynchosporium), such as barley leaf-streak bacteria (Rhynchosporium secalis); septoria species (Septoria), such as Septoria apiacea (Septoria apii), Septoria lycopersici (Septoria lycopersici); corallina species (Typhyla), such as Scleronaria carolina (Typhyla incarnata); venturia species (Venturia), such as Venturia inaequalis (Venturia inaqualis);

root and stem diseases caused by, for example, the following pathogens: species of the genus humicola (cornium), such as the species humicola (cornium graminearum); fusarium species, such as Fusarium oxysporum (Fusarium oxysporum); gaeumannomyces species, such as wheat take-all (Gaeumannomyces graminis); rhizoctonia species (Rhizoctonia), such as, for example, Rhizoctonia solani (Rhizoctonia solani); for example, a Scopulariopsis (Sarocladium) disease caused by Sarocladium oryzae (Sarocladium oryzae); sclerotium (Sclerotium) disease caused by, for example, Sclerotium oryzae (Sclerotium oryzae); tapesia species, such as Tapesia acuformis; species of the genus Rhinocerotis (Thielavirosis), such as, for example, Rhizoctonia solani (Thielavirosis basicola);

panicle and panicle diseases (including corn cobs) caused by, for example, the following pathogens: alternaria species, such as Alternaria species (Alternaria ssp.); aspergillus species (Aspergillus), such as Aspergillus flavus; cladosporium species (Cladosporium), such as Cladosporium cladosporioides (Cladosporium cladosporioides); species of the genus ergot (Claviceps), such as, for example, purple ergot (Claviceps purpurea); fusarium species, such as Fusarium culmorum (Fusarium culmorum); gibberella species, such as Gibberella zeae; small-picture line shells belong to the genus of monographeella, such as snow rot small-picture line shells (Monographella nivalis); septoria species (Septoria), such as Septoria nodorum (Septoria nodorum);

diseases caused by, for example, smut: neisseria species (Sphacelotheca), such as Sphacelotheca zeae (Sphacelotheca reiliana); tilletia species (Tilletia), such as Tilletia tritici (Tilletia caries), Tilletia controversa (T.contrivarsa); ustilago species, such as Ustilago graminis (Urocystis occulta); smut species (Ustilago), such as, for example, Ustilago nuda (Ustilago nuda), Sphaerotheca paniculata (U.nuda tritici);

fruit decay caused by pathogens such as: aspergillus species (Aspergillus), such as Aspergillus flavus; botrytis species, such as Botrytis cinerea (Botrytis cinerea); penicillium species (Penicillium species), such as Penicillium expansum (Penicillium expansum) and Penicillium purpurogenum (p.purpurogenum); sclerotinia species, such as Sclerotinia sclerotiorum (Sclerotinia sclerotiorum); verticillium species, such as Verticillium alboatrum;

seed-and soil-borne spoilage, mildew, wilting, rot and cataplexy diseases caused by, for example, the following pathogens: alternaria species (Alternaria), such as that caused by Alternaria brassicae (Alternaria brassicolo); species of the genus Aphanomyces (Aphanomyces), such as that caused by rhizopus oryzae (Aphanomyces euteiches); ascochyta (Ascochyta) species, for example caused by Ascochyta lentis (Ascochyta lentis); aspergillus species (Aspergillus species), such as those caused by Aspergillus flavus; species of the genus mycobacteria (Cladosporium), such as caused by the species Cladosporium (Cladosporium herbarum); species of the genus Sporotrichum (Cochliobolus), such as that caused by Sporotrichum gramineum (Cochliobolus sativus); (conidia form: Dermatopteria (Drechslera), Bipalea (Bipolaris), synonym: Helminthosporium (Helminthosporium)); colletotrichum species, such as caused by anthrax (Colletotrichum coccoides); fusarium (Fusarium) species, such as that caused by Fusarium culmorum; gibberella species (Gibberella), for example caused by Gibberella zeae; species of the genus ascochyta (macrophospora), such as caused by ascochyta phaseoloides (macrophospora phaseolina); the small line shells belong to the species monographeella, for example, caused by the snow rot small line shells (Monographella nivalis); penicillium (Penicillium) species, such as caused by Penicillium expansum (Penicillium expansum); phoma species (Phoma), such as caused by Phoma nigricans (Phoma linggam); species of the genus Phomopsis (Phomopsis), for example caused by Phomopsis sojae; phytophthora species (Phytophthora), such as caused by Phytophthora infestans (Phytophthora cactorum); pyrenophora species, for example caused by Pyrenophora graminea (Pyrenophora graminea); pyricularia species (Pyricularia species), such as those caused by Pyricularia oryzae (Pyricularia oryzae); pythium species, for example caused by Pythium ultimum; rhizoctonia species (Rhizoctonia), for example caused by Rhizoctonia solani (Rhizoctonia solani); rhizopus species (Rhizopus), for example, caused by Rhizopus oryzae (Rhizopus oryzae); sclerotium species (Sclerotium), such as caused by Sclerotium rolfsii (Sclerotium rolfsii); septoria species (Septoria), for example caused by Septoria nodorum (Septoria nodorum) tritici; corallina species (Typhyla), for example, those caused by Corallina carnosa (Typhyla incarnate); verticillium species, for example caused by Verticillium dahlia;

cancers (cancer), galls and broom diseases (witches' brooms) caused by, for example, the following pathogens: species of the genus Nectria (Nectria), such as, for example, Nectria carinata (Nectria galligena);

wilting diseases caused by pathogens such as: species of the genus sclerotinia (Monilinia), such as, for example, sclerotinia sclerotiorum (Monilinia laxa);

blistering or leaf curl diseases caused by, for example, the following pathogens: exobasidium (Exobasidium) species, such as the species ectobasidium destructor (Exobasidium vexans);

species of the genus exocystium (Taphrina), such as, for example, exocystis malformation (Taphrina deformans);

degenerative diseases of woody plants: asca (Esca) diseases caused by, for example, Rhizopus oryzae (Phaemoniella clavulosa), Acremonium fulvum (Phaeoacremonium aleophilum) and Porphyromonas mediterrae (Fomitosporia mediterrana); grape blight (Eutypa dyseback) caused by, for example, grapevine damping-off (Eutypa lata); diseases of the genus Ganoderma (Ganoderma) caused by, for example, Ganoderma islet boninense (Ganoderma lucidum); scleroderma (Rigidoporus) disease caused by, for example, scleroderma (Rigidoporus lignosus);

flower and seed diseases caused by pathogens such as: botrytis species, such as Botrytis cinerea (Botrytis cinerea);

diseases of plant tubers caused by, for example, the following pathogens: rhizoctonia species (Rhizoctonia), such as Rhizoctonia solani (Rhizoctonia solani); helminthosporium species, such as Helminthosporium solani (Helminthosporium solani);

clubroot diseases caused by, for example, the following pathogens: plasmodiophora species, such as Plasmodiophora brassicae;

diseases caused by bacterial pathogens such as: species of the genus Xanthomonas (Xanthomonas), such as Xanthomonas oryzae P.solani var alba (Xanthomonas campestris pv. oryzae); pseudomonas species, such as Pseudomonas syringae Cucumaria var syringae (Pseudomonas syringae pv. Lachrymans); erwinia species (Erwinia), for example, Erwinia amylovora (Erwinia amylovora).

The following diseases of soybean are preferably controlled:

fungal diseases located on leaves, stems, pods and seeds caused by pathogens such as: alternaria leaf spot (Alternaria spec. atrans tenezia), anthracnose (Colletotrichum gloeosporides dematum var. trmonocatum), brown spot (Septoria Septoria), Cercospora leaf spot and leaf blight (Cercospora kikuchi), Chondrus leaf spot (Chondrococcus inulinus) (synonym), Rhizoctonia solani (Rhizoctonia solani), Rhizoctonia solani (Dactylophora lanuginosa), downy mildew (Peronospora manshurica), Dermata (Drhsizaeus solani), Graptospira trichoderma (Pseudochinensis (Drhslera Septoria), Graptospira leaf spot (Cercospora septentrionalis) (Cercospora microphylla Septoria), and leaf blight (Pseudoseptoria Septoria), Graptospira purpurea (Cercospora Septoria), Graptospira purpurea (Pseudoseptoria Septoria), and phomophora Chaetosa (Phosphonospora Chalcospira), and leaf spot (Phosphonospora Chaetomium globerulina), and leaf spot (Pseudosepedospora Chaetosa), and leaf spot, respectively, and leaf spot of soybean, Rhizoctonia overground part, leaf blight and damping off (web blast) (Rhizoctonia solani), rust (Phakopsora pachyrhizi, Phakopsora meibomiae), scab (Sphaceloma esculenta), Stemphylium (stemphyium) leaf blight (stemphyium thyosum)), target spot (Corynespora cassiicola)).

Fungal diseases of roots and stem bases caused by pathogens such as: black root rot (cupricola cristata); charcoal rot (ascochyta phaseoloides (macrophosphoma phaseolina)); fusarium wilt or wilting, root rot, and pod and root neck rot (Fusarium oxysporum), Fusarium tricholobus (Fusarium orthoceras), Fusarium semitectum (Fusarium semitectum), and Fusarium equiseti (Fusarium equiseti)); mycobacteriosis root rot (mycobacteriosis terrestris); neocastanospora (neocomospora) (invasion of neocastanospora vasicifolia (neocomospora vasicifolia)); pod and stem blight (Diaporthe phaseolorum); stem ulcers (northern stem canker of soybean var. calulivora)); phytophthora rot (Phytophthora megasporum); brown stem rot (phaophora gregata) of soybean; pythium rot (Pythium aphanidermatum), Pythium irregulare (Pythium irregularis), Pythium debaryanum (Pythium debaryanum), Pythium aggregatum (Pythium myrinotum), Pythium ultimum (Pythium ultimum)); rhizoctonia root rot, stem rot and damping-off (Rhizoctonia solani); sclerotinia stem rot (Sclerotinia sclerotiorum); sclerotinia sclerotiorum (sclerotiotinia rolfsii) Sclerotinia sclerotiorum; root Beadina spp.root rot (Thielavirosis basicola).

The fungicidal compositions of the invention are useful for the therapeutic or protective/prophylactic control of phytopathogenic fungi. The invention therefore also relates to a therapeutic and protective method for controlling phytopathogenic fungi by using the compositions according to the invention, wherein the compositions are applied to the seeds, to the plants or to parts of plants, to the fruits or to the soil in which the plants are growing.

The fact that the composition is well tolerated by plants at the concentrations required for controlling plant diseases makes it possible to treat the aerial parts of plants, the propagating rhizomes and seeds and the soil.

All plants and plant parts can be treated according to the invention, including cultivars and plant varieties (whether protected by plant varieties or plant breeders' rights). Cultivars and plant varieties may be plants obtained by conventional propagation and breeding methods, which may be assisted or supplemented by one or more of the following biotechnological methods, for example: by using doubled haploids, protoplast fusion, random and directed mutagenesis, molecular or genetic markers, or by bioengineering and genetic engineering methods.

In certain aspects, the compositions of the invention are used at about 1X 10 per hectare⁸To about 1X 10¹⁴A Colony Forming Unit (CFU) of the fungicidal paenibacillus species strain NRRL B-50972 or a fungicidal mutant strain thereof. In other aspects, the compositions of the invention are used at a rate of about 1X 10 per hectare⁹To about 1X 10¹³A Colony Forming Unit (CFU) of the fungicidal paenibacillus species strain NRRL B-50972 or a fungicidal mutant strain thereof. In yet other aspects, the compositions of the invention are used at a rate of about 1X 10 per hectare¹⁰To about 1X 10¹²A Colony Forming Unit (CFU) of the fungicidal paenibacillus species strain NRRL B-50972 or a fungicidal mutant strain thereof.

In some embodiments, the compositions of the present invention are applied at from about 0.1kg to about 10kg of fermentation solids per hectare. In other embodiments, the compositions of the present invention are applied at from about 0.25kg to about 7.5kg of fermentation solids per hectare. In yet other embodiments, the compositions of the present invention are applied at from about 0.5kg to about 5kg of fermentation solids per hectare. The compositions of the present invention may also be applied at from about 1kg to about 2kg of fermentation solids per hectare.

The compositions of the invention have advantageous warm blooded animal toxicity when well tolerated by plants, are environmentally well tolerated, are suitable for protecting plants and plant organs, for increasing harvest yield, for improving the quality of the harvested material. They are preferably used as crop protection compositions. They are active against generally sensitive and resistant species and active against all or some developmental stages.

Plants which may be treated according to the invention include the following major crop plants: corn, soybean, alfalfa, cotton, sunflower, Brassica oilseed (Brassica) such as Brassica napus (e.g. canola (canola), rapeseed), turnip (Brassica rapa), mustard (b.juncea) (e.g. (field) mustard) and Brassica juncea (Brassica carinata), areca (arecae sp.) (e.g. oil palm, coconut), rice, wheat, sugar beet, sugarcane, oat, rye, barley, millet and sorghum, triticale, flax, nuts, grapes and vines, and various fruits and vegetables from various plant taxonomic units, for example rosaceous species (Rosaceae sp.) (e.g. pome fruits (e.g. apple and pear), and stone fruits (e.g. apricot, cherry, almond, peach and strawberry), and berry fruits (e.g. raspberry, red currant) and black currant berries), and currants (e.g. currant, currant and black currant) and black currant fruits (e.g. currant) and black currant), Tea (subfamily sp.), Juglandaceae (Juglaceae sp.), Betulaceae (Betulaceae sp.), Anacardiaceae (Anacardiaceae sp.), Fagaceae (Fagaceae sp.), Moraceae (Moraceae sp.), Oleaceae (Oleaceae sp.), such as Olive tree, Actinidiaceae (Actinidiaceae sp.), Lauraceae (Lauraceae sp.), such as avocado, cinnamon, camphor, Musaceae (Musaceae sp.), such as banana tree and plantain, Rubiaceae (Rubiaceae sp.), such as coffee, Theaceae (Theaceae sp.), Theaceae (Sterculiaceae sp.), Citrus (Sterculiaceae sp.), Citrus (Citrus sp.), Citrus aurantifolia, Citrus aurantium, Citrus aurantifolia, etc.); solanaceae (Solanaceae sp.) (e.g., tomatoes, potatoes, peppers, eggplant, tobacco), Liliaceae (Liliaceae sp.), Compositae (Compositae sp.) (e.g., lettuce, artichoke and chicory (chicory) including root chicory, chicory (endive) or common chicory), Umbelliferae (Umbeferae sp.) (e.g., carrots, parsley and celeries), Cucurbitaceae (Cucurbitaceae sp.) (e.g., cucumbers including gherkin, squash, watermelon, cucurbit and melon), Alliaceae (Alliaceae sp.) (e.g., leeks and onions), Cruciferae (Cruciferae sp.) (e.g., white cabbage, red cabbage, broccoli, kohlrabi, cabbage, blue bean, wasabi, watercress and radish (e.g., beans, legume pea, legume beans, canavalia bean and fava bean), Chenopodiaceae (Chenopodiaceae sp.) (e.g., swiss chard, fodder beet, spinach, beetroot), Linaceae (Linaceae sp.) (e.g., hemp), cannabiaceae (canabiaceae sp.) (e.g., indian hemp), Malvaceae (Malvaceae sp.) (e.g., okra, cacao), Papaveraceae (Papaveraceae) (e.g., poppy), asparagus (Asparagus) (e.g., asparagus); useful plants and ornamentals in horticulture and forests, including lawn, grassland, pasture and Stevia (Stevia rebaudiana); and in each case genetically modified versions of these plants.

In certain aspects, the fermentation product further comprises formulation ingredients. The formulation ingredients may be wetting agents, extenders, solvents, spontaneous promoters, emulsifiers, dispersants, anti-freeze agents, thickeners and/or adjuvants. In one embodiment, the formulation ingredient is a wetting agent. In other aspects, the fermentation product is a lyophilized powder or a spray-dried powder.

The compositions of the present invention may include formulation ingredients that are added to the compositions of the present invention to improve recovery, efficacy or physical properties and/or aid in processing, packaging and application. Such formulation ingredients may be added alone or in combination.

Formulation ingredients may be added to compositions comprising cells, cell-free preparations, isolated compounds and/or metabolites to improve efficacy, stability and physical properties, availability and/or facilitate processing, packaging and end-use applications. Such formulation ingredients may include agriculturally acceptable carriers, inert materials, stabilizers, preservatives, nutrients or physical property modifiers, which may be added alone or in combination. In some embodiments, the carrier may include liquid materials, such as water, oils, and other organic or inorganic solvents, as well as solid materials, such as minerals, polymers, or polymer composites obtained by biological methods or chemical synthesis. In some embodiments, the formulation ingredient is a binder, adjuvant, or adhesive that promotes adhesion of the composition to a plant part (e.g., leaf, seed, or root). See, for example, Taylor, a.g. et al, "Concepts and Technologies of Selected Seed Treatments," annu.rev.phytopathohol, 28: 321-339(1990). Stabilizers may include anti-caking agents, antioxidants, anti-settling agents, anti-foaming agents, drying agents, protective agents or preservatives. Nutrients may include sources of carbon, nitrogen and phosphorus such as sugars, polysaccharides, oils, proteins, amino acids, fatty acids and phosphates. The physical property modifier can include a filler, a wetting agent, a thickener, a pH modifier, a rheology modifier, a dispersion, an adjuvant, a surfactant, a film former, a water aidSolvents, builders, antifreeze or colorants. In some embodiments, the composition comprising cells produced by fermentation, cell-free preparations, and/or metabolites may be used directly, with or without water as a diluent, without any other preparation. In a particular embodiment, a wetting agent or dispersing agent is added to the fermentation solid, e.g. a lyophilized or spray-dried powder. Wetting agents enhance the diffusion and penetration properties of the active ingredient when applied to a surface, or dispersing agents may enhance the dispersibility and solubility of the active ingredient (once diluted). Exemplary wetting agents are known to those skilled in the art and include sulfosuccinates and derivatives thereof, such as muliwet^TMMO-70R (Croda Inc., Edison, NJ); siloxanes, e.g.(Evonik, Germany); non-ionic compounds, e.g. ATLOX^TM4894(Croda inc., Edison, NJ); alkyl polyglucosides, e.g.3001(Huntsman International LLC，The Woodlands，Texas)；C₁₂-C₁₄Alcohol ethoxylates, e.g.15-S-15(The Dow Chemical Company, Midland, Michigan); phosphoric esters, e.g.BG-510(Rhodia, Inc.); and alkyl ether carboxylates, e.g. EMULSOGEN^TM LS(Clariant Corporation，North Carolina)。

The invention further provides formulations as crop protection agents and/or insecticides and application forms (e.g. drench, dip and spray solutions) prepared therefrom, which comprise at least one active compound according to the invention. The application forms may further comprise further crop protection agents and/or pesticides and/or activity-enhancing adjuvants (for example penetrants, examples being vegetable oils (for example rapeseed oil, sunflower oil), mineral oils (for example liquid paraffin), alkyl esters of vegetable fatty acids (for example rapeseed oil methyl ester or soybean oil methyl ester) or alkanol alkoxylates)), and/or spreaders (for example alkylsiloxanes and/or salts, examples being organic or inorganic ammonium or phosphorus salts, examples being ammonium sulfate or diammonium phosphate), and/or retention promoters (for example dioctyl sulfosuccinate or hydroxypropyl guar polymers), and/or humectants (for example glycerol), and/or fertilizers (for example ammonium, potassium or phosphate fertilizers).

The formulations or application forms in question preferably comprise auxiliaries, such as extenders (extenders), solvents, spontaneous promoters, carriers, emulsifiers, dispersants, antifreeze agents, biocides (biocides), thickeners and/or further auxiliaries, such as adjuvants. In this context, an adjuvant is a component that enhances the biological effect of a formulation, and the component itself has no biological effect. Examples of adjuvants are agents that promote retention, spreading, adhesion to the leaf surface or penetration.

These formulations are prepared in a known manner, for example by mixing the active compounds with auxiliaries (for example extenders, solvents and/or solid carriers) and/or with further auxiliaries (for example surfactants).

The adjuvants used may be substances which are suitable for imparting specific properties (e.g. certain physical, technical and/or biological properties) to the formulations of the active compounds or to the application forms prepared from these formulations (e.g. useful crop protection agents, such as spray liquors or seed dressing products).

Suitable extenders are, for example, water, polar and nonpolar organic chemical liquids, for example from the group of aromatic and nonaromatic hydrocarbons (for example paraffins, alkylbenzenes, alkylnaphthalenes, chlorobenzenes), alcohols and polyols (which may also be substituted, etherified and/or esterified, if appropriate), ketones (for example acetone, cyclohexanone), esters (including fats and oils) and (poly) ethers, unsubstituted and substituted amines, amides, lactams (for example N-alkylpyrrolidones) and lactones, sulfones and sulfoxides (for example dimethyl sulfoxide).

If the extender used is water, it is also possible to use, for example, organic solvents as cosolvents. Suitable liquid solvents are mainly: aromatic compounds such as xylene, toluene or alkylnaphthalene; chlorinated aromatic compounds and chlorinated aliphatic hydrocarbons, such as chlorobenzene, vinyl chloride or dichloromethane; aliphatic hydrocarbons, such as cyclohexane or paraffins, such as petroleum fractions, mineral oils and vegetable oils; alcohols (e.g., butanol or ethylene glycol) and ethers and esters thereof; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone; strongly polar solvents, such as dimethylformamide and dimethyl sulfoxide, and water.

In principle, all suitable solvents can be used. Suitable solvents are: for example, an aromatic hydrocarbon such as xylene, toluene or alkylnaphthalene; for example, chlorinated aromatic hydrocarbons or aliphatic hydrocarbons, such as chlorobenzene, vinyl chloride or dichloromethane; for example, aliphatic hydrocarbons such as cyclohexane, e.g., paraffin, petroleum fractions, mineral oils, and vegetable oils; alcohols, such as methanol, ethanol, isopropanol, butanol or ethylene glycol and ethers and esters thereof; ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone; such as strongly polar solvents, for example dimethyl sulfoxide and water.

In principle, all suitable carriers can be used. Suitable carriers are in particular: for example, ammonium salts and ground natural minerals (e.g. kaolin, clay, talc, chalk, quartz, attapulgite, montmorillonite or diatomaceous earth), and ground synthetic minerals (e.g. finely divided silica, alumina) and natural or synthetic silicates, resins, waxes and/or solid fertilizers. Mixtures of such carriers may also be used. Suitable carriers for granules include: for example, natural ores (e.g., calcite, marble, pumice, sepiolite, dolomite), as well as synthetic granules of inorganic and organic flours, and granules of organic materials (e.g., sawdust, paper, coconut shells, corn cobs, and tobacco stalks) that are crushed and classified.

Liquefied gaseous extenders or solvents may also be used. Particularly suitable are those extenders or carriers which are gaseous at standard temperature and at standard pressure, examples being aerosol propellants such as halogenated hydrocarbons, and also butane, propane, nitrogen and carbon dioxide.

Examples of emulsifiers and/or foaming agents, dispersants or wetting agents, or mixtures of these surface-active substances, which have ionic or nonionic character, are: salts of polyacrylic acids; salts of lignosulfonic acid; salts of phenolsulfonic or naphthalenesulfonic acids; polycondensates of ethylene oxide with fatty alcohols or with fatty acids or with fatty amines, with substituted phenols, preferably alkylphenols or arylphenols; a salt of sulfosuccinic acid ester; taurine derivatives (preferably alkyl taurates); phosphoric esters of polyethoxylated alcohols or phenols; fatty acid esters of polyhydric alcohols; and derivatives of sulfate-, sulfonate-and phosphate-containing compounds, such as alkylaryl polyglycol ethers, alkylsulfonates, alkyl sulfates, arylsulfonates, protein hydrolysates, lignosulfite waste liquors and methylcellulose. The presence of a surface-active substance is advantageous if one of the active compounds and/or one of the inert carriers is insoluble in water and when applied in water.

Further auxiliaries which may be present in the formulations and in the application forms obtained therefrom include colorants, for example inorganic pigments, such as iron oxide, titanium oxide, prussian blue, and organic dyes, for example alizarin dyes, azo dyes and metal phthalocyanine dyes, and also nutrients and micronutrients, such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc.

Further components which may be present are stabilizers (e.g. low-temperature stabilizers), preservatives, antioxidants, light stabilizers or other agents which improve the chemical and/or physical stability. A foaming or defoaming agent may also be present.

Furthermore, the formulations and the application forms obtained therefrom may also comprise as further auxiliaries: stickers such as carboxymethyl cellulose; and natural and synthetic polymers in the form of powders, granules or latices, such as gum arabic, polyvinyl alcohol, polyvinyl acetate; and natural phospholipids (e.g., cephalins and lecithins) and synthetic phospholipids. Other adjuvants may include mineral and vegetable oils.

Other adjuvants may be present in the formulations and the application forms obtained therefrom. Examples of such additives include fragrances, protective colloids, binders, adhesives, thickeners, thixotropic substances, penetrants, retention promoters, stabilizers, sequestering agents, complexing agents, humectants, and spreading agents. In general, the active compounds may be combined with any solid or liquid additive commonly used for formulation purposes.

Suitable retention promoters include all those that reduce dynamic surface tension (e.g., dioctyl sulfosuccinate) or increase viscoelasticity (e.g., hydroxypropyl guar polymer).

Suitable penetrants in the context of the present invention include all those substances that are commonly used to enhance the penetration of active agrochemical compounds into plants. The osmotic agent is defined herein as follows: from (usually aqueous) application liquors and/or from spray coatings, they are able to penetrate the epidermis of the plant and thus increase the flowability of the active compound in the epidermis. This property can be determined using methods described in the literature (Baur et al 1997, Pesticide Science 51, 131-. Examples include: alcohol alkoxylates (e.g., coconut fatty ethoxylate (10) or isotridecyl ethoxylate (12)); fatty acid esters (e.g., rapeseed oil methyl ester or soybean oil methyl ester); fatty amine alkoxylates (e.g., tallow amine ethoxylate (15)) or ammonium and/or phosphorus salts (e.g., ammonium sulfate or diammonium phosphate).

Preservation information

Samples of the Paenibacillus species strains of the present invention were deposited under the Budapest treaty at the Culture Collection of Agricultural Research institutes (Agricultural Research Service Culture Collection) located at North university street 1815, department of Agricultural Research, the national center for Agricultural applications Research (NRRL), zip code 61604, of Pionella, Ill. Paenibacillus sp strain NRRL B-50972 was deposited at 2014 at 28/8. Paenibacillus sp strain NRRL B-67129 was deposited at 9/1 of 2015. Paenibacillus sp strain NRRL B-67304 and Paenibacillus sp strain NRRL B-67306 are both deposited at 2016, 7, 22 days. Paenibacillus species NRRL B-67615 was deposited at 2018 on 3/5.

Paenibacillus species strains are maintained under conditions that ensure that qualified persons, as identified by patent and trademark specialists in accordance with 37 c.f.r. § 1-14 and 35 u.s.c. § 122, are able to obtain cultures during the prosecution of the present patent application. It should be understood, however, that the availability of a deposit is not an admission that the invention may be practiced in the event of a breach of patent rights granted by the government.

The following examples are given for illustrative and non-limiting purposes of the present invention.

Example 1 antifungal Activity of Stable Paenibacillus formulations

Paenibacillus sp strain NRRL B-50972 strain was grown in soy-based medium to produce a whole broth culture. The whole broth culture was then centrifuged and concentrated to yield a Broth Concentrate (BC). To evaluate the effect of various compounds as stabilizers, a sample of Paenibacillus sp strain NRRL B-50972BC was taken and mixed with urea, guanidine hydrochloride, triethanolamine or ethyl glycerinooxalate at a dilution of 60: 40 (BC: water) to a final concentration of 2% (i.e., 20 mg/mL). Samples were divided into three groups and stored under one of the following storage conditions: 1) ambient temperature (23 ℃) for 2 weeks; 2) 2 weeks at 40 ℃; or 3) at 54 ℃ for 2 weeks. A60: 40 (BC: water) dilution of Paenibacillus sp strain NRRL B-50972BC without any stabilizer was included in each group as a control.

At the end of 2 weeks of storage, samples were applied to tomato plants at the concentrations shown in table 2. The concentration (i.e. application rate) is the dilution of the reference treatment in water. After each treatment, the solution containing the inoculum of Alternaria sonneri was sprayed onto the plants. Several days after exposure to the plant pathogen inoculum, each plant was scored as a percentage of pathogen control relative to untreated control plants. The incidence of disease in untreated control plants was between 75% and 100% in each assay. Treatments were evaluated in triplicate or quadruplicate and the mean percent control and standard deviation reported (see table 2). 0% means an efficacy comparable to the untreated control group, while an efficacy of 100% means that no disease was observed. Each assay included the use of a reagent known to have activity on Alternaria solaniASO (Bacillus subtilis (Bac)Bacillus subtilis) QST 713). In addition, a blank containing only urea diluted in water was applied to the plants, and no activity on alternaria solani was observed.

A60: 40 (BC: water) dilution sample of Paenibacillus sp strain NRRL B-50972BC mixed with urea, guanidine hydrochloride or triethanolamine was placed under the following storage conditions: 1) 8 weeks at 4 ℃; 2) ambient temperature (23 ℃) for 8 weeks; or 3) at 40 ℃ for 8 weeks. The samples were diluted in water and evaluated for activity against alternaria solani in tomato plants by assay as described above. The results of the second set of measurements are shown in table 3.

The third set of assays was performed using a 60: 40 (BC: water) dilution sample of Paenibacillus sp strain NRRL B-50972BC mixed with urea, guanidine hydrochloride, triethanolamine or ethyl glycerinooxalate, placed under the following storage conditions: 1) 6 months at 4 ℃; or 2) ambient temperature (23 ℃) for 6 months. The results of these measurements are shown in Table 4.

In each of these three assays, urea, guanidine hydrochloride, and triethanolamine retained the antifungal activity of paenibacillus strain NRRL B-50972BC, with little effect of the addition of ethyl glycerinooxalate. As expected, the stabilization of the compounds was most pronounced in the samples subjected to the most severe storage conditions (i.e., 2 weeks at 54 ℃ as shown in Table 2, 8 weeks at 40 ℃ in Table 3, and 6 months at ambient temperature (23 ℃) in Table 4).

Example 2 quantification of Fusarium A in Stable Bacteroides formulations

Paenibacillus cells are known to produce several antifungal compounds including fusaricidin A. To determine the effect of the stabilizer on fusaricidin a levels in samples of the paenibacillus species NRRL B-50972BC mixed with urea, guanidine hydrochloride, triethanolamine or ethyl glycerinooxalate analyzed in example 1, the samples were extracted with an organic solvent to produce a cell extract.

The relative level of fusaricidin a in these extracts was quantified by chromatographic methods using high performance liquid chromatography/mass spectrometry time of flight (HPLC/MS TOF) as follows: column: YMC^TMBasic 4.6X 250mm, 5 μm; water (0.1% FA) and acetonitrile (0.1% Formic Acid (FA)); gradient (% B): 28-30% in 0-9 min; 30-33% in 9-14 min; 33-50% in 14-34 min; and (5) flushing.

A standard sample stored at-80 ℃ containing a whole broth culture of paenibacillus sp strain NRRL B-50972 was run for each HPLC/MS-TOF analysis and the peak areas in the remaining samples were normalized based on the peak area of fusaricidin a in the standard sample and the reported relative amounts were calculated.

The relative amounts of fusaricidin A in samples of Paenibacillus sp strain NRRL B-50972BC with and without stabilizer stored at 54 ℃ for 2 weeks or at 40 ℃ for 8 weeks are shown in FIG. 1 and FIG. 2, respectively. The Paenibacillus sp strain NRRL B-50972BC stored at 40 ℃ was sampled and analyzed at 0, 2, 4 and 8 weeks of storage.

Urea, guanidine hydrochloride and triethanolamine inhibited the degradation of fusaricidin a in samples of paenibacillus sp. In contrast, the addition of ethyl glycerinooxalate in these samples had little effect on the stability of fusaricidin a. The relative quantification of fusaricidin A in samples of Paenibacillus sp strain NRRL B-50972BC stored at ambient temperature (23 ℃) for 6 months confirms the trend observed in FIGS. 1 and 2.

These results are consistent with the data given in tables 2-4, where urea, guanidine hydrochloride and triethanolamine retained antifungal activity against alternaria solani with little effect on ethyl glycerate after storage of paenibacillus sp strain NRRL B-50972BC for extended periods of time and/or at elevated temperatures.

Example 3 evaluation of other Stable Paenibacillus preparations

Other stable formulations of Paenibacillus strain NRRL B-50972BC containing one of several candidate stabilizers were prepared and evaluated for antifungal activity against Alternaria solani as described in example 1. Candidate stabilizers were added to a 70: 30 (BC: water) dilution of Paenibacillus sp strain NRRL B-50972BC to a final concentration of 2% or 0.5%. The stable formulation was then stored at 54 ℃ for 2 weeks or at 40 ℃ for 8 weeks and compared to Paenibacillus sp strain NRRL B-50972BC without any stabilizer.

Experiments were performed to determine the antifungal activity of the different stable formulations as described in example 1. The activity against alternaria solani or botrytis cinerea was measured at the application rates indicated in tables 5 to 8. The concentration (i.e. application rate) is the dilution of the reference treatment in water. 0% means an efficacy comparable to the untreated control group, while an efficacy of 100% means that no disease was observed.

Betaine hydrochloride together with urea showed a strong effect in stabilizing the antifungal activity of paenibacillus sp strain NRRL B-50972BC against alternaria solani and botrytis cinerea. The stabilization of potassium phosphate (dibasic) is less than that of urea and betaine hydrochloride. Several other candidate stabilizers had no significant effect on the antifungal activity of Paenibacillus sp strain NRRL B-50972 BC.

Example 4 quantification of Fusarium A in other Stable Bacteroides formulations

The relative amount of fusaricidin a present in the samples prepared in example 3 was determined using the quantitative method described in example 2. The relative amounts of fusaricidin a in these other samples of paenibacillus sp strain NRRL B-50972BC with and without stabilizer after 2 weeks storage at 54 ℃ or 8 weeks storage at 40 ℃ are shown in fig. 3 and 4, respectively.

Urea and betaine hydrochloride retained fusaricidin a in the paenibacillus strain NRRL B-50972BC samples, whereas potassium phosphate (binary) had less of an effect on stabilizing fusaricidin a in these samples. Samples of Paenibacillus sp strain NRRL B-50972BC mixed with candidate stabilizers that do not retain antifungal activity were also analyzed. The degradation of fusaricidin A in these samples was similar to that of Paenibacillus sp strain NRRL B-50972BC, which did not contain any stabilizers.

These results are consistent with the data given in tables 5-8, where the addition of urea and betaine hydrochloride maintained the antifungal activity against Alternaria solani and Botrytis cinerea after storage of Paenibacillus sp strain NRRL B-50972BC for 2 weeks at 54 ℃ or 8 weeks at 40 ℃, while the stabilization of potassium phosphate (binary) was less.

Example 5 evaluation of Stable Paenibacillus preparations with Potassium phosphate (Mono), Potassium phosphate (binary) or Potassium citrate (ternary)

After storage for 2 weeks at 54 ℃, several closely related compounds were tested for their potential to stabilize paenibacillus sp strain NRRL B-50972 BC. Samples were prepared and analyzed as described in example 3 and contained urea, potassium phosphate (monobasic), potassium phosphate (dibasic) or potassium citrate (tribasic) at 2% in a 70: 30 (BC: water) dilution of paenibacillus sp. Samples of Paenibacillus sp strain NRRL B-50972 Whole Broth (WB) were included as a control, then centrifuged and concentrated. After 2 weeks of storage at 54 ℃, the antifungal activity in the WB control samples decreased, so that the disease appeared on WB-treated plants indistinguishable from untreated plants (data not shown).

The antifungal activity of the Paenibacillus strain NRRL B-50972BC samples mixed with urea, potassium phosphate (monobasic), potassium phosphate (dibasic) or potassium citrate (tribasic) after storage for 2 weeks at 54 ℃ is shown in Table 9. The concentration (i.e. application rate) is the dilution of the reference treatment in water. 0% means an efficacy comparable to the untreated control group, while an efficacy of 100% means that no disease was observed. Surprisingly, potassium phosphate (dibasic) stabilized the antifungal activity, whereas potassium phosphate (monobasic) did not. Potassium citrate (ternary) was also shown to have a stabilizing effect on Paenibacillus sp strain NRRL B-50972 BC.

Example 5 determination of the Effect of solvents on Paenibacillus preparations

Paenibacillus sp strain NRRL B-50972 was grown on soy-based medium to produce a whole broth culture. The whole broth culture was then centrifuged and concentrated to yield a Broth Concentrate (BC). A50: 50 dilution of the Paenibacillus sp strain NRRL B-50972BC in water or propylene glycol was prepared and analyzed for antifungal activity and relative fusarium A fusarium oxysporum levels as described in examples 1 and 2. The concentration (i.e. application rate) is the dilution of the reference treatment in water. 0% means an efficacy comparable to the untreated control group, while an efficacy of 100% means that no disease was observed.

The antifungal activity of Paenibacillus sp strain NRRL B-50972BC was retained by dilution in propylene glycol to a greater extent than in water after storage for 2 weeks at 54 ℃ or 2, 4 or 8 weeks at 40 ℃ (compare tables 10 and 11). Furthermore, the rate of fusaricidin A degradation was slower for the Paenibacillus sp strain NRRL B-50972BC diluted in propylene glycol than for the Paenibacillus sp strain NRRL B-50972BC diluted in water after storage at 54 ℃ for 2 weeks or after storage at 40 ℃ for 2, 4 or 8 weeks (compare FIGS. 5 and 6).

Example 6 improvement of physical Properties of Stable Paenibacillus preparations

Paenibacillus sp strain NRRL B-50972BC was prepared and diluted with a mixture of propylene glycol and water. A portion of the diluted Paenibacillus sp strain NRRL B-50972BC was mixed with 2% urea, the remainder being urea-free.

A formulation is considered physically stable if little or no syneresis or sedimentation of the formulation ingredients is observed over an extended period of time. The longer it takes for syneresis or settling to occur, the more physically stable the formulation. The formulation is considered to be physically stable if little or no syneresis or sedimentation is observed after 2 weeks at 54 ℃.

Diluted samples of Paenibacillus sp strain NRRL B-50972BC with or without urea were stored at 54 ℃ for 2 weeks. No syneresis or sedimentation of the samples was observed prior to storage. Photographs of the samples after storage showed little or no syneresis or sedimentation of the urea-containing diluted paenibacillus strain NRRL B-50972 BC. In contrast, photographs of diluted Paenibacillus strain NRRL B-50972BC without urea showed that the formulation separated into distinct layers, indicating syneresis and sedimentation. The results show that the physical stability of Paenibacillus sp strain NRRL B-50972BC is improved due to the addition of urea.

Example 7 relative Fusarium A levels and antifungal Activity of Bacteroides formulations stabilized after 2 years at 23 deg.C

Paenibacillus sp strain NRRL B-50972 Broth Concentrate (BC) with and without 2% urea was prepared and after storage for 2 years at 23 ℃ their antifungal activity against Alternaria solani was measured as described in example 1. The concentration (i.e. application rate) is the dilution of the reference treatment in water. 0% means an efficacy comparable to the untreated control group, while an efficacy of 100% means that no disease was observed. The relative level of fusaricidin a in the broth concentrate was also determined as described in example 2.

The antifungal activity of the paenibacillus strain NRRL B-50972BC containing 2% urea remained at about 90% even after storage for 2 years at 23 ℃, while the antifungal activity of the paenibacillus strain NRRL B-50972BC without urea decreased to less than half of this level (see Table 12). Similarly, the level of fusaricidin a remained relatively constant in the 2% urea containing paenibacillus strain NRRL B-50972BC stored at 23 ℃ for 2 years, while the level of fusaricidin a in the urea free paenibacillus strain NRRL B-50972BC decreased to about one-fourth of the initial level during the same storage period (see figure 8).

Example 8 relative Fusarium A levels and antifungal Activity in Stable formulations from various Bacillus species strains

As described in U.S. patent application No. 62/671,067, Paenibacillus sp.NRRL B-50972 is related to Paenibacillus sp.NRRL B-67304, Paenibacillus sp.NRRL B-67306 and Paenibacillus sp.NRRL B-67615. The paenibacillus strain NRRL B-50972 is the parent strain from which each of the other strains was obtained by multiple rounds of mutagenesis and screening for increased yields of fusaricidin and fusaricidin-like compounds and decreased viscosity in liquid culture. Paenibacillus species strain NRRL B-67304, paenibacillus species strain NRRL B-67306 and paenibacillus species strain NRRL B-67615 each have a mutation in the degU and/or degS genes resulting in a non-slime-like colony morphology on solid media and reduced viscosity in liquid cultures.

The urea-containing broth concentrates of Paenibacillus sp.NRRL B-50972, Paenibacillus sp.NRRL B-67304 and Paenibacillus sp.NRRL B-67306 were prepared and measured for antifungal activity against Alternaria solani as described in example 1 after 8 weeks of storage at 40 ℃ and after 1 year of storage at 23 ℃. The concentration (i.e. application rate) is the dilution of the reference treatment in water. 0% means an efficacy comparable to the untreated control group, while an efficacy of 100% means that no disease was observed. The relative level of fusaricidin a in the broth concentrate was also determined as described in example 2.

When applied to plants at 0.625% or 0.312%, the antifungal activity of the urea-free paenibacillus strain NRRL B-50972BC was not significantly different from the untreated control after 8 weeks of storage at 40 ℃, while the antifungal activity of paenibacillus strain NRRL B-50972BC, paenibacillus strain NRRL B-67304 BC and paenibacillus strain NRRL B-67306 BC, each mixed with urea, remained between 79% and 89% (see table 13). Similar results were observed for broth concentrates stored for 1 year at 23 ℃ except that under these conditions, the antifungal activity of the urea-free paenibacillus strain NRRL B-50972BC was detectable at low levels above the untreated control (see table 14).

After 8 weeks of storage at 40 ℃ and 1 year of storage at 23 ℃, the level of fusaricidin a in each of the bacillus species NRRL B-50972BC, bacillus species NRRL B-67304 BC and bacillus species NRRL B-67306 BC mixed with urea remained relatively constant, whereas the level of fusaricidin a in the bacillus species NRRL B-50972BC without urea decreased to about one third of the initial level during these same storage periods (see fig. 9 and 10).

Example 9 relative Fusarium A levels and antifungal Activity of Stable formulations of Paenibacillus sp strains NRRL B-67304, NRRL B-67306, and NRRL B-67615

Whole broth and broth concentrates of Paenibacillus species NRRL B-67304, Paenibacillus species NRRL B-67306, and Paenibacillus species NRRL B-67615 were prepared as described in example 1. The whole broth was not urea added, whereas the broth concentrate was stabilized with urea. After 8 weeks of storage at 40 ℃ and 1 year of storage at 23 ℃, the antifungal activity of the whole broth and stable broth concentrates on botrytis cinerea and alternaria sonnei was measured as described in examples 1 and 3. In the antifungal assay, the whole broth samples were applied at twice the rate as the broth concentrate samples to adjust for dilution of the antifungal chemicals in the whole broth. The concentration (i.e. application rate) is the dilution of the reference treatment in water. 0% means an efficacy comparable to the untreated control group, while an efficacy of 100% means that no disease was observed. The relative levels of fusaricidin a in the whole broth and broth concentrates were also determined as described in example 2.

The antifungal activity of the whole broth samples of the three urea-free paenibacillus species strains was similar to or slightly higher than the untreated control. In contrast, the antifungal activity of the urea-stabilized broth concentrates of the three paenibacillus species strains remained relatively high and more similar to the positive control: (b) ((b))ASO (bacillus subtilis QST 713)). This positive control had no storage conditions for stability determination and was known to have relatively high antifungal activity. For two fungal pathogens (i.e., Botrytis cinerea and Alternaria solani) and at two time periodsThe stabilizing effect of urea was consistent (i.e. 8 weeks at 40 ℃ and 1 year at 23 ℃) (see tables 15-18).

The levels of fusaricidin a in the whole broth samples and urea-stabilized broth concentrate samples of paenibacillus species NRRL B-67304, paenibacillus species NRRL B-67306 and paenibacillus species NRRL B-67615 during 8 weeks of storage at 40 ℃ and during 1 year of storage at 23 ℃ are shown in fig. 11 and 12, respectively. Although fusaricidin a levels in unstable whole broth samples decreased significantly over these time periods, fusaricidin a levels in urea-containing broth concentrate samples remained relatively constant.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patents, and patent publications cited are hereby incorporated by reference in their entirety for all purposes.

It is to be understood that the disclosed invention is not limited to the particular methodology, protocols, and materials described, as these can 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 limit the scope of the present invention, which will be limited only by the appended claims.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.