METHODS AND COMPOSITIONS TO IMPROVE LETTUCE PRODUCTION

专利摘要:
the present invention provides both compositions comprising methylobacterium and compositions comprising methylobacterium which are depleted of substances that promote the cultivation of microorganisms residing in a lettuce plant or seed. methods for improving lettuce production, methods for producing the compositions and methods for treating a lettuce plant or seed with the compositions comprising methylobacterium are also provided.
公开号:BR112016012870B1
申请号:R112016012870-2
申请日:2014-12-04
公开日:2021-06-22
发明作者:Rachel DiDonato Floro；Justin Lee；Gregg Bogosian；Doug Bryant
申请人:Newleaf Symbiotics, Inc；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[0001] This International Patent Application claims the benefit of Provisional Patent Application No. US 61/954840, filed March 18, 2014, and Provisional Patent Application No. US 61/911516, filed December 4, 2013, which are incorporated herein by reference. SEQUENCE LISTING STATEMENT
[0002] A string listing containing the file named 53907-137549_SL.txt, which is 15,167,308 bytes (measured in MS-Windows®) and created on November 28, 2014, comprises 10,250 strings, is provided with the document through the USPTO's EFS system and is incorporated herein by reference in its entirety. BACKGROUND
[0003] Organic compounds of one carbon, such as methane and methanol, are found extensively in nature and are used as carbon sources by bacteria classified as methanotrophic and methylotrophic. Methanotrophic bacteria include species in the genera Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, Methylosinus, Methylocystis, Methylosphaera, Methylocaldum and Methylocella (Lidstrom, 2006). Methanotrophics have the enzyme methane monooxygenase, which incorporates an O2 oxygen atom into methane, which forms methanol. All methanotrophics are obligatory carbon users who do not have the ability to use compounds that contain carbon-carbon bonds. Methylotrophs, on the other hand, can also use more complete organic compounds, such as organic acids, higher alcohols, sugars, and the like. Thus, methylotrophic bacteria are facultative methylotrophic. Methylotrophic bacteria include species in the genera Methylobacterium, Hyphomicrobium, Methylophilus, Methylobacillus, Methylophaga, Aminobacter, Methylorhabdus, Methylopila, Methylosulfonomonas, Marino sulphonomonas, Paracoccus, Xanthobacter, Ancylobacter, Rhobacter, Bactobacterillus (also known as Microbacterillus) Mycobacterium, Arthobacter and Nocardia (Lidstrom, 2006).
[0004] Most methylotrophic bacteria of the genus Methylobacterium are pink pigmented. They are conventionally termed as PPFM bacteria, which are pink pigmented facultative methylotrophs. Green (2005, 2006) identified twelve validated species in the Methylobacterium genus, specifically M. aminovorans, M. chloromethanicum, M. dichloromethanicum, M. extorquens, M. fujisawaense, M. mesophilicum, M. organophilum, M. radiotolerans, M. rhodesianum, M. rhodesianum, M. rhodinum, M. thiocyanatum and M. zatmanii. However, M. nidulans is a nitrogen-fixing Methylobacterium that is not a PPFM (Sy et al, 2001). Methylobacterium are ubiquitous in nature, found in soil, dust, fresh water, sediment and leaf surfaces, as well as in industrial and clinical environments (Green, 2006). SUMMARY
[0005] This document provides compositions comprising Methylobacterium, which are depleted of substances that promote the cultivation of bacteria residing in the plant or seed, compositions comprising a solid substance with adherent Methylobacterium cultivated on it or a emulsion that has Methylobacterium grown in it, compositions that comprise certain strains of Methylobacterium, methods of using the compositions to improve lettuce production, and methods of making the compositions. Such compositions are, in certain cases, referred to herein simply as "Methylobacterium-containing compositions". In certain embodiments, the Methylobacterium in the composition, or which is used, is the NLS0020, NLS0066, NLS0017, NLS0065, NLS0089, NLS0042 or NLS0068 strain. In certain modalities, the Methylobacterium in the composition, or that is used, is the Methylobacterium that is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 ( NRRL B-50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936), NLS0089 (NRRL B-50933) and derivatives thereof. In certain embodiments, the Methylobacterium in the composition, or that is used, is a Methylobacterium that has at least one gene that encodes a protein that is orthologous to a protein that has an amino acid sequence of SEQ ID NO: 1-5125. In certain embodiments, the Methylobacterium has at least one gene that encodes a protein that is orthologous to a reference protein in Table 7. In certain embodiments, the Methylobacterium species can contain at least one gene that encodes a protein that is orthologous to a protein which has the amino acid sequence of 13, 14, 23, 27, 28, 30, 40, 43, 44, 51, 52, 57, 76, 85, 127, 197, 198, 199, 1094, 1100, 1106 , 1114,1116, 1117, 1120, 1180, 2180, 2190, 2463, 2467, 2468, 2471, 2510, 2515, 2676, 2971, 3357, 3370, 3372, 3394, 3427, 3429, 3430, 3950, 3952, 3968 , 3987, 3996, 4004, 4006 and/or 4067 of Table 7. In certain embodiments, Methylobacterium has at least one gene encoding a protein that is orthologous to a reference protein is selected from the group consisting of SEQ ID NO: 13, 14, 23, 1094, 1100, 1106, 2467, 2468, 3357, 3370 and/or 3968. In certain embodiments, Methylobacterium has at least one gene that encodes a protein that and is orthologous to a reference protein is selected from the group consisting of SEQ ID NO: 1100, 1116, 2471 and/or 3950. In certain embodiments, the Methylobacterium in the composition, or which is used, is the strain NLS0020, NLS0066 , NLS0017, NLS0065 or NLS0089 and the composition is used to treat a lettuce seed.
[0006] Methods for improving lettuce production comprise applying a coating or a partial coating of a composition comprising Methylobacterium to a lettuce plant, a part thereof or to a lettuce seed, wherein said composition comprises: ( a) a solid substance with adherent Methylobacterium cultivated on it; (b) an emulsion that has Methylobacterium grown in it; (c) certain Methylobacterium strains selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL B- 50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936), NLS0089 (NRRL B-50933) and derivatives thereof and an agriculturally acceptable adjuvant, an agriculturally acceptable excipient or combination thereof; or (d) a Methylobacterium that has at least one gene that encodes at least one protein that is orthologous to a protein that has an amino acid sequence of SEQ ID NO: 1-5125 and an agriculturally acceptable adjuvant, an agriculturally acceptable excipient or combination of the same; and wherein said lettuce plant or lettuce plant grown from said seed exhibits an improvement trait selected from the group consisting of an increased rate of leaf cultivation, an increased rate of root cultivation, biomass production increased total, increased seed yield, decreased cycle time and combinations thereof, when compared to an untreated control lettuce plant or a control lettuce plant grown from an untreated seed, is provided herein. . In certain embodiments, the composition comprises Methylobacterium at a titer of about 1 x 106 CFU/g to about 1 x 1014 CFU/g for a solid composition or at a titer of about 1 x 106 CFU/ml to about 1 x 1011 CFU/ml for a liquid composition containing the solid substance or for the emulsion. In certain embodiments, the Methylobacterium has at least one gene that encodes at least one protein that is orthologous to a protein that has an amino acid sequence of SEQ ID NO: 1-5125. In certain embodiments, the Methylobacterium has at least one gene that encodes a protein that is orthologous to a reference protein in Table 7. In certain embodiments, the Methylobacterium species can contain at least one gene that encodes a protein that is orthologous to a protein which has the amino acid sequence of 13, 14, 23, 27, 28, 30, 40, 43, 44, 51, 52, 57, 76, 85, 127, 197, 198, 199, 1094, 1100, 1106 , 1114,1116, 1117, 1120, 1180, 2180, 2190, 2463, 2467, 2468, 2471.2510, 2515, 2676, 2971.3357, 3370, 3372, 3394, 3427, 3429, 3430, 3950, 3952, 3968 , 3987, 3996, 4004, 4006 and/or 4067 of Table 7. In certain embodiments, Methylobacterium has at least one gene encoding a protein that is orthologous to a reference protein is selected from the group consisting of SEQ ID NO: 13, 14, 23, 1094, 1100, 1106, 2467, 2468, 3357, 3370 and/or 3968. In certain embodiments, Methylobacterium has at least one gene that encodes a protein that is orthologous to a reference protein is selected from the group consisting of SEQ ID NO: 1100, 1116, 2471 and/or 3950. In certain embodiments, the Methylobacterium in the composition, or that is used, is selected from the group that consists of NLS0017 (NR L B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932) , NLS0046(NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936), NLS0089 (NRRL B-50933) and derivatives thereof. In certain embodiments, the Methylobacterium in the composition, or that is used, is selected from the group consisting of NLS0017, NLS0037, NLS0066, NLS0020, NLS0042, NLS0065, NLS0089, NLS0046,NLS0021, NLS0069, NLS0068, NLS0064, NLS0062, NLS0038 and derivatives thereof. In certain embodiments, the Methylobacterium has at least one polymorphic DNA element that is present in at least one Methylobacterium strain, selected from the group consisting of NLS0020, NLS0066, NLS0017, NLS0065, NLS0089, NLS0042 and NLS0068, provided herein document that improves lettuce production, but is absent from the Methylobacterium species that does not improve lettuce production. In certain embodiments, the composition is applied to a lettuce seed and at least one polymorphic DNA element is present in at least one Methylobacterium strain selected from the group consisting of NLS0020, NLS0066, NLS0017, NLS0065 and NLS0089. In certain modalities, the composition is applied to a lettuce seed and the Methylobacterium is selected from the group consisting of NLS0020, NLS0066, NLS0017, NLS0065 and NLS0089. In certain embodiments, the composition is applied to a lettuce plant or a part thereof and at least one polymorphic DNA element is present in at least one Methylobacterium strain selected from the group consisting of NLS0042, NLS0017, NLS0020 and NLS0068. In certain embodiments, the composition is applied to a lettuce plant or a part thereof and the Methylobacterium is selected from the group consisting of NLS0042, NLS0017, NLS0020 and NLS0068. In certain embodiments, the applied composition coats or partially coats said plant or a part thereof, or said seed. In certain embodiments, the methods further comprise: (i) cultivating said lettuce plant or the lettuce plant grown from said seed; and/or (ii) harvesting leaves or seed from said lettuce plant or from the lettuce plant grown from said seed. In certain modalities, the solid substance with adherent Methylobacterium is not a substance that promotes the cultivation of microorganisms residing in the lettuce plant, in its part or in the lettuce seed. In certain embodiments, the composition comprises an agriculturally acceptable adjuvant and/or excipient. In certain modalities of any of the aforementioned methods, the composition is depleted of substances that promote the cultivation of microorganisms residing in said plant or seed. Also provided are lettuce plant parts or lettuce seeds obtained by any of the above methods and which are coated or partially coated with a composition comprising Methylobacterium.
[0007] Methods for improving lettuce plant production comprising applying a composition comprising Methylobacterium to a lettuce plant, a part thereof or a lettuce seed, wherein said composition is depleted of substances that promote the cultivation of microorganisms residing on said plant or seed and wherein said plant or plant cultivated from said seed exhibits an improvement of trait selected from the group consisting of an increased rate of leaf cultivation, an increased rate of cultivation of root, increased total biomass production, increased seed yield, decreased cycle time, and combinations thereof when compared to an untreated control lettuce plant or a control lettuce plant grown from an untreated seed. In certain embodiments, the composition comprises a solid substance with adherent Methylobacterium grown thereon. In certain modalities, the solid substance is not a substance that promotes the cultivation of microorganisms residing in the lettuce plant, in its part or in the lettuce seed. In certain embodiments, the composition comprises Methylobacterium at a titer of about 1 x 106 CFU/g to about 1 x 1014 CFU/g. In certain embodiments, the composition comprises a liquid, a solid substance with Methylobacterium adhered thereto in a liquid, a solid substance with Methylobacterium adhered thereto in an emulsion or an emulsion. In certain embodiments, the composition comprises Methylobacterium at a titer of about 1x106 CFU/ml to about 1x1011 CFU/ml. In certain embodiments, the methods further comprise: (i) cultivating said lettuce plant or the lettuce plant grown from said seed; and/or (ii) harvesting leaves or seed from said lettuce plant or from the lettuce plant grown from said seed. In certain embodiments, the Methylobacterium has at least one gene that encodes at least one protein that is orthologous to a protein that has an amino acid sequence of SEQ ID NO: 1-5125. In certain embodiments, the Methylobacterium has at least one gene that encodes a protein that is orthologous to a reference protein in Table 7. In certain embodiments, the Methylobacterium species can contain at least one gene that encodes a protein that is orthologous to a protein which has the amino acid sequence of 13, 14, 23, 27, 28, 30, 40, 43, 44, 51.52, 57, 76, 85, 127, 197, 198, 199, 1094, 1100, 1106 , 1114,1116, 1117, 1120, 1180, 2180, 2190, 2463, 2467, 2468, 2471, 2510, 2515, 2676, 2971,3357, 3370, 3372, 3394, 3427, 3429, 3430, 3950, 3952, 3968 , 3987, 3996, 4004, 4006 and/or 4067 of Table 7. In certain modalities, Methylobacterium has at least one gene encoding a protein that is orthologous to a reference protein is selected from the group consisting of SEQ ID NO : 13, 14, 23, 1094, 1100, 1106, 2467, 2468, 3357, 3370 and/or 3968. In certain embodiments, the Methylobacterium has at least one gene that encodes a protein that is orthologous to a reference protein is selected from the group consisting of SEQ ID NO: 1100, 1116, 2471 and/or 3950. In certain embodiments, the Methylobacterium in the composition, or that is used, is selected from the group that consists of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069 ( NRRL B-50936), NLS0089(NRRL B-50933) and derivatives thereof. In certain embodiments, the Methylobacterium in the composition, or that is used, is selected from the group consisting of NLS0017, NLS0037, NLS0066, NLS0020, NLS0042, NLS0065, NLS0089, NLS0046, NLS0021, NLS0069, NLS0068, NLS0064,NLS0062, NLS0038 and derivatives thereof. In certain embodiments the reference protein is selected from the group consisting of SEQ ID NO: 11000, 1116, 2471 and/or 3950. In certain embodiments, aMethylobacterium has at least one polymorphic DNA element that is present in at least one Methylobacterium strain, selected from the group consisting of NLS0020, NLS0066, NLS0017, NLS0065, NLS0089, NLS0042 and NLS0068, provided in this document that improves lettuce production, but which is absent from the Methylobacterium species that does not improve lettuce production lettuce. In certain embodiments, the composition is applied to a lettuce seed and at least one polymorphic DNA element is present in at least one Methylobacterium strain selected from the group consisting of NLS0020, NLS0066, NLS0017, NLS0065 and NLS0089. In certain embodiments, the composition is applied to a lettuce seed and the Methylobacterium is selected from the group consisting of NLS0020, NLS0066, NLS0017, NLS0065 and NLS0089. In certain modalities, the composition is applied to a lettuce plant or a part thereof and at least one polymorphic DNA element is present in at least one Methylobacterium strain selected from the group consisting of NLS0042, NLS0017, NLS0020 and NLS0068 . In certain modalities, the composition is applied to a lettuce plant or a part of it and the Methylobacterium is selected from the group consisting of NLS0042, NLS0017, NLS0020 and NLS0068. In certain embodiments of any of the aforementioned methods, the composition coats or partially coats said plant or a part thereof, or said seed. Also provided are lettuce plant parts or lettuce seeds obtained by any of the above methods and which are coated or partially coated with a composition comprising Methylobacterium.
[0008] Compositions comprising: (i) a solid substance with adherent Methylobacterium grown on it wherein said Methylobacterium has at least one gene encoding a protein that is orthologous to a protein that has an amino acid sequence of SEQ ID NO: 1-5125; (ii) an emulsion with Methylobacterium grown in it wherein said Methylobacterium has at least one gene encoding a protein that is orthologous to a protein that has an amino acid sequence of SEQ ID NO: 1-5125 ; or (iii) certain Methylobacterium strains selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), NLS0038 ( NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935), NLS0066 (NRRL B -50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936), NLS0089 (NRRL B-50933) and derivatives thereof, and an agriculturally acceptable adjuvant, an agriculturally acceptable excipient or a combination thereof are provided in the this document. Also provided herein are compositions comprising: (a)(i) a solid substance with adherent Methylobacterium grown thereon; (ii) an emulsion with Methylobacterium grown therein; or (iii) a Methylobacterium that has at least one gene that encodes a protein that is orthologous to a protein that has an amino acid sequence of SEQ ID NO: 1-5125; and (b) an agriculturally acceptable excipient, an agriculturally acceptable adjuvant or a combination thereof. In certain embodiments, Methylobacterium has at least one gene encoding a protein that is orthologous to a reference protein of Table 7. In certain embodiments, the reference protein is selected from the group consisting of SEQ ID NO: 13, 14, 23, 1094, 1100, 1106, 2467, 2468, 3357, 3370 and/or 3968. In certain embodiments, the Methylobacterium is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B- 50930), NLS0037 (NRRL B-50941), NLS0042 (NRRL B-50932), NLS0065 (NRRL B-50935), NLS0066 (NRRL B-50940) and derivatives thereof. Compositions are also provided which comprise: (i) a solid substance with adherent Methylobacterium grown thereon; or (ii) an emulsion with Methylobacterium grown therein, wherein said Methylobacterium has at least one polymorphic DNA element that is present in at least one Methylobacterium strain, selected from the group consisting of NLS0020, NLS0066, NLS0017, NLS0065, NLS0089, NLS0042 and NLS0068, provided herein which improves lettuce production, but which is absent from the Methylobacterium species which does not improve lettuce production. In certain embodiments, the at least one polymorphic DNA element is present in at least one Methylobacterium strain selected from the group consisting of NLS0020, NLS0066, NLS0017, NLS0065, and NLS0089. In certain embodiments, at least one polymorphic DNA element is present in at least one Methylobacterium strain selected from the group consisting of NLS0042, NLS0017, NLS0020, and NLS0068. In certain embodiments, Methylobacterium is selected from the group consisting of NLS0020, NLS0066, NLS0017, NLS0065, NLS0089, NLS0042, and NLS0068. In certain modalities, the composition is depleted of substances that promote the cultivation of microorganisms residing in a plant or seed. In certain modalities, the substance that promotes the cultivation of microorganisms residing in a plant or seed is selected from the group consisting of a carbon source, a nitrogen source, a phosphorus source, a sulfur source, a source of magnesium and combinations thereof. In certain embodiments, the compositions additionally comprise an agriculturally acceptable adjuvant and/or excipient. In certain embodiments, the solid substance with adherent Methylobacterium grown thereon has a Methylobacterium titer of at least about 5 x 108 CFU/g to at least about 1 x 1014 CFU/g. In certain embodiments, Methylobacterium is selected from the group consisting of NLS0020, NLS0066, NLS0017, NLS0065, and NLS0089. In certain embodiments, the aforementioned compositions are adapted for use in treating a plant or seed or are used to treat a plant or seed. Also provided herein is a lettuce plant part or a lettuce seed which is coated or partially coated with any of the aforementioned compositions. In certain embodiments, the coated or partially coated lettuce plant part or lettuce seed is obtained by any of the above methods.
[0009] An isolated Methylobacterium selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), is also provided. NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B-50935), NLS0066 ( NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069(NRRL B-50936), NLS0089 (NRRL B-50933) and derivatives thereof.
[0010] Compositions are also provided comprising: (i) an isolated Methylobacterium selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 ( NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042(NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B -50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069 (NRRL B-50936), NLS0089(NRRL B-50933), derivatives thereof; and (ii) an agriculturally acceptable adjuvant, an agriculturally acceptable excipient or a combination thereof.
[0011] Also provided are plants, plant parts and plant seeds that are coated or partially coated with any of the aforementioned compositions. In certain embodiments, a lettuce plant, a plant part or a plant seed is coated or partially coated with the aforementioned compositions.
[0012] Methods for identifying compositions, plant parts, plant seeds or processed plant products comprising the Methylobacterium species NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0037 ( NRRL B-50941), NLS0042 (NRRL B-50932), NLS0065 (NRRL B-50935), or NLS0066 (NRRL B-50940) by evaluating the presence of nucleic acid sequences contained in SEQ ID NO: 5126-10250 in those materials. In certain embodiments, such methods may comprise submitting a sample suspected of containing the Methylobacterium species NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0037 (NRRL B-50941), NLS0042 (NRRL B-50932), NLS0065(NRRL B-50935) or NLS0066 (NRRL B-50940) by a nucleic acid analysis technique and determine that the sample contains one or more nucleic acids that contain a sequence of at least about 20, 50, 100, 200 , 500 or 1000 nucleotides that is identical to at least one of SEQ ID NO: 5126-10250, wherein the presence of a sequence that is identical to at least one of SEQ ID NO: 5126-6211 is indicative of the presence of NLS017, wherein the presence of a sequence that is identical to at least one of SEQ ID NO: 6212-7301 is indicative of the presence of NLS020, wherein the presence of a sequence that is identical to at least one of SEQ ID NO: 7302- 7586 is indicative of the presence of NLS037, wherein the presence of a sequence that is identical to at least one of SEQ ID NO: 7587-8472 is indicative of the presence of NLS042, wherein the presence of a sequence that is identical to at least one of SEQ ID NO: 84739074 is indicative of the presence of NLS065, and wherein the presence of a sequence that is identical to at least one of SEQ ID NO: 9075-10250 is indicative of the presence of NLS066. Such nucleic acid analyzes include, but are not limited to, techniques based on nucleic acid hybridization, polymerase chain reactions, mass spectroscopy, nanopore-based detection, branched DNA analyses, combinations thereof, and the like.
Methods for identifying Methylobacterium species that may impart useful traits to plants are also provided herein by evaluating the presence of nucleic acid sequences contained in SEQ ID NO: 512610250 in Methylobacterium species. In certain embodiments, such methods may comprise subjecting a candidate Methylobacterium species to a nucleic acid analysis technique and determining that the sample contains one or more nucleic acids that contain a sequence of at least about 20, 50, 100, 200, 500 or 1000 nucleotides which is identical to at least one of SEQ ID NO: 5126-10250 which indicates that the candidate Methylobacterium species can confer useful traits on a plant. Such nucleic acid analyzes include, but are not limited to, techniques based on nucleic acid hybridization, polymerase chain reactions, mass spectroscopy, nanopore-based detection, branched DNA analyses, combinations thereof, and the like. DESCRIPTION DEFINITIONS
[0014] As used herein, the terms "adhered thereto" and "adherent" refer to Methylobacterium that is associated with a solid substance by cultivation, or that has been cultivated, in a solid substance.
[0015] As used herein, the term "agriculturally acceptable adjuvant" refers to a substance that enhances the performance of an active agent in a composition for treating plants and/or plant parts. In certain compositions, an active agent can comprise a monoculture or a coculture of Methylobacterium.
[0016] As used herein, the term "agriculturally acceptable excipient" refers to an essentially inert substance that can be used as a diluent and/or carrier for an active agent in a composition for treating plants and/or parts of plant. In certain compositions, an active agent can comprise a monoculture or a coculture of Methylobacterium.
[0017] As used herein, the term "Methylobacterium" refers to bacteria that are facultative methylotrophic bacteria of the genus Methylobacterium. The term Methylobacterium, as used herein, therefore, does not encompass species in the genera Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, Methylosinus, Methylocystis, Methylosphaera, Methylocaldum and Methylocella, which are mandatory methanotrophic.
As used herein, the term "Methylobacterium coculture" refers to a culture of Methylobacterium comprising at least two strains of Methylobacterium or at least two species of Methylobacterium.
[0019] As used herein, the term "contamination microorganism" refers to microorganisms in a culture, fermentation broth, fermentation broth product or composition that were not identified prior to introduction into the culture, in the fermentation broth, in the fermentation broth product or in the composition.
[0020] As used herein, the term "derivatives thereof", when used in the context of a Methylobacterium isolate, refers to any strain that is obtained from the Methylobacterium isolate. Derivatives of a Methylobacterium isolate include , but without limitation, strain variants obtained by selection, strain variants selected by mutagenesis and selection, and a genetically transformed strain obtained from the Methylobacterium isolate.
[0021] As used herein, the term "emulsion" refers to a colloidal mixture of two immiscible liquids, where one liquid is the continuous phase and the other liquid is the dispersed phase. In certain embodiments, the continuous phase is an aqueous liquid and the dispersed phase is a liquid that is not miscible, or is partially miscible, in the aqueous liquid.
[0022] As used herein, the term "essentially free of contaminating microorganisms" refers to a culture, a fermentation broth, a fermentation product or a composition in which at least about 95% of the micro- organisms present by amount or type in the culture, fermentation broth, fermentation product or composition are the desired Methylobacterium or other desired microorganisms of predetermined identity.
[0023] As used herein, the term "inanimate solid substance" refers to a substance that is insoluble or partially soluble in water or aqueous solutions and that is both non-living and is not a part of an organism still alive to from which it was derived.
[0024] As used herein, the term "Methylobacterium monoculture" refers to a Methylobacterium culture that consists of a single Methylobacterium strain.
As used herein, the term "peptide" refers to any polypeptide of 50 amino acid residues or less.
[0026] As used herein, the term "lettuce" refers to plants of the species Lactuca. Plants of Lactuca species include, but are not limited to, Lactuca biennis, Lactuca canadensis, Lactuca floridana, Lactuca graminifolia, Lactuca hirsuta, Lactuca indica, Lactuca ludoviciana, Lactuca saligna, Lactuca sativa, Lactuca serriola, Lactuca terrae-novae, Lactuca virosae, species Lactuca X morssii.
As used herein, the term "protein" refers to any polypeptide that has 51 or more amino acid residues.
[0028] As used herein, a "pesticide" refers to an agent that is an insecticide, fungicide, nematicide, bactericide, or any combination thereof.
[0029] As used herein, the term "bacteriostatic agent" refers to agents that inhibit the growth of bacteria but do not kill the bacteria.
[0030] As used herein, the term "pesticide does not substantially inhibit the cultivation of a said Methylobacterium" refers to any pesticide which, when provided in a composition comprising a fermentation product comprising a solid substance, wherein a monoculture or a coculture of Methylobacterium is adhered thereto, results in no more than 50% inhibition of Methylobacterium cultivation when the composition is applied to a plant or plant part compared to a composition lacking the pesticide. In certain embodiments, the pesticide results in no more than a 40%, 20%, 10%, 5% or 1% inhibition of Methylobacterium cultivation when the composition is applied to a plant or plant part compared to a composition. devoid of the pesticide.
[0031] As used herein, the term "PPFM bacteria" refers, without limitation, to bacterial species in the genus Methylobacterium other than M. nodularis.
[0032] As used herein, the term "solid substance" refers to a substance that is insoluble or partially soluble in water or aqueous solutions.
[0033] As used herein, the term "solid phase that can be suspended therein" refers to a solid substance that can be distributed throughout a liquid by agitation.
[0034] As used herein, the term "non-regenerable" refers to both a plant part and a processed plant product that cannot be regenerated into a complete plant.
[0035] As used herein, the term "substantially all of the solid phase is suspended in the liquid phase" refers to media in which at least 95%, 98% or 99% of the solid substance(s) comprises (m) the solid phase are distributed throughout the liquid by stirring.
[0036] As used herein, the expression "substantially all of the solid phase is not suspended in the liquid phase" refers to media in which less than 5%, 2% or 1% of the solid is in a particulate form that is distributed by all means by agitation.
[0037] As used herein, the term "resident microorganism" refers to resident bacteria, fungi or yeast.
[0038] As used herein, the term "substance that promotes the cultivation of microorganisms residing in a plant or seed" refers to a source of carbon, a source of nitrogen, a source of phosphorus and combinations thereof.
[0039] To the extent that any of the foregoing definitions are inconsistent with the definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the foregoing definition will be used in this document.COMPOSITIONS CONTAINING METHYLOBACTERIUM EXHAUSTED FROM SUBSTANCES THAT PROMOTE THE CULTIVATION OF BACTERIA RESIDENT IN A PLANT OR A SEED, METHODS OF USE AND PRODUCTION METHODS
[0040] Compositions comprising Methylobacterium which are depleted of substances that promote the cultivation of bacteria residing in a plant or a seed, methods of using the compositions to improve lettuce production and methods of producing the compositions are provided herein. In certain embodiments of any of the aforementioned compositions, the composition comprises a solid substance in which a monoculture or coculture of Methylobacterium is adhered thereto. In certain embodiments where the Methylobacterium is adhered to a solid substance, the composition comprises a colloid formed by the solid substance to which a monoculture or coculture of Methylobacterium is adhered and a liquid. In certain modalities, colloid is a gel. In certain embodiments of certain of the aforementioned compositions, the composition is an emulsion that does not contain a solid substance.
Compositions comprising a solid substance with adherent Methylobacterium cultivated thereon are provided. In certain embodiments, the adherent Methylobacterium can be at a titer of at least about 5 x 108 CFU/g to at least about 5 x 1013 CFU/g or about 1 x 1014 CFU/g and the composition is depleted of substances that promote the cultivation of microorganisms residing in a plant or seed.
[0042] In certain modalities, the compositions containing Methylobacterium provided or used in this document are depleted of substances that promote the cultivation of resident microorganisms when one or more of these substances are absent or are essentially absent. In certain modalities, the composition is depleted of substances that promote the cultivation of resident microorganisms when these substances are present at a percentage of no more than about 5%, 2%, 1%, 0.5%, 0.2 % or 0.1% of the total mass, the total mass/volume or the total volume of the composition. In certain modalities, the substance that promotes the cultivation of microorganisms residing in a plant or seed is selected from the group consisting of a carbon source, a nitrogen source, a phosphorus source, a sulfur source, a source of magnesium and combinations thereof. Carbon sources include, but are not limited to, alcohols, monosaccharides, disaccharides, polysaccharides, lipids, fatty acids, and the like. Alcohols that are depleted include, but are not limited to, methanol, ethanol, glycerol, and the like. Nitrogen sources include, but are not limited to, ammonia and various compounds that contain amino groups that can be metabolized by microorganisms. In certain embodiments, the substance that is depleted is a source of two or more of a carbon source, a nitrogen source, a phosphorus source, a sulfur source, and a magnesium source. For example, a composition that is depleted of amino acids or peptides, and is devoid of other sources of carbon or nitrogen, is depleted of both a source of carbon and nitrogen. In certain embodiments, the composition comprises an agriculturally acceptable adjuvant and/or excipient.
Plant or seed resident microorganisms include, but are not limited to bacteria, fungi and yeast. Substances that promote the cultivation of such microorganisms can be identified by methods that include, but are not limited to, evaluating the surface of the plant or seed for the amount or number of microorganisms present prior to exposure of the plant or seed to the substance. (or a composition containing the substance), expose the evaluated plant or seed to the substance or composition in parallel with a control composition devoid of the substance, and then re-evaluate the surface of the plant or seed for the amount or number of micro -organisms present after a suitable period of time and under suitable temperature conditions to allow the cultivation of resident microorganisms. Assessments for numbers of microorganisms include, but are not limited to, determinations of colony forming units by an amount of plant or seed exposed to the substance and control.
[0044] Without being limited by theory, it is believed that compositions containing Methylobacterium provided or used herein that are depleted of substances that promote the cultivation of resident microorganisms may result in superior results compared to other compositions containing such substances when applied to plants, plant parts or seeds. Such superior results are believed to include, but are not limited to, improved plant yield, pathogen resistance, insect resistance, fruit ripening, and the like. Although not limited by theory, it is believed that compositions containing Methylobacterium that are depleted of substances that promote the cultivation of resident microorganisms allow for a more effective and/or extensive colonization of the plant, part of it or seed, depending on competition by one or more of space or nutrients by the resident microorganisms is reduced.
[0045] Also provided herein are methods for improving a lettuce production comprising applying any of the aforementioned compositions of Methylobacterium provided herein to a lettuce plant, lettuce plant part or lettuce seed, and, optionally, cultivating the plant and/or harvesting leaves or seed from the plant or a plant grown from the seed. In certain embodiments, the composition coats or partially coats the lettuce plant, plant part or seed. The lettuce plant or the treated plant grown from seed exhibits an increased rate of root cultivation, an increased rate of leaf cultivation, an increased seed production yield, a decreased cycle time (from seed planting to seed planting). seed yield) and/or an increased total biomass when compared to an untreated control lettuce plant or a control lettuce plant grown from untreated seed, thus obtaining an improved lettuce yield. In certain modalities, the application of the composition allows an increase of at least about 5%, 10%, 15%, 20%, 30% or 40% in the root cultivation rate, leaf cultivation rate, seed production and /or increased total biomass in the lettuce plant, lettuce plant part or a lettuce plant derived therefrom, compared to an untreated control lettuce plant or a control lettuce plant grown from an unseeded seed. treated. In certain modalities, the application of the composition allows an increase of about 5% or 10% up to about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60% or 70 % in root cultivation rate, leaf cultivation rate, seed production and/or increased total biomass in the plant, plant part or a plant derived therefrom compared to an untreated control lettuce plant or a control lettuce plant grown from an untreated seed. In certain modalities, the application of the composition allows a reduction of at least about 5%, 10%, 15%, 20%, 30% or 40% in cycle time in the treated lettuce plant or in a lettuce plant grown at from a treated seed, compared to the untreated control lettuce plant or control lettuce plant grown from an untreated seed. In certain modalities, the application of the composition allows a reduction of about 5% or 10% to about 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% in cycle time in the treated lettuce plant or on a lettuce plant grown from a treated seed as compared to an untreated control lettuce plant or a control lettuce plant grown from an untreated seed. In certain embodiments, the lettuce plant part is a leaf, stem, flower, root, tuber, or seed. In certain embodiments, the method further comprises the steps of cultivating the plant and/or the step of harvesting at least one plant part selected from the group consisting of a leaf, a stem, a flower, a root, a tuber or a seed of the lettuce plant or plant part. In certain embodiments of any of the aforementioned methods, the methods further comprise obtaining a processed food or feed composition from the plant or plant part. In certain embodiments, the processed food composition comprises chopped or chopped lettuce leaves.
[0046] Methods are also provided for producing a lettuce plant or a plant seed treatment composition that comprises Methylobacterium and is depleted of substances that promote the cultivation of bacteria residing on a plant or seed that is provided herein. Such method comprises (i) cultivating a monoculture or coculture of Methylobacterium in media comprising an aqueous phase, a liquid phase and a solid phase or an emulsion, thereby obtaining media containing Methylobacterium; (ii) separating the Methylobacterium from at least one other portion of the Methylobacterium containing media; and (iii) reconstitute Methylobacterium in a matrix devoid of substances that promote the cultivation of bacteria residing in a plant or seed. In certain modalities, the separation step is carried out by centrifuging, filtering or settling the media containing Methylobacterium and removing excess liquid or emulsion from them. In certain embodiments, the substance that promotes the cultivation of bacteria residing in a plant or seed is selected from the group consisting of a carbon source, a nitrogen source, a phosphorus source, and combinations thereof. In certain embodiments, the matrix is a liquid, an emulsion, or one or more solids, and comprises an agriculturally acceptable adjuvant and/or excipient. Also in certain embodiments, Methylobacterium is cultivated in media comprising a liquid phase and a solid substance with adherent Methylobacterium cultivated thereon. The solid substance is separated from the liquid phase of the media containing Methylobacterium and the solid substance with adherent Methylobacterium grown on it is reconstituted in the aforementioned matrix. In certain embodiments of the methods, the Methylobacterium species is selected from the group consisting of aminovorans, M. extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, M. rhodesianum, M. nodularis, M. phyllosphaerae, M. thiocyanatum and oryzae. In certain embodiments of methods, the Methylobacterium is not M. radiotolerans or M. oryzae. In certain modalities of methods, Methylobacterium is adhered to a solid substance. In certain embodiments of the methods, Methylobacterium is adhered to the solid substance and is combined with a liquid to form a composition that is a colloid. In certain modalities of methods, the colloid is a gel. In certain modalities of methods, the Methylobacterium adhered to the solid substance is provided by cultivating the Methylobacterium in the presence of the solid substance. In certain embodiments of the methods, the composition comprises an emulsion. In certain embodiments of the methods, Methylobacterium is provided by cultivating Methylobacterium in an emulsion.
[0047] In methods in which Methylobacterium is cultivated in biphasic media that comprise a liquid phase and a solid substance, a significant increase in the resulting yield of Methylobacterium was found in relation to methods in which Methylobacterium is cultivated only in liquid media. In certain embodiments, the methods may comprise culturing the Methylobacterium in liquid media with a solid particulate substance that can be suspended in the liquid by agitation under conditions that allow for the cultivation of Methylobacterium. In certain embodiments where solid particulate substances are used, at least substantially all of the solid phase can thereby be suspended in the liquid phase upon stirring. Such solid particulate substances can comprise materials that are about 1 millimeter or less in length or diameter. In certain embodiments, the degree of agitation is sufficient to allow for uniform distribution of the solid particulate substance in the liquid phase and/or optimal levels of culture aeration. However, in other embodiments provided herein, at least substantially all of the solid phase is not suspended in the liquid phase, or portions of the solid phase are suspended in the liquid phase and portions of the solid phase are not suspended in the liquid phase. Solid non-particulate substances can be used in certain biphasic media where the solid phase is not suspended in the liquid phase. Such solid non-particulate substances include, but are not limited to, materials that are greater than about 1 millimeter in length or diameter. Such solid particulate and non-particulate substances also include, but are not limited to, materials that are porous, fibrous, or otherwise configured to allow for increased surface areas for adherent cultivation of Methylobacterium. Biphasic media in which portions of the solid phase are suspended in the liquid phase and portions of the solid phase are not suspended in the liquid phase may comprise a mixture of solid particulate and non-particulate substances. Such solid particulate and non-particulate substances used in any of the aforementioned biphasic media also include, but are not limited to, materials that are porous, fibrous, or otherwise configured to allow for increased surface areas for adherent cultivation of Methylobacterium. In certain embodiments, the media comprises a colloid formed from a solid and a liquid phase. A colloid comprising a solid and a liquid can be preformed and added to liquid media or can be formed in media containing a solid and a liquid. Colloids comprising a solid and a liquid can be formed by subjecting certain solid substances to a chemical and/or thermal change. In certain modalities, colloid is a gel. In certain embodiments, the liquid phase of the media is an emulsion. In certain embodiments, the emulsion comprises an aqueous liquid and a liquid that is not miscible, or is only partially miscible, in the aqueous liquid. Liquids that are not miscible, or are only partially miscible, with water include, but are not limited to, any of the following: (1) liquids that have a miscibility in water that is equal to or less than that of pentanol, hexanol or heptanol at 25°C; (2) liquids comprising an alcohol, an aldehyde, a ketone, a fatty acid, a phospholipid or any combination thereof; (3) alcohols selected from the group consisting of aliphatic alcohols containing at least 5 carbons and sterols; (4) an animal oil, a microbial oil, a synthetic oil, a vegetable oil, or a combination thereof; and/or, (5) a vegetable oil is selected from the group consisting of corn, soybean, cotton, peanut, sunflower, olive, flax, coconut, palm, rapeseed, sesame seed, safflower and combinations thereof. In certain embodiments, the immiscible or partially immiscible liquid can comprise at least about 0.02% to about 20% of the liquid phase by mass. In certain embodiments, the methods may comprise obtaining biphasic culture media comprising liquid, solid and Methylobacterium, and incubating the culture under conditions that allow for the cultivation of Methylobacterium. Biphasic culture media comprising liquid, solid and Methylobacterium may be obtained by a variety of methods including, but not limited to, any of: (a) inoculating biphasic media comprising liquid and solid substance with Methylobacterium ; (b) inoculate the solid substance with Methylobacterium and then introduce the solid substance comprising the Methylobacterium into the liquid media; (c) inoculate the solid substance with Methylobacterium, incubate the Methylobacterium in the solid substance, and then introduce the solid substance comprising the Methylobacterium into the liquid media; or (d) any combination of (a), (b) or (c). Methods and compositions for cultivating Methylobacterium in biphasic media comprising a liquid and a solid are disclosed in Patent Application assigned to the same assignee in US 13/907161, filed March 31, 2013, which is incorporated herein by way of reference in its entirety, and in the International Patent Application assigned to the same assignee in PCT/US13/43722, filed on May 31, 2013, which is incorporated herein by way of reference in its entirety.
[0048] In methods in which Methylobacterium is cultivated in media that comprise an emulsion, a significant increase in the resulting yield of Methylobacterium was also found in relation to methods in which Methylobacterium is cultivated only in liquid media. In certain embodiments, the methods for producing the compositions provided herein may comprise culturing the Methylobacterium agent in an emulsion under conditions that allow for the cultivation of Methylobacterium. Media comprising the emulsion and Methylobacterium can be obtained by a variety of methods including, but not limited to, any of: (a) inoculating media comprising the emulsion with Methylobacterium; (b) inoculate the aqueous liquid with the Methylobacterium, introduce the non-aqueous liquid and mix to form an emulsion; (c) inoculate the aqueous liquid with the Methylobacterium, introduce the non-aqueous liquid and mix to form an emulsion; or (d) any combination of (a), (b) or (c). In certain embodiments, the emulsion comprises an aqueous liquid and a liquid that is not miscible, or is only partially miscible, in the aqueous liquid. Non-aqueous liquids that are not miscible, or are only partially miscible, with water include, but are not limited to, any of the following: (1) liquids that have a miscibility in water that is equal to or less than that of n-pentanol , n-hexanol or n-heptanol at 25°C; (2) liquids comprising an alcohol, an aldehyde, a ketone, a fatty acid, a phospholipid or any combination thereof; (3) alcohols that are selected from the group consisting of aliphatic alcohols that contain at least 5, 6 or 7 carbons and sterols; (4) an animal oil, a microbial oil, a synthetic oil, a vegetable oil, or a combination thereof; and/or (5) a vegetable oil that is selected from the group consisting of corn, soybean, cotton, peanut, sunflower, olive, flax, coconut, palm, rapeseed, sesame seed, safflower and combinations thereof. In certain embodiments, the immiscible or partially immiscible non-aqueous liquid can comprise at least about 0.02% to about 20% of the emulsion by mass. In certain embodiments, the immiscible or partially immiscible non-aqueous liquid can comprise at least about any one of about 0.05%, 0.1%, 0.5% or 1% to about 3%, 5%, 10 % or 20% of the emulsion by mass. Methods and compositions for cultivating Methylobacterium in media comprising an emulsion are disclosed in the International Patent Application assigned to the same grantee in PCT/US2014/040218, filed May 30, 2014, which is incorporated herein by title reference in its entirety.
In certain embodiments, the fermentation broth, fermentation broth product or compositions comprising the Methylobacterium species may further comprise one or more introduced microorganisms of predetermined identity other than Methylobacterium. Other microorganisms that can be added include, but are not limited to, microorganisms that are biopesticides or that provide some other benefit when applied to a plant or plant part. Biopesticidal or otherwise beneficial microorganisms thus include, but are not limited to, various Bacillus species, Pseudomonas species, Coniothyrium species, Pantoea species, Streptomyces species, and Trichoderma species. Microbial biopesticides can be a bacterium, a fungus, a virus or a protozoan. Particularly useful biopesticidal microorganisms include various strains of Bacillus subtilis, Bacillus thuringiensis, Bacillus pumilis, Pseudomonas syringae, Trichoderma harzianum, Trichoderma virens, and Streptomyces lydicus. Other microorganisms that are added can be genetically modified or naturally occurring isolates that are available as pure cultures. In certain embodiments, it is anticipated that the bacterial or fungal microorganism may be provided in the fermentation broth, in the fermentation broth product or in the composition in the form of a spore.
[0050] In certain embodiments, the liquid culture medium is prepared from inexpensive and readily available components, which include, but are not limited to, inorganic salts such as potassium phosphate, magnesium sulfate and the like, carbon sources such as such as glycerol, methanol, glutamic acid, aspartic acid, succinic acid and the like, and amino acid mixtures such as peptone, tryptone and the like. Exemplary liquid media that can be used include, but are not limited to, ammonium mineral salts (AMS) medium (Whittenbury et al, 1970), Vogel-Bonner minimal culture medium (VB) (Vogel and Bonner, 1956) and LB broth ("Luria-Bertan broth").
[0051] In general, the solid substance used in the methods and compositions that enable the effective cultivation of Methylobacterium can be any suitable solid substance that is insoluble or only partially soluble in water or aqueous solutions. Such suitable solid substances are also non-bactericidal or non-bacteriostatic with respect to Methylobacterium when solid substances are provided in liquid culture media. In certain embodiments, such suitable solid substances are also solid substances that are readily obtained in sterile form or made sterile. The solid substances used herein may be sterilized by any method that makes the removal of contaminating microorganisms possible and thus include, but are not limited to, methods such as autoclaving, irradiation, chemical treatment, and any combination thereof. These solid substances include natural substances of animal, vegetable, microbial, fungal or mineral origin, man-made substances, or combinations of natural and man-made substances. In certain embodiments, solid substances are solid inanimate substances. Inanimate solid substances of animal, vegetable, microbial or fungal origin can be obtained from animals, plants, microbes or fungi that are non-viable (ie, no longer alive) or that have been rendered non-viable. Diatom shells are thus solid inanimate substances when previously associated diatom algae have been removed or otherwise rendered unviable. Since diatom shells are solid inanimate substances, they are not considered as photosynthetic organisms or photosynthetic microorganisms. In certain embodiments, solid substances include, but are not limited to, sand, silt, earth, clay, ash, coal, diatomaceous earth and other similar minerals, ground glass or glass spheres, ground ceramic materials, ceramic spheres, bentonite, kaolin, talc, perlite, mica, vermiculite, silicas, quartz powder, montmorillonite and combinations thereof. In certain embodiments, the solid substance can be a polymer or polymeric spheres. Polymers that can be used as a solid substance include, but are not limited to, various polysaccharides, such as cellulosic polymers and chitinous polymers, which are insoluble or only partially soluble in water or aqueous solutions, agar (i.e., galactants) and combinations of the same. In certain embodiments, the solid substance can be an insoluble or only partially soluble salt crystal. Salt crystals that can be used include, but are not limited to, insoluble or only partially soluble carbonates, chromates, sulfites, phosphates, hydroxides, oxides and sulfides. In certain embodiments, the solid substance can be a microbial cell, fungal cell, microbial spore, or fungal spore. In certain embodiments, the solid substance can be a microbial cell or a microbial spore, wherein the microbial cell or microbial spore is not a photosynthetic microorganism. In certain embodiments, the microbial cell or microbial spore is not a photosynthetic microorganism, wherein the photosynthetic microorganism is selected from the group consisting of algae, cyanobacteria, diatoms, Botryococcus braunii, Chlorella, Dunaliella tertiolecta, Gracilaria, Pleurochry carterae, Sargassum and Ulva. In still other embodiments, the solid substance can be a microbial cell, a fungal cell, a microbial spore, or an inactivated (i.e., non-viable) fungal spore. In still other embodiments, the solid substance can be a microbial cell, a fungal cell, a microbial spore, or a quiescent (i.e., viable, but not actively dividing) fungal spore. In still other embodiments, the solid substance can be cellular fragments of microbial origin. In still other embodiments, the solid substance can be a particulate matter from any part of a plant. Plant parts that can be used to obtain the solid substance include, but are not limited to, ears, bark, hooves, leaves, roots, flowers, stems, tree bark, seeds and combinations thereof. Products obtained from processed plant parts include, but are not limited to, bagasse, wheat bran, soybeans, crushed seed cake, stubble and the like can also be used. Such plant parts, processed plants and/or processed plant parts can be crushed to obtain the solid material in a particulate form that can be used. In certain embodiments, wood or a wood product that includes, but is not limited to, wood pulp, sawdust, chips and the like can be used. In certain embodiments, the solid substance can be a particulate matter from an animal(s), which includes, but is not limited to, bone meal, gelatin, ground or powdered shells, fur, ground leather, and the like.
[0052] In certain embodiments, the solid substance is provided in a particulate form that enables the distribution of the solid substance in the culture media. In certain embodiments, the solid substance comprises a particle of about 2 microns to about 1000 microns in average length or average diameter. In certain embodiments, the solid substance comprises a particle of about 1 micron to about 1000 microns in average length or average diameter. In certain embodiments, the solid substance is a particle of about 1, 2, 4, 10, 20 or 40 microns to any one of about 100, 200, 500, 750 or 1000 microns in average length or average diameter. Desirable characteristics of particles used in the methods and compositions provided herein include adequate wettability so that the particles can be suspended by all means upon agitation.
[0053] In certain embodiments, the solid substance is supplied in the media as a colloid in which the continuous phase is a liquid and the dispersed phase is the solid. Suitable solids that can be used to form colloids in liquid media used to cultivate Methylobacterium include, but are not limited to, various solids that are termed hydrocolloids. Such hydrocolloids used in the media, methods and compositions provided herein may be hydrophilic polymers of plant, animal, microbial or synthetic origin. The hydrocolloid polymers used in the methods can contain many hydroxyl groups and/or can be polyelectrolytes. Hydrocolloid polymers used in the compositions and methods provided herein include, but are not limited to, agar, alginate, arabinoxylan, carrageenan, carboxymethylcellulose, cellulose, curdlan, gelatin, gellan, β-glucan, guar gum, gum arabic, gum locust bean, pectin, starch, xanthan gum and mixtures thereof. In certain embodiments, the colloid used in the media, methods and compositions provided herein can comprise a hydrocolloid polymer and one or more proteins.
[0054] In certain embodiments, the solid substance can be a solid substance that enables the adherent cultivation of Methylobacterium in the solid substance. Methylobacterium that is adhered to a solid substance is a Methylobacterium that cannot be substantially removed by simply washing the solid substance with the adherent Methylobacterium with culture media, while the non-adherent Methylobacterium can be substantially removed by washing the solid substance with liquid culture media. In this context, "substantially removed" means that at least about 30%, 40%, 50%>, 60%>, 70%> or 80%> of the Methylobacterium present is removed when the solid substance is washed with three volumes of media of liquid cultivation. Such washing can be accomplished by a variety of methods including, but not limited to, decanting the liquid from a washed solid phase or passing liquid through a solid phase in a filter that allows flow through of bacteria in the liquid. In certain modalities, the adherent Methylobacterium that is associated with the solid may include a Methylobacterium that is directly attached to the solid and/or a Methylobacterium that is indirectly attached to the solid substance. Methylobacterium that is indirectly fixed to the solid substance includes, but is not limited to, Methylobacterium that is fixed to another Methylobacterium or to another microorganism that is fixed to the solid substance, Methylobacterium that is fixed to the solid substance being fixed to another substance that is fixed to solid substance and the like. In certain modalities, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5% or 99.9 % of the Methylobacterium in the fermentation broth, fermentation broth product or compositions is a Methylobacterium that is adhered to the solid substance. In certain embodiments, the adherent Methylobacterium may be present on the surface of the solid substance in the fermentation broth, fermentation broth product or composition at a density of at least about 1 Methylobacterium/20 square micrometers, of at least about 1 Methylobacterium/ 10 square micrometers of at least about 1 Methylobacterium/10 square micrometers of at least about 1 Methylobacterium/ 5 square micrometers of at least about 1 Methylobacterium/2 square micrometers or of at least about 1 Methylobacterium/ square micrometer. In certain embodiments, adherent Methylobacterium may be present on the surface of the solid substance in the fermentation broth, fermentation broth product or composition at a density of at least about 1 Methylobacterium/20 square micrometers to about 1 Methylobacterium/micrometer square, from at least about 1 Methylobacterium / 10 square micrometers to about 1 Methylobacterium / square micrometer, from at least about 1 Methylobacterium/10 square micrometers to about 1 Methylobacterium / square micrometer, from at least about 1 Methylobacterium / 5 square micrometers to about 1 Methylobacterium/ square micrometer or at least about 1 Methylobacterium/2 square micrometers to about 1 Methylobacterium / square micrometer. In certain embodiments, the adherent Methylobacterium may be present on the surface of the solid substance in the fermentation broth, fermentation broth product or composition at a density of at least about 1 Methylobacterium/20 square micrometers to about 1 Methylobacterium/2 square micrometers, from at least about 1 Methylobacterium/10 square micrometers to about 1 Methylobacterium/2 square micrometers, from at least about 1 Methylobacterium/10 square micrometers to about 1 Methylobacterium/2 square micrometers or at least about 1 Methylobacterium/5 square micrometers to about 1 Methylobacterium/2 micrometer square. The biphasic fermentation broths provided herein may comprise a liquid phase which contains non-adherent Methylobacterium. In certain embodiments, liquid phase non-adherent Methylobacterium titers may be less than about 100,000, 10,000, or 1,000 CFU/ml.
[0055] The biphasic culture methods provided can yield Methylobacterium fermentation broths at a titer greater than about 5 x 108 colony forming units per millimeter, at a titer greater than about 1 x 109 colony forming units per millimeter, at a titer greater than about 1 x 1010 colony forming units per millimeter, at a titer of at least about 3 x 1010 colony forming units per millimeter. In certain embodiments, the fermentation broths provided herein may comprise Methylobacterium at a titer of at least about 5 x 108 colony forming units per millimeter to at least about 3 x 1010 colony forming units per millimeter, at minus about 5 x 108 colony forming units per millimeter to at least about 4 x 1010 colony forming units per millimeter or at least about 5 x 108 colony forming units per millimeter to at least about 6 x 1010 colony formation units per millimeter. In certain embodiments, the fermentation broths provided herein may comprise Methylobacterium at a titer of at least about 1 x 109 colony forming units per millimeter to at least about 3 x 1010 colony forming units per millimeter, at minus about 1 x 109 colony forming units per millimeter to at least about 4 x 1010 colony forming units per millimeter or at least about 1 x 109 colony forming units per millimeter to at least about 6 x 1010 colony formation units per millimeter. In certain embodiments, the fermentation broths provided herein will comprise Methylobacterium at a titer of at least about 1 x 1010 colony forming units per millimeter to at least about 3 x 1010 colony forming units per millimeter, at minus about 1 x 1010 colony forming units per millimeter to at least about 4 x 1010 colony forming units per millimeter, or at least about 1 x 1010 colony forming units per millimeter to at least about 6 x 1010 colony forming units per millimeter. In certain embodiments, the fermentation broths provided herein will comprise Methylobacterium at a titer of at least about 3 x 1010 colony forming units per millimeter to at least about 4 x 1010 colony forming units per millimeter or at least minus about 3 x 1010 colony forming units per millimeter to at least about 6 x 1010 colony forming units per millimeter.
[0056] Solid substances with adherent Methylobacterium can be obtained as fermentation products that can be used to produce various compositions useful to treat plants or plant parts to improve plant yield, plant insect resistance, disease resistance plant fungus and/or to improve lettuce production. In certain embodiments, the composition comprises Methylobacterium and is depleted of substances that promote the cultivation of resident bacteria. Compositions provided herein which comprise Methylobacterium, solid substances with Methylobacterium grown therein or which comprise emulsions with Methylobacterium grown therein can be used to treat plants or plant parts. Plants, plant parts and, in particular, plant seeds which have been at least partially coated or coated with the fermentation broth products or compositions comprising Methylobacterium are thus provided. Processed plant products are also provided which contain the fermentation broth products or compositions with adherent Methylobacterium or Methylobacterium. Solid substances with adherent Methylobacterium can be used to produce various compositions that are particularly useful for treating plant seeds. Seeds which have been at least partially coated with the fermentation broth products or compositions are thus provided. Processed seed products are also provided which include, but are not limited to, bran, flour, feed and flakes containing the fermentation broth products or compositions provided herein. In certain embodiments, the processed plant product will be non-regenerable (that is, it will not have the ability to develop into a plant). In certain embodiments, the solid substance used in the fermentation product or composition which at least partially coats the plant, plant part or plant seed or which is contained in the processed plant, plant part or seed product comprises a solid substance and associated or adherent Methylobacterium that can be readily identified by comparing a treated and untreated plant, plant part, plant seed or processed product. Partial coating of a plant, a plant part or a seed includes, but is not limited to, coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or about 99.5% of the surface area of the plant, plant part or plant seed.
[0057] Methods for preparing a plant or plant seed treatment composition that comprises Methylobacterium and is depleted of substances that promote the cultivation of bacteria residing in a plant or seed are also provided herein. Such methods may comprise (i) cultivating a monoculture or coculture of Methylobacterium in media comprising: (a) an aqueous phase; (b) a liquid phase and a solid phase; or (c) an emulsion, thereby obtaining media containing Methylobacterium; (ii) separating the Methylobacterium from at least one other portion of the Methylobacterium containing media; and (iii) reconstitute Methylobacterium in a matrix devoid of substances that promote the cultivation of bacteria residing in a plant or seed. In certain modalities, the separation step is carried out by centrifuging, filtering or settling the media containing Methylobacterium and removing excess liquid or emulsion from them. In certain modalities where Methylobacterium is grown in the presence of a solid substance, separation will provide a fraction that contains Methylobacterium with culture adherent to the solid substance and some non-adherent Methylobacterium that can be reconstituted in the matrix. In certain modalities, the substance that promotes the cultivation of bacteria residing in a plant or seed is selected from the group consisting of a carbon source, a nitrogen source, a phosphorus source, a sulfur source, a source of magnesium and combinations thereof. In certain embodiments, the matrix is a liquid, an emulsion, or one or more solids, and comprises an agriculturally acceptable adjuvant and/or excipient. In certain modalities; Methylobacterium is grown in media comprising a liquid phase and a solid substance with adherent Methylobacterium grown on top of it. The solid substance is separated from the liquid phase of the media containing Methylobacterium and the solid substance with adherent Methylobacterium grown on it is reconstituted in the aforementioned matrix. In certain embodiments, the matrix can be a liquid that includes, but is not limited to, an aqueous and water buffer depleted of substances that promote the cultivation of bacteria residing in a plant or seed, or an aqueous solution depleted of substances that promote the cultivation of bacteria residing in a plant or seed.
[0058] In certain embodiments, the Methylobacterium species that enhances lettuce production can be identified by testing a newly isolated candidate Methylobacterium species for the presence of polymorphic nucleic acid sequences that are present in an exemplary Methylobacterium species provided herein that improves lettuce production and which are absent from the Methylobacterium species provided herein that do not improve lettuce production. In certain embodiments, polymorphic nucleic acid sequences that are present in the identified Methylobacterium species that enhance lettuce production are also present in one or more of the exemplary Methylobacterium species isolates NLS0020, NLS0066, NLS0017, NLS0065, NLS0089, NLS0042, and NLS0068 provided herein that enhance lettuce production, but are absent from one or more of the Methylobacterium species isolates provided herein that do not improve lettuce production. In certain embodiments, polymorphic nucleic acid sequences that are present in the identified Methylobacterium species that enhance lettuce production are also present in one or more of the exemplary Methylobacterium species isolates NLS0020, NLS0066, NLS0017, NLS0065 and/or NLS0089 provided herein that improve lettuce production when applied as seed treatments, but are absent from one or more of the Methylobacterium species isolates provided herein that do not improve lettuce production when applied as seed treatments. In certain embodiments, polymorphic nucleic acid sequences that are present in the identified Methylobacterium species that enhance lettuce production are also present in one or more of the exemplary Methylobacterium species isolates NLS0020, NLS0017, NLS0042, and NLS0068 provided herein that enhance the lettuce production when applied as foliar treatments, but are absent from one or more of the Methylobacterium species isolates provided herein that do not improve lettuce production when applied as foliar treatments. In certain embodiments, polymorphic nucleic acid sequences that are present in the identified Methylobacterium species that enhance lettuce production are also present in two or more of the exemplary Methylobacterium species isolates NLS0020, NLS0066, NLS0017, NLS0065, NLS0089, NLS0042, and NLS0068 provided herein that improve lettuce production, but are absent in two or more of the Methylobacterium species isolates provided herein that do not improve lettuce production. In certain embodiments, polymorphic nucleic acid sequences that are present in the identified Methylobacterium species that enhance lettuce production are also present in one or more of the exemplary Methylobacterium species isolates NLS0020, NLS0066, NLS0017, NLS0065, NLS0089, NLS0042 and/or NLS0068 provided herein that improve lettuce production, but are absent from all Methylobacterium species isolates provided herein that do not improve lettuce production. In certain embodiments, polymorphic nucleic acid sequences present in the identified Methylobacterium species that enhance lettuce production are present in all exemplary Methylobacterium species isolates NLS0020, NLS0066, NLS0017, NLS0065, NLS0089, NLS0042, and NLS0068 provided herein that enhance the lettuce production, but are absent in all Methylobacterium species isolates provided herein that do not improve lettuce production. Such nucleic acid polymorphisms that occur in the Methylobacterium species that enhance lettuce production may include, but are not limited to, single nucleotide polymorphisms, RFLP, AFLP and/or other DNA variations such as repetitive sequences, insertion sequences, transposons and genomic islands that occur as a result of insertions, deletions, and substitutions (Indels) in the bacterial genome that include both chromosomal DNA and any extrachromosomal nucleic acid elements that may be present in the Methylobacterium species that enhance lettuce production. Such extrachromosomal nucleic acid elements include, but are not limited to, plasmids, bacteriophage DNA or RNA, and the like. Methods used to identify such nucleotide polymorphisms include, but are not limited to, single base extension (SBE) techniques, allele-specific hybridization (ASH), real-time PCR detection (ie, TaqMan™; US Patent 5,804,375 ; 5,538,848; 5,487,972; and 5,210,015, which are each incorporated herein by reference in their entirety), combinations of ASH and RT-PCR (KASP™ detection systems, LGC Genomics , Middlesex, UK) and deep sequencing techniques (US Patent Application No. 20120264632, incorporated herein by reference in its entirety.
[0059] Also provided herein are compositions, methods for making the compositions, and methods for using the compositions to improve improved lettuce yield, wherein the compositions or methods comprise or use any of the Methylobacterium a species isolates a provided in Table 1 below or derivatives of the isolates. In certain embodiments, such derivatives may include, but are not limited to, variants of the isolates obtained by selection, variants of the isolates selected by mutagenesis and selection, and genetically transformed isolates obtained from the isolates.
[0060] TABLE 1. ISOLATES OF METHYLOBACTERIUM sp.


[0061] 1Deposit number for strain to be deposited with the AGRICULTURAL RESEARCH SERVICE CULTURE COLLECTION (NRRL) of the National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604 USA under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. Subject to 37 CFR § 1.808(b), that all restrictions imposed by the applicant on the public availability of the deposited material will be irrevocably removed upon the grant of any patent under this patent application.
[0062] Patent applications assigned to the same concessionaire that reveal additional specific uses of the Methylobacterium strains in Table 1, such as: (1) increasing corn yield (US 61/911780, filed on 4/12/2013; and Application International claiming the benefit thereof, filed on 4/12/2014); (2) increase soybean yield (US 61/911698, filed 4/12/2013; and International Application claiming the benefit thereof, filed 4/12/2014); (3) increase tomato cultivation (US 61/954390, filed 3/17/2014; and International Application claiming the benefit thereof, filed 4/12/2014); (4) improve fruit ripening (US 61/911577, filed 4/12/2013; and International Application claiming the benefit thereof, filed 4/12/2014); (5) provide resistance to fungal disease (US 62/045950, filed 4/9/2014; US 62/013,464, filed 6/17/2014) and are each incorporated herein by reference in their wholes. Specifically incorporated herein by reference in their entirety are the amino acid and genomic nucleic acid sequences of NLS017 and NLS066, disclosed in the International Application for Compositions And Methods For Improved Tomato Growth, filed on 4/12/2014 and which claims the benefit of US 61/954390, filed on 3/17/2014.
[0063] This document also provides Methylobacterium species that enable the production of improved lettuce, wherein the Methylobacterium species has any one of: (i) at least one gene encoding at least one protein that is orthologous to a protein that has an amino acid sequence of SEQ ID NO: 1-5125; or (ii) at least one gene that encodes at least one protein that is orthologous to a reference protein of Table 7. The Methylobacterium species has at least one gene that is orthologous to a protein that has an amino acid sequence of at least one from SEQ ID NO: 1-5125, or to the corresponding SEQ ID NO of a reference protein of Table 7, when a chromosome and/or any extrachromosomal DNA in which the Methylobacterium species contains a gene encoding a protein having at least 50 %, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or 100% sequence identity throughout the length of the amino acid sequence of at least one of SEQ ID NO: 1-5125. The Methylobacterium species may also have at least two, three, four, six, eight, 10, 15, or 20 genes that encode proteins that are orthologous to proteins that have an amino acid sequence of SEQ ID NO: 1-5125 or that encode proteins which are orthologous to the corresponding SEQ ID NO of a reference protein of Table 7. In certain embodiments, the Methylobacterium species may contain at least one gene that encodes a protein that is orthologous to a reference protein that has the amino acid sequence of 13 , 14, 23, 27, 28, 30, 40, 43, 44, 51, 52, 57, 76, 85, 127, 197, 198, 199, 1094, 1100, 1106, 1114,1116, 1117, 1120, 1180 , 2180, 2190, 2463, 2467, 2468, 2471, 2510, 2515, 2676, 2971, 3357, 3370, 3372, 3394, 3427, 3429, 3430, 3950, 3952, 3968, 3987, 3996, 4004, 4006 and/ or 4067 of Table 7. In certain embodiments, the Methylobacterium species may contain at least one gene that encodes a protein that is orthologous to a reference protein that has the amino acid sequence. SEQ ID NO: 13, 14, 23, 1094, 1100, 1106, 2467, 2468, 3357, 3370 and/or 3968 of Table 7. In certain embodiments, the Methylobacterium species can contain at least one gene encoding a protein. which is orthologous to a reference protein having the amino acid sequence of SEQ ID NO: 1100, 1116, 2471, 2971 and/or 3950 of Table 7. Examples of proteins that are orthologous to SEQ ID NO: 1094 include, but without limitation, the orthologous proteins identified as transcriptional regulators that are provided in Table 7. Examples of proteins that are orthologous to SEQ ID NO: 23 include, but are not limited to, the orthologous proteins identified as transcriptional regulatory XRE family proteins that are provided in Table 7. Examples of proteins that are orthologous to SEQ ID NO: 1100 include, but are not limited to, proteins that have the amino acid sequence of SEQ ID NO: 17, 1110, 2179, 2484 and 3367 that are similar to identified proteins like trans-type proteins carrier ABC. Examples of proteins that are orthologous to SEQ ID NO: 1116 include, but are not limited to, proteins that have the amino acid sequence of SEQ ID NO: 37, 1116, 2182, and 2521 that are similar to proteins identified as MatE multidrug carriers. Examples of proteins that are orthologous to SEQ ID NO: 2471 include, but are not limited to, proteins that have the amino acid sequence of SEQ ID NO: 10, 2471, 3356 and 3958 that are similar to proteins identified as ACR pump pump proteins. arsenite efflux. Examples of proteins that are orthologous to SEQ ID NO: 2971 include, but are not limited to, proteins that have the amino acid sequence of SEQ ID NO: 250, 1309, 2263 and 2971 that are similar to proteins identified as members of the transcriptional regulators of LysR family. In certain embodiments, the Methylobacterium species has at least one gene that is orthologous to a protein that has an amino acid sequence of at least one of SEQ ID NO: 1-5125 or to the corresponding SEQ ID NO of a reference protein of Table 7 , with the exception that the gene is not found in AMI extorquens, M. extorquens PA1 or M. extorquens ME4. Compositions comprising any of the aforementioned Methylobacterium species and an agriculturally acceptable excipient, an agriculturally acceptable adjuvant or a combination thereof are also provided together with lettuce seeds or leaves that are at least partially coated with such compositions, and methods for use such compositions as seed or foliar treatments to improve lettuce production.
A Methylobacterium species can be determined to contain a gene encoding a protein that is orthologous to a protein that has an amino acid sequence of SEQ ID NO: 1-5125 by a variety of different techniques. In certain embodiments, a Methylobacterium species can be determined to contain a gene encoding a protein that is orthologous to a protein that has an amino acid sequence of SEQ ID NO: 1-5125 by assembling a complete electronic genomic sequence comprising sequences of chromosomal and extrachromosomal DNA present in that Methylobacterium species with a computer and associated software, and determining whether any of the open reading frames (ORF) present in that DNA sequence encodes a protein that has the sequence identity in percent above. In such embodiments, ORFs can be identified by performing a six-way translation of the electronically assembled sequence and querying the translation with an amino acid sequence of SEQ ID NO: 1-5125 or with the corresponding SEQ ID NO: of a Table 7 reference protein. In other embodiments, the presence or absence of a given sequence within a Methylobacterium species, an amino acid sequence of SEQ ID NO: 15125 or the corresponding SEQ ID NO: of a Table reference protein 7, can be determined by a nucleic acid analysis or protein analysis technique. Examples of nucleic acid sequences encoding the proteins of SEQ ID NO: 1-5125 include, but are not limited to, SEQ ID NO: 5126-10250, respectively. Such nucleic acid analyzes include, but are not limited to, techniques based on nucleic acid hybridization, polymerase chain reactions, mass spectroscopy, nanopore-based detection, combinations thereof, and the like. Protein analysis techniques include, but are not limited to, immunodetection, mass spectroscopy, combinations thereof, and the like.
[0065] Compositions provided herein that are useful for treating lettuce plants or plant parts that comprise Methylobacterium, and/or are depleted of substances that promote the cultivation of bacteria residing in a plant or seed, contain a solid substance with Adherent Methylobacterium grown thereon, or which comprise emulsions with Methylobacterium grown thereon, may also additionally comprise an agriculturally acceptable adjuvant or an agriculturally acceptable excipient. An agriculturally acceptable adjuvant or agriculturally acceptable excipient is typically an ingredient that does not cause undue phytotoxicity or other adverse effects when exposed to a plant or plant part. In certain embodiments, the solid substance itself can be an agriculturally acceptable adjuvant or an agriculturally acceptable excipient, so long as it is not bactericidal or bacteriostatic to Methylobacterium. In other embodiments, the composition further comprises at least one of an agriculturally acceptable adjuvant or agriculturally acceptable excipient. Any of the aforementioned compositions may also additionally comprise a pesticide. Pesticides used in the composition include, but are not limited to, an insecticide, a fungicide, a nematicide and a bactericide. In certain embodiments, the pesticide used in the composition is a pesticide that does not substantially inhibit the cultivation of Methylobacterium. As Methylobacterium are gram-negative bacteria, suitable bactericides used in the compositions may include, but are not limited to, bactericides that exhibit activity against gram-positive bacteria, but not against gram-negative bacteria. The compositions provided herein may also comprise a bacteriostatic agent that does not substantially inhibit the cultivation of Methylobacterium. Suitable bacteriostatic agents for use in compositions provided herein include, but are not limited to, those that exhibit activity against gram-positive bacteria, but not against gram-negative bacteria. Any of the aforementioned compositions can also be an essentially dry product (i.e., having about 5% or less water content), a mixture of the composition with an emulsion, or a suspension. Any of the compositions provided herein can be used to coat or partially coat a plant, a plant part or a plant seed. Partial coating of a plant, a plant part or a seed includes, but is not limited to, coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or about 99.5% of the surface area of the plant, plant part or plant seed.
Agriculturally acceptable adjuvants used in compositions comprising Methylobacterium include, but are not limited to, components that improve the effectiveness of the product and/or products that improve the ease of application of the product. Adjuvants that improve product effectiveness can include various wetters/dispersers that promote adhesion to the composition in plant parts and dispersion thereof, adhesives that promote adhesion to the plant part, penetrators that can promote contact of the active agent with interior tissues , extenders that increase the active agent's half-life by inhibiting environmental degradation, and wetting agents that increase the density or drying time of sprayed compositions. Wetters/dispersers used in the compositions may include, but are not limited to, nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, organic silicate surfactants and/or acidified surfactants. Adhesives used in the compositions can include, but are not limited to, latex-based substances, terpene/pinolene and pyrrolidone-based substances. Penetrators can include mineral oil, vegetable oil, esterified vegetable oil, organic silicate surfactants and acidified surfactants. Extenders used in the compositions can include, but are not limited to, ammonium sulfate or menthene-based substances. Humectants used in the compositions can include, but are not limited to, glycerol, propylene glycol and diethyl glycol. Adjuvants that improve product ease of application include, but are not limited to, acidifying/buffering agents, defoaming/defoaming agents, compatibility agents, drift reducing agents, paints and water conditioners. Defoamers/defoamers used in the compositions can include, but are not limited to, dimethopolyoxyloxane. Compatibility agents used in the compositions can include, but are not limited to, ammonium sulfate. Bypass reducing agents used in the compositions can include, but are not limited to, polyacrylamides and polysaccharides. Water conditioners used in the compositions can include, but are not limited to, ammonium sulfate.
[0067] Methods for treating plants and/or plant parts with fermentation broths, fermentation broth products and compositions comprising Methylobacterium are also provided herein. Treated plants and treated plant parts obtained therefrom include, but are not limited to, a pepper, tomato, berry or banana plant. Plant parts that are treated include, but are not limited to, leaves, stems, flowers, roots, seeds, fruits, tubers, coleoptiles and the like. Seeds or other propagules from any of the aforementioned plants can be treated with the fermentation broths, fermentation broth products, fermentation products and/or compositions provided herein.
In certain embodiments, plants and/or plant parts are treated by applying fermentation broths, fermentation broth products, fermentation products and compositions comprising Methylobacterium as a spray. Such spray applications include, but are not limited to, single plant part treatments or any combination of plant parts. The spraying can be carried out with any device that delivers the fermentation broths, fermentation broth products, fermentation products and compositions to the plant and/or plant part(s). Useful spray devices include a boom sprinkler, a hand or backpack sprinkler, planting sprayers (i.e., aerial spray) and the like. Spray devices and/or methods enable the application of fermentation broths, fermentation broth products, fermentation products and compositions to either or both the adaxial surface and/or the abaxial surface can also be used. Plants and/or plant parts that are at least partially coated with any one of a biphasic fermentation broth, a fermentation broth product, a fermentation product or compositions, which comprise a solid substance with Methylobacterium adhered thereto, also are provided in this document. Also provided herein are processed plant products which comprise a solid substance with Methylobacterium adhered thereto. Any of the compositions provided herein can be used to coat or partially coat a plant, a plant part or a plant seed. Partial coating of a plant, a plant part or a seed includes, but is not limited to, coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or about 99.5% of the surface area of the plant, plant part or plant seed.
[0069] In certain embodiments, lettuce seeds are treated by exposing the seeds to fermentation broths, fermentation broth products, fermentation products and compositions comprising Methylobacterium. Seeds can be treated with the fermentation broths, fermentation broth products and compositions provided herein by methods including, but not limited to, absorption, coating, spraying and the like. In certain embodiments, surface sterilized seeds are treated with a composition comprising Methylobacterium. In certain embodiments, unsterilized seeds (i.e., seeds that have not undergone surface sterilization) are treated with a composition comprising Methylobacterium that has been depleted of substances that promote the cultivation of micro-organisms residing in the seed. Seed treatments can be carried out with continuous and/or batch seed treaters. In certain embodiments, coated seeds can be prepared by forming a slurry of seeds with a coating composition containing a fermentation broth, a fermentation broth product or compositions comprising the solid substance with Methylobacterium and drying the air the resulting product. Air drying can be carried out at any temperature that is not harmful to the seed or Methylobacterium, but will typically not be higher than 30 degrees centigrade. The proportion of coating comprising a solid substance and Methylobacterium includes, but is not limited to, a range of 0.1 to 25% by weight of the seed, 0.5 to 5% by weight of the seed and 0.5 to 2.5% by seed weight. Partial coating of a seed may include, but is not limited to, coating at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or about 99.5% of the seed surface area. In certain embodiments, a solid substance used in seed coating or treatment will have Methylobacterium adhered to it. In certain embodiments, a solid substance used in seed coating or treatment will be associated with the Methylobacterium and will be a fermentation broth, a fermentation broth product or a composition obtained by the methods provided herein. Various seed treatment compositions and methods for seed treatment disclosed in US Patents 5,106,648, 5,512,069 and 8,181,388 are incorporated herein by reference in their entirety and may be adapted for use with an active agent. which comprises the fermentation broths, fermentation broth products or compositions provided herein. In certain embodiments, the composition used to treat the seed can contain agriculturally acceptable excipients including, but not limited to, wood flours, clays, activated carbon, diatomaceous earth, fine-grained inorganic solids, calcium carbonate, and the like. Clays and inorganic solids that can be used with the fermentation broths, fermentation broth products or compositions provided herein include, but are not limited to, calcium bentonite, kaolin, Chinese clay, talc, perlite, mica , vermiculite, silicas, quartz powder, montmorillonite and mixtures thereof. Agriculturally acceptable seed adhesion promoting adjuvants that can be used include, but are not limited to, polyvinyl acetates, polyvinyl acetate copolymers, hydrolyzed polyvinyl acetates, polyvinylpyrrolidone-vinyl acetate copolymer, polyvinyl alcohols, polyvinyl alcohol copolymers , polyvinyl methyl ether, methyl ether maleic anhydride copolymer, wax, latex polymers, celluloses including ethylcelluloses and methylcelluloses, hydroxy methylcelluloses, hydroxy xypropylcellulose, hydroxymethylpropylcelluloses, polyvinyl pyrrolidones, alginates, dextrins, fat, maltodextrins , proteins, karaya gum, jaguar gum, tragacanth, polysaccharide gums, mucilage, arable gum, shellac, vinylidene chloride polymers and copolymers, soy protein polymers and copolymers, lignosulfonates, acrylic copolymers, starches, polynylacrylates, zeins, gelatin, carboxymethylcellulose, chitosan, oxide of polyethylene, acrylamide polymers and copolymers, polyhydroxyethyl acrylate, methacrylamide monomers, alginate, ethylcellulose, polychloroprene and syrups or mixtures thereof. Other useful agriculturally acceptable adjuvants that can promote coating include, but are not limited to, vinyl acetate polymers and copolymers, polyvinylpyrrolidone-vinyl acetate copolymer, and water-soluble waxes. Various surfactants, dispersants, anti-caking agents, foam control agents and paints disclosed herein and in US Patent 8,181,388 can be adapted for use with an active agent comprising fermentation broths, fermentation broth products or the compositions provided in this document.
There is provided herein compositions comprising Methylobacterium which provides improved lettuce production over untreated plants which have not been exposed to the compositions. In certain embodiments, plant parts including, but not limited to, a seed, leaf, fruit, stem, root, tuber, or coleoptile can be treated with the compositions provided herein to improve lettuce production. . Treatments or applications may include, but are not limited to, spraying, coating, partially coating, immersing and/or absorbing the plant or plant parts with the compositions provided herein. In certain embodiments, a seed, leaf, fruit, stem, root, tuber, or coleoptile can be immersed and/or absorbed with a liquid, semi-liquid, emulsion, or slurry of a composition provided herein. document. Such seed immersion or absorption may be sufficient to enable improved lettuce production on a treated plant or plant part compared to an untreated plant or plant part. Improved lettuce production includes, but is not limited to, increased root cultivation, increased leaf cultivation, increased seed production and/or increased total biomass relative to untreated plants. In certain embodiments, plant seeds can be immersed and/or absorbed for at least 1, 2, 3, 4, 5 or 6 hours. Such immersion and/or absorption may, in certain embodiments, be conducted at temperatures that are not harmful to the plant seed or Methylobacterium. In certain embodiments, seeds can be treated at about 15 to about 30 degrees centigrade or about 20 to about 25 degrees centigrade. In certain modalities, seed absorption and/or immersion can be performed with gentle agitation.
[0071] It is therefore expected that compositions provided herein comprising Methylobacterium will be useful for improving lettuce production.
[0072] In certain embodiments, an amount of a composition provided herein that is sufficient to enable improved lettuce production may be a composition with Methylobacterium at a titer of at least about 1 x 106 colony forming units per millimeter , at least about 5 x 106 colony forming units per millimeter, at least about 1 x 107 colony forming units per millimeter, at least about 5 x 108 colony forming units per millimeter, at least about 1 x 109 colony forming units per millimeter, at least about 1 x 1010 colony forming units per millimeter, or at least about 3 x 1010 colony forming units per millimeter. In certain embodiments, an amount of a composition provided herein that is sufficient to enable improved lettuce production may be a composition with Methylobacterium at a titer of about at least about 1 x 106 colony forming units per millimeter, at least about 5 x 106 colony forming units per millimeter, at least about 1 x 107 colony forming units per millimeter, or at least about 5 x 108 colony forming units per millimeter for at least about 6 x 1010 colony forming units per millimeter of a liquid or an emulsion. In certain embodiments, an amount of a composition provided herein that is sufficient to enable improved lettuce production may be a fermentation broth product with a solid phase Methylobacterium titer of that product that is at least about 5x 108 colony forming units per millimeter for at least about 5 x Methylobacterium colony forming units per gram of solid phase. In certain embodiments, an amount of a composition provided herein that is sufficient to enable improved lettuce production may be a composition having a Methylobacterium titer of at least about 1 x 106 colony forming units per gram, at least about 5 x 106 colony forming units per gram, at least about 1 x 107 colony forming units per gram, or at least about 5 x 108 colony forming units per gram for at least about 6 x 1010 colony forming units of Methylobacterium per gram of particles in the composition containing particles comprising a solid substance, wherein a monoculture or coculture of Methylobacterium is adhered thereto. In certain embodiments, an amount of a composition provided herein that is sufficient to enable improved lettuce production may be a composition having a Methylobacterium titer of at least about 1 x 106 colony forming units per ml, at least about 5 x 106 colony forming units per ml, at least about 1 x 107 colony forming units per ml, or at least about 5 x 108 colony forming units per ml for at least about 6 x 1010 colony forming units of Methylobacterium per ml in a composition comprising an emulsion, wherein a monoculture or a coculture of a Methylobacterium adhered to a solid substance is provided therein or cultivated therein. In certain embodiments, an amount of a composition provided herein that is sufficient to enable improved lettuce production may be a composition having a Methylobacterium titer of at least about 1 x 106 colony forming units per ml, at least about 5 x 106 colony forming units per ml, at least about 1 x 107 colony forming units per ml, or at least about 5 x 108 colony forming units per ml for at least about 6 x 1010 colony forming units of Methylobacterium per ml of a composition comprising an emulsion, wherein a monoculture or a coculture of a Methylobacterium is provided therein or cultivated therein.
[0073] In certain embodiments, an amount of a composition provided herein that is sufficient to enable the production of improved lettuce may be a composition with a Methylobacterium species at a titer of at least about 1 x104 colony forming units per millimeter, at least about 1 x105 colony strain units per millimeter, at least about 1 x106 colony strain units per millimeter, at least about 5x106 colony strain units per millimeter, at least about 1 x107 colony forming units colony per millimeter, at least about 5 x 10 colony strain units per millimeter, at least about 1 x 109 colony strain units per millimeter, at least about 1 x 1010 colony strain units per millimeter, or at least about 3 x 1010 colony deformation units per millimeter. In certain embodiments, an amount of a composition provided herein that is sufficient to enable the production of improved lettuce may be a composition with Methylobacterium species at a titer of at least about 1 x109 colony forming units per millimeter, at least about 1 x105 colony forming units per millimeter, at least about 1 x106 colony forming units per millimeter, at least about 5x106 colony forming units per millimeter, at least about 1 x107 colony forming units per millimeter per millimeter or at least about 5 x 108 colony forming units per millimeter to at least about 6 x 1010 colony forming units per millimeter of a liquid or an emulsion. In certain embodiments, an amount of a composition provided herein that is sufficient to enable improved lettuce production may be a fermentation broth product with a Methylobacterium species titer of a solid phase of that product that is at least about 1 x104 colony forming units per gram, at least about 1 x105 colony forming units per gram, at least about 1 x106 colony forming units per gram, at least about 5x106 colony forming units per gram, at least about 1x107 colony forming units per gram, at least about 5x108 colony forming units per gram, at least about 1x109 colony forming units per gram, or at least about 5x109 forming units of colony per gram to at least about 6 x 1010 Methylobacterium colony forming units per gram, at least about 1x 1011 colony forming units Methylobacterium colony per gram, at least about 1x 1012 Methylobacterium colony forming units per gram, at least about 1x 1013 Methylobacterium colony forming units per gram, or at least about 5x1013 Methylobacterium colony forming units per gram per gram of solid phase. In certain embodiments, an amount of a composition provided herein that is sufficient to enable improved lettuce production may be a composition having a Methylobacterium titer of at least about 1 x106 colony forming units per gram, at least about 5x106 colony forming units per gram, at least about 1x107 colony forming units per gram, at least about 5x108 colony forming units per gram, at least about 1x109 colony forming units per gram gram or at least about 5x109 colony forming units per gram to at least about 6x1010 Methylobacterium colony forming units per gram, at least about 1x1011 Methylobacterium colony forming units per gram, at least about 1 x 1012 Methylobacterium colony forming units per gram, at least about 1 x1013 Methylobact colony forming units erium per gram or at least about 5 x1013 colony forming units of Methylobacterium per gram of particles in the composition containing the particles comprising a solid substance, wherein a monoculture or coculture of Methylobacterium species is adhered thereto. In certain embodiments, an amount of a composition provided herein that is sufficient to enable improved lettuce production may be a composition having a Methylobacterium titer of at least about 1 x106 colony forming units per ml, at least about from 5x106 colony forming units per ml, at least about 1x107 colony forming units per ml, or at least about 5x108 colony forming units per ml for at least about 6x1010 colony forming units per ml. colony of Methylobacterium per ml in a composition comprising an emulsion, wherein a monoculture or a coculture of a Methylobacterium species adhered to a solid substance is provided therein or cultivated therein. In certain embodiments, an amount of a composition provided herein that is sufficient to enable improved lettuce production may be a composition having a Methylobacterium titer of at least about 1 x106 colony forming units per ml, at least about from 5x106 colony forming units per ml, at least about 1x107 colony forming units per ml, or at least about 5x108 colony forming units per ml for at least about 6x1010 colony forming units per ml. Methylobacterium colony per ml in a composition comprising an emulsion, wherein a monoculture or a coculture of a Methylobacterium species is provided therein or cultivated therein.
[0074] In certain embodiments, compositions with a Methylobacterium species at a titer of at least about 1 x104 colony forming units per millimeter, at least about 1 x105 colony forming units per millimeter, at least about 1 x106 colony forming units per millimeter, at least about 5x106 colony forming units per millimeter, at least about 1 x107 colony forming units per millimeter, at least about 5 x 108 colony forming units per millimeter, at least minus about 1 x 109 colony forming units per millimeter, at least about 1 x 1010 colony forming units per millimeter, or at least about 3 x 1010 colony forming units per millimeter are provided or used. In certain embodiments, compositions with Methylobacterium species at a titer of at least about 1 x104 colony forming units per millimeter, at least about 1 x105 colony forming units per millimeter, at least about 1 x106 colony forming units per millimeter. colony per millimeter, at least about 5x106 colony forming units per millimeter, at least about 1x107 colony forming units per millimeter, or at least about 5x108 colony forming units per millimeter at least about 6x1010 colony forming units per millimeter of a liquid or an emulsion are provided. In certain embodiments, fermentation broth products with a Methylobacterium species titer of a solid phase of that product that is at least about 1 x104 colony forming units per gram, at least about 1 x105 colony forming units per gram. gram, at least about 1x106 colony forming units per gram, at least about 5x106 colony forming units per gram, at least about 1x107 colony forming units per gram, at least about 5x108 colony forming units per gram, at least about 1x109 colony forming units per gram or at least about 5x109 colony forming units per gram for at least about 6x1010 Methylobacterium colony forming units per gram , at least about 1x 1011 Methylobacterium colony forming units per gram, at least about 1x1012 Methylobacterium colony forming units per gram, at least about 1x1013 Methylobacterium colony forming units per gram or at least about 5x1013 Methylobacterium colony forming units per gram of solid phase are provided. In certain embodiments, compositions with a Methylobacterium titer of at least about 1 x106 colony forming units per gram, at least about 5x106 colony forming units per gram, at least about 1 x107 colony forming units per gram , at least about 5x108 colony formation units per gram, at least about 1x109 colony formation units per gram, or at least about 5x109 colony formation units per gram for at least about 6x1010 formation units of Methylobacterium colony per gram, at least about 1 x 1011 Methylobacterium colony forming units per gram, at least about 1 x 1012 Methylobacterium colony forming units per gram, at least about 1 x1013 of Methylobacterium colony per gram or at least about 5 x1013 Methylobacterium colony forming units per gram of particles in the composition containing are particles comprising a solid substance, in which a monoculture or a coculture of Methylobacterium species is adhered thereto, are provided. In certain embodiments, compositions with a Methylobacterium titer of at least about 1 x106 colony forming units per ml, at least about 5x106 colony forming units per ml, at least about 1 x107 colony forming units per ml per ml or at least about 5 x 108 colony forming units per ml to at least about 6 x 1010 colony forming units of Methylobacterium per ml in a composition comprising an emulsion, in which a monoculture or coculture of a Methylobacterium species adhered to a solid substance is provided therein or cultivated therein, are provided. In certain embodiments, compositions with a Methylobacterium titer of at least about 1 x106 colony forming units per ml, at least about 5x106 colony forming units per ml, at least about 1 x107 colony forming units per ml ml or at least about 5 x 108 colony forming units per ml to at least about 6 x 1010 Methylobacterium colony forming units per ml of a composition comprising an emulsion, wherein a monoculture or a coculture of a Methylobacterium species is provided therein or grown in it, are provided. In certain embodiments of any of the aforementioned compositions, the Methylobacterium species is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), NLS0038(NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B- 50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069(NRRL B-50936), NLS0089 (NRRL B-50933) and derivatives thereof. In certain embodiments of any of the aforementioned compositions, the composition can further comprise an agriculturally acceptable adjuvant, an agriculturally acceptable excipient, or a combination thereof. In certain embodiments of any of the aforementioned compositions, the Methylobacterium species is selected from the group consisting of NLS0017 (NRRL B-50931), NLS0020 (NRRL B-50930), NLS0021 (NRRL B-50939), NLS0037 (NRRL B-50941), NLS0038 (NRRL B-50942), NLS0042 (NRRL B-50932), NLS0046 (NRRL B-50929), NLS0062 (NRRL B-50937), NLS0064 (NRRL B-50938), NLS0065 (NRRL B- 50935), NLS0066 (NRRL B-50940), NLS0068 (NRRL B-50934), NLS0069(NRRL B-50936), NLS0089 (NRRL B-50933), derivatives thereof; and also comprises an agriculturally acceptable adjuvant, an agriculturally acceptable excipient or a combination thereof. EXAMPLES
[0075] The following examples are included to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples below represent techniques found by the Applicants to function well in the practice of the invention and, therefore, may be considered to constitute preferred modes for such practice. However, those skilled in the art should, in light of the present disclosure, consider that many changes can be made in the specific modalities that are disclosed while still obtaining a similar or similar result without departing from the scope of the invention**. EXAMPLE 1. CULTIVATION OF PPFM stumps IN LIQUID CULTIVATION MEDIA COMPLEMENTED WITH A SOLID SUBSTANCE
[0076] The liquid culture medium used to cultivate the PPFM cultures was a medium of basic salts supplemented with glycerol, peptone and diatomaceous earth. The basic salts medium used was the ammonium mineral salts medium (AMS). AMS medium contains, per liter, 700 milligrams of dibasic potassium phosphate anhydride, 540 milligrams of monobasic potassium phosphate anhydride, one gram of magnesium sulfate heptahydrate, 500 milligrams of ammonium chloride anhydride and 200 milligrams of calcium chloride dihydrate.
[0077] The basic AMS medium was prepared from three stock solutions listed below: MOTHER SOLUTION I: FOR A LITER AT A CONCENTRATION OF 50X
[0078] 35 grams of dibasic potassium phosphate anhydride
[0079] 27 grams of monobasic potassium phosphate anhydride MOTHER SOLUTION II: FOR A LITER AT A CONCENTRATION OF 50X
[0080] 50 grams of magnesium sulfate heptahydrate
[0081] 25 grams of ammonium chloride anhydride MOTHER SOLUTION III: FOR ONE LITER AT A CONCENTRATION OF 50X
[0082] 10 grams of calcium chloride dihydrate
[0083] Stock solutions I, II and III were autoclaved separately.
[0084] To prepare one liter of liquid AMS medium with glycerol, peptone and diatomaceous earth, the following were added to 920 ml of distilled water:
[0085] 20 ml of stock solution I
[0086] 20 ml of stock solution II
[0087] 20 ml of stock solution III
[0088] 20 ml of a 50% glycerol stock solution
[0089] 10 grams of peptone
[0090] 2 grams of diatomaceous earth
[0091] The resulting solution with suspended diatomaceous earth was sterilized by autoclaving.
[0092] Two liters of the above AMS medium were placed in a four liter bottle. Two milliliters of liquid culture PPFMs were added to the inoculation media. The vial was then placed on an incubated shaker set at 240 RPM and 30 oC. Cultures were grown for six days and then stored at 4 oC for future use. EXAMPLE 2. INOCULATION OF “REX” LETTUCE SEED TO IDENTIFY PPFMS THAT IMPROVE ROOT AND SPROUT GROWING.
An Oasis HorticubeXL™ cell 104 (with bottom grooves, single tap; Smithers-Oasis North America, Kent, OH, USA) was placed in a 1020 tray without holes. Four cubes were removed from the center of the net to allow for lower irrigation. The Oasis HorticubeXL™ was irrigated in such a way that it was completely saturated, where the setting of a tempered water bath was used. One seed was placed in each cell for a total of 100 seeds per group. LETTUCE SEED INOCULATION
[0094] The PPFM strains to be tested were grown, as described in Example 1, in a liquid medium supplemented with a solid substance. In the laboratory hood, the desired amount of PPFM solution was pipetted into conical tubes (be sure to rotate/shake the bottle vigorously before pipetting to suspend particulates). A centrifuge was used to centrifuge at 3500 RPM for 15 minutes at 23 °C. While the tubes were spun, a volume of lukewarm tap water was measured to bring the volume of each sample up to the total volume.
[0095] The liquid was carefully poured from each tube, taking care to keep the granule intact. The appropriate volume of tap water was added to each tube to match its initial volume of PPFM solution. PPFMS resuspended with water were used as soon as possible.
[0096] 100 microliters of solution (PPFM solution for treated groups and tap water for control groups) were pipetted onto the top of each seed. After every 3 rows, the tube was covered and shaken to resuspend any PPFMs that may have settled to the bottom. Pipette tips were changed between each group to avoid cross contamination. Labels have been marked and dated for each tray and clear moisture domes placed on top of the tray. The tray was placed in the culture chamber with temperature settings at 20°C and 12-hour days with 200 micromole lighting. CULTIVATION
[0097] After five to six days, the cups were removed after the seeds were germinated. The trays were bottom irrigated only and fertilized with Jack's™ 15-16-17 (JR PETERS, Inc. Allentown, PA, USA) at each irrigation (approximately every other day).
[0098] Daily repositioning of the trays was performed to prevent potential effects on cultivation due to variations in light conditions in the culture chamber. PROCESSING
[0099] The harvest was carried out in the trays between two and three weeks. Clear moisture domes were placed on each tray to prevent evapotranspiration during transport. The cups were left in place until the tray was being processed. Each plant was cut directly below the cotyledons and immediately weighed on an analytical balance. COMMENTS
[0100] It was observed that some strains repeatedly showed an increase in shoot biomass from lettuce seedlings when a seed was treated at the time of planting. Visual observations of mass and root development were also made, where treated groups showed more growth at harvest. Due to the natural variation of biological systems, all sample sizes were at least 98 to 100 plants and any difference below 12% was not considered significant. CONCLUSION
[0101] It was evident that strains NLS0017, NLS0020, NLS0066, NLS0065 and NLS0089 showed an increase in wet weight of lettuce seedlings after seed treatment. Strains NLS0069, NLS0037, NLS0038 and NLS0062 exhibited negligible increases in wet weight compared to controls. It was also observed, together with an increase in shoot biomass, a corresponding increase in root development.
1 Each row represents data obtained from separate plant trays obtained from treated seed versus control seed. 2 NA: not determined. EXAMPLE 3. LEAF APPLICATION OF 'REX' LETTUCE TO OBSERVE HOW PPFMS AFFECT ROOT AND SPROUT FARMING.SEEDURA
[0102] A cell 104 of Oasis HorticubeXL (with lower grooves, single tappet) was placed in a 1020 holeless tray. Four cubes were removed in the center of the net to allow for lower irrigation. The Oasis was irrigated in such a way that it was completely saturated, in which the definition of a temperate water bath was used. One seed was placed in each cell for a total of 100 seeds per group. Labels have been marked and dated for each tray and clear moisture domes placed on top of the tray. The tray was placed in the culture chamber with temperature settings at 20°C and 12-hour days with 200 micromole lighting. LETTUCE SOWING INOCULATION
[0103] After five to six days, the cups were removed after the seeds had germinated. The plants were inoculated at that time, when only the cotyledons emerged. The PPFM strains to be tested were grown, as described in Example 1, in a liquid medium supplemented with a solid substance. The PPFM strains to be tested were grown, as described in Example 1, in a liquid medium supplemented with a solid substance. In the laboratory hood, the desired amount of PPFM solution was pipetted into conical tubes (be sure to rotate/shake the bottle vigorously before pipetting to suspend particulates). A centrifuge was used to centrifuge at 3500 RPM for 15 minutes at 23 °C. While the tubes were spun, a volume of lukewarm tap water was measured to bring the volume of each sample up to the total volume.
[0104] The liquid was carefully poured from each tube, taking care to keep the granule intact. The appropriate volume of tap water was added to each tube to match its initial volume of PPFM solution. PPFMS resuspended with water were used as soon as possible.
[0105] 100 ml of PPFM solution (control tap water) was poured into a 1 L Solo™ Manual Sprinkler (Solo™, Newport News, VA, USA). The tray has been removed from the group to avoid cross contamination. The finer mist setting was used and an even coating of solution was sprayed over the top of the seedlings, which ensured an even coverage across the tray. For each group, this was prepared using appropriate treatment. CULTIVATION
[0106] The trays were bottom irrigated only and fertilized with Jack's™ 15-16-17 (JR PETERS, Inc. Allentown, PA, USA) at each irrigation (approximately every other day). Daily repositioning of trays was performed to prevent potential effects on cultivation due to variations in light conditions in the cultivation chamber. PROCESSING
[0107] Harvesting from trays took place between two and three weeks. Clear moisture domes were placed on each tray to prevent evapotranspiration during transport. The cups were left in place until the tray was being processed. Each plant was cut directly below the cotyledons and immediately weighed on an analytical balance. COMMENTS
[0108] It was observed that some strains repeatedly showed an increase in shoot biomass of lettuce seedlings when the seedling was treated at the cotyledon stage. Visual observations of mass and root development were also made, where treated groups showed more growth at harvest. Due to the natural variation of biological systems, all sample sizes were a minimum of 98 to 100 plants and any difference below 12% was not considered significant. CONCLUSION
[0109] It is evident that strains NLS0042, NLS0017, NLS0020 and NLS0068 show an increase in wet weight of lettuce seedlings after foliar application. Strains NLS0069, NLS0037, NLS0038 and NLS0062 exhibited negligible increases in wet weight compared to controls. It was also observed, together with an increase in shoot biomass, a corresponding increase in root development.

1 Each row represents data obtained from a separate tray of treated versus control plants.2 NA: not determined. EXAMPLE 4. IDENTIFICATION OF NUCLEICO ACID POLYMORPHISMS PRESENTED IN METHYL BACTERIA THAT IMPROVE LETTUCE PRODUCTION
[0110] The complete genome sequencing libraries for the Illumina™ High Throughput Sequencing Platform are generated for Methylobacterium species isolates provided in Table 1 using Illumina TRUSEQ™ or NEXTERA™ DNA Sample Preparation Kits (described at internetres.illumina.com/documents/products/datasheets/datasheet_truseq_dna_sampl e_prep_kits.pdf and res.illumina.com/documents/products/datasheets/datasheet_nextera_dna_sample_prep.pdf) using the methods described by the manufacturer. The resulting libraries are then subjected to pyrogen sequencing (Siqueira JF et al. J Oral Microbiol. 2012; 4: 10.3402/jom.v4i0.10743). Genomic sequence data generated by raw pyrogen sequencing is subjected to adapter- and quality-based trimming for quality control. Full Genome Shotgun Sequence Montage (1) is achieved by assembling past quality data using the Velvet assembler again (2). For gene annotation and finding, reference training data are leveraged from TIGRFAM (9), Pfam, COG (10) and UniReflOO (11). rRNAs are identified with RNAmmer (5), protein encoding genes are identified with Glimmer (3) or Maker (6), and tRNAs are identified with tRNAscan-SE (4). Gene functions are assigned with blastx (7), blastp (7), HMMER (8) and InterProScan against the comprehensive protein databases described above (Reference data).
[0111] The detection of polymorphisms (SNP or other DNA variations that occur as a result of insertions, deletions and substitutions (Indels)) in the Methylobacterium species isolates from Table 1 is performed with BWA (12) and the Samtools set (in internet at samtools.sourceforge.net/), the structural variation is identified with BreakDancer (on the internet at breakdancer.sourceforge.net/) and CoGE (on the internet at genomevolution.org/CoGe/). Diagnosis of polymorphisms for Methylobacterium that enable improved lettuce production is identified by sequence comparisons of exemplary Methylobacterium isolates NLS0020, NLS0066, NLS0017, NLS0065, NLS0089, NLS0042 and/or NLS0068 that improve lettuce production but are absent from one or more Methylobacterium isolates that do not improve lettuce production. Polymorphisms present in exemplary Methylobacterium isolates NLS0020, NLS0066, NLS0017, NLS0065, NLS0089, NLS0042 and/or NLS0068 that improve lettuce production, but which are absent in exemplary Methylobacterium isolates that do not improve lettuce production, are then , used to identify other Methylobacterium isolates that enhance lettuce production. REFERENCES FOR EXAMPLE 41. Miller JR, Koren S, Sutton G (2010) Assembly algorithms for next-generation sequencing data. Genomics 95: 315 to 327.2. Zerbino DR, Birney E (2008) Velvet: algorithms is again short read assembly using de Bruijn graphs. Genome Res 18: 821 to 829.3. Delcher AL, Bratke KA, Powers EC, Salzberg SL (2007) Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673 to 679.4. Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of RNA gene transfer in genomic sequence. Nucleic Acids Res 25: 955 to 964.5. Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T, et al. (2007) RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 35: 3100 A 3108.6. Cantarel B, Korf I, Robb S, et al. (2008) MAKER: An easy-to-use annotation pipeline designed for emerging model organism genomes. Genome Research 18: 188 to 196.7. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389 to 3.402.8. Eddy SR (2009) A new generation of homology search tools based on probabilistic inference. Genome Inform 23: 205 to 211.9. Haft DH, Selengut JD, White O (2003) The TIGRFAMs database of protein families. Nucleic Acids Res 31: 371 to 373.10. Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, et al. (2003) The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4: 41.11. Suzek BE, Huang H, McGarvey P, Mazumder R, Wu CH (2007) UniRef: comprehensive and non-redundant UniProt reference clusters. Bioinformatics 23: 1282 to 1,288.12. Li H. and Durbin R. (2009) Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics, 25: 1754 to 60.EXAMPLE 5. FLANDRIA LETTUCE SEED INOCULATION WITH PPFMS INCREASES SPROUT AND ROOT BIOMASS.SEEDING A sheet of Oasis HORTICUBES® 276 (1-inch Thin-Cut; Smithers-Oasis North) cell America, Kent, OH, USA) was placed in a 1020 mesh tray. The tray was split in half with a piece of plastic to allow for two groups per tray. The Oasis HORTICUBES® were irrigated until complete saturation. Flandria lettuce seed from Rijk Zwaan USA (Salinas, CA, USA) was used. One seed was placed in each cell for a total of 132 or 144 seeds per group. LETTUCE SEED INOCULATION
[0112] The PPFM strains to be tested were grown, as described in Example 1, in a liquid medium supplemented with diatomaceous earth at 2 grams/liter. On the bench, the desired amount of PPFM solution was pipetted into conical tubes (ensuring to rotate/shake the bottle vigorously before pipetting to suspend the particulates). A centrifuge was used to granulate the cells at 7,500 RPM for 5 minutes at 23°C. The supernatant was discarded and the PPFM granules were resuspended in an equal volume of water.
[0113] 100 microliters of solution (PPFM solution for treated groups and tap water for control groups) were pipetted over the top of each seed. The tube was periodically shaken to keep the PPFM cells in suspension. Clear moisture domes were placed over each tray. The trays were placed in an oven with temperature settings of 30 °C during the day, 28 °C at night and with a day length of 16 hours achieved using supplemental light as needed. CULTIVATION
[0114] After two to three days after planting, the seeds germinated and the moisture domes were removed. The trays were top irrigated and fertilized with Jack's™ 15-16-17 (JR PETERS, Inc. Allentown, PA, USA) at each irrigation. Daily repositioning of trays was performed to prevent potential effects on cultivation due to variations in light conditions in the cultivation chamber. PROCESSING
[0115] The lettuce seedlings were harvested 10 days after planting. Each plant was cut directly below the cotyledons and immediately weighed on an analytical balance. COMMENTS
[0116] It was observed that some strains repeatedly showed an increase in shoot biomass of lettuce seedlings after seed treatment. Visual observations of mass and root development were also made and it was noted that treated groups showed more growth at harvest. The outer row of each group was not cropped in order to eliminate any edge effects on the trays. CONCLUSION
[0117] It was evident that the PPFM strains NLS0017, NLS0020, NLS0066 and NLS0068 showed a reproducible and statistically significant increase in the wet weight of lettuce seedlings after seed treatment. It was also observed, together with an increase in shoot biomass, a corresponding increase in root development. TABLE 4. RESULTS FOR FLANDRIA LETTUCE SEED INOCULATION


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"Bergey's Manual of Systematic Bacteriology. Volume two, The Proteobacteria. Part C, The alpha-, beta-, delta-, and epsilonproteobacteria." Second edition. Springer, New York. Pages 567 to 571.7. Green, PN 2006. Methylobacterium. In Dworkin, M., S. Falkow, E. Rosenberg, K.-H. Schleifer, and E. Stackebrandt (eds.). "The Prokaryotes. A Handbook on the Biology of Bacteria. Volume 5. Proteobacteria: Alpha and Beta Subclasses." Third edition. Springer, New York. Pages 257 to 265.8. Holland, MA 1997. Methylobacterium and plants. Recent. Debt Res. in Plant Physiol. 1,207 to 213.9. Holland, MA, and JC Polacco. 1994. PPFMs and other covert contaminants: Is there more to plant physiology than just plant Annu. Rev. Plant Physiol. Plant Mol. Biol. 45: 197 to 209.10. Kutschera, U. 2007. Plant-associated methylobacteria as co-evolved phytosymbionts. The hypothesis. Plant Signal Behav. 2: 74 to 78.11. Lidstrom, ME 2006. Aerobic methylotrophic prokaryotes. In Dworkin, M., S. Falkow, E. Rosenberg, K.-H. Schleifer, and E. Stackebrandt (eds.). "The Prokaryotes. A Handbook on the Biology of Bacteria. Volume 2. Ecophysiology and biochemistry." Third edition. Springer, New York. Pages 618 to 634.12. Madhaiyan, M., S. Poonguzhali, HS Lee, K. Hari, SP Sundaram, and TM Sa. 2005. Pink-pigmented facultative methylotrophic bacteria accelerate germination, growth and yield of sugarcane clone Co86032 (Saccharum officinarum L.) Biol. Fertile. Soils 41: 350 to 358. 13. Madhaiyan, M., S. Poonguzhali, M. Senthilkumar, S. Seshadri, H. Chung, J. Yang, S. Sundaram and T Sa. 2004. Growth promotion and induction of systemic resistance in rice cultivar CO-47 (Oryza sativa L.) by Methylobacterium spp. Bot. Bull. Academic Sin. 45: 315 to 324.14. Madhaiyan, M., S. Poonguzhali, and T. Sa. 2007. Influence of plant species and environmental conditions on epiphytic and endophytic pink-pigmented facultative methylotrophic bacterial populations associated with field-grown rice cultivars. J Microbiol Biotechnol. October 17, 2007 (10): 1645 to 54.15. Stanier, RY, NJ Palleroni, and M. Doudoroff. 1966. The aerobic pseudomonads: A taxonomic study. J. Gen. Microbiol. 43: 159 to 271.16. Sy, A., Giraud, E., Jourand, P., Garcia, N., Willems, A., De Lajudie, P., Prin, Y., Neyra, M., Gillis, M., Boivin-Masson, C, and Dreyfus, B. 2001. Methylotrophic Methylobacterium Bacteria Nodulate and Fix Nitrogen in Symbiosis with Legumes. Journal Bacteriol. 183(1):214 to 220.17. Sy, A., ACJ Timmers, C. Knief, and JA Vorholt. 2005. Methylotrophic metabolism is advantageous for Methylobacterium extorquens during colonization of Medicago truncatula under competitive conditions. Appl. Environ. Microbiol. 71, 7245 to 7252.18. Vogel, HJ, and DM Bonner. 1956. Acetylornithinase of Escherichia coli: Partial purification and some properties. J. Biol. Chem., 218: 97 to 106.19. Vogel, HJ 1956. A convenient growth medium for Neurospora (Medium N). Microbial Genet Bull 13: 42 to 4320. Whittenbury, R., SL Davies, and JF Wilkinson. 1970. Enrichment, isolation and some properties of methane-using bacteria. J. Gen. Microbiol. 61. 205 to 218. 21. Vuilleumier S, Chistoserdova L, Lee MC, Bringel F, Lajus A, Zhou Y, Gourion B, Barbe V, Chang J, Cruveiller S, Dossat C, Gillett W, Gruffaz C, Haugen E, Hourcade E, Levy R, Mangenot S, Muller E, Nadalig T, Pagni M, Penny C, Peyraud R, Robinson DG, Roche D, Rouy Z, Saenampechek C, Salvignol G, Vallenet D, Wu Z, Marx CJ, Vorholt JA , Olson MV, Kaul R, Weissenbach J, Medigue C, Lidstrom ME.Methylobacterium genome sequences: a reference blueprint to investigate microbial metabolism of CI compounds from natural and industrial sources. PLoS One. 2009;4(5): e5584. doi: 10.1371/journal.pone.0005584. May 18, 2009 Epub PubMed PMID: 19440302; PubMed Central PMCID: PMC2680597.22. Marx CJ, Bringel F, Chistoserdova L, Moulin L, Farhan Ul Haque M, Fleischman DE, Gruffaz C, Jourand P, Knief C, Lee MC, Muller EE, Nadalig T, Peyraud R, Roselli S, Russ L, Goodwin LA, Ivanova N, Kyrpides N, Lajus A, Land ML, Medigue C, Mikhailova N, Nolan M, Woyke T, Stolyar S, Vorholt JA, Vuilleumier S. Complete genome sequences of six strains of the genus Methylobacterium. J Bacteriol. September 2012;194(17):4746 to 8. doi: 10.1128/JB.01009-12. PubMed PMID: 22887658; PubMed Central PMCID: PMC3415506.23. Knief C, Frances L, Vorholt JA. Competitiveness of diverse Methylobacterium strains in the phyllosphere of Arabidopsis thaliana and identification of representative models, including M. extorquens PA1. Microb Eco. August 2010;60(2):440-52. doi: 10.1007/s00248-010-9725-3. August 11, 2010Epub. PubMed PMID: 20700590. EXAMPLE 6. INOCULATION OF “REX” LETTUCE SEED WITH PPFMS INCREASES SPROUT AND ROOT BIOMASS. SEEDING
[0118] A sheet of Oasis HORTICUBES® 276 cell (1-inch Thin-Cut; Smithers-Oasis North America, Kent, OH, USA) was placed in a 1020 mesh tray. The tray was split in half with a piece of plastic to allow two groups per tray. The Oasis HORTICUBES® were irrigated until complete saturation. Rex lettuce seed was used. One seed was placed in each cell for a total of 132 or 144 seeds per group. LETTUCE SEED INOCULATION
[0119] The PPFM strains to be tested were grown, as described in Example 1, in a liquid medium supplemented with diatomaceous earth at 2 grams/liter. On the bench, the desired amount of PPFM solution was pipetted into conical tubes (ensuring to rotate/shake the bottle vigorously before pipetting to suspend the particulates). A centrifuge was used to granulate the cells at 7,500 RPM for 5 minutes at 23°C. The supernatant was discarded and the PPFM granules were resuspended in an equal volume of water.
[0120] 100 microliters of solution (PPFM solution for treated groups and tap water for control groups) were pipetted over the top of each seed. The tube was periodically shaken to keep the PPFM cells in suspension. Clear moisture domes were placed over each tray. The trays were placed in an oven with temperature settings of 30 °C during the day, 28 °C at night and with a day length of 16 hours achieved using supplemental light as needed. CULTIVATION
[0121] After two to three days after planting, the seeds germinated and the moisture domes were removed. The trays were top irrigated and fertilized with Jack's™ 15-16-17 (JR PETERS, Inc. Allentown, PA, USA) at each irrigation. Daily repositioning of trays was performed to prevent potential effects on cultivation due to variations in light conditions in the cultivation chamber. PROCESSING
[0122] The lettuce seedlings were harvested 10 days after planting. Each plant was cut directly below the cotyledons and immediately weighed on an analytical balance. COMMENTS
[0123] It was observed that some strains repeatedly showed an increase in shoot biomass of lettuce seedlings after seed treatment. Visual observations of mass and root development were also made and it was noted that treated groups showed more growth at harvest. The outer row of each group was not cropped in order to eliminate any edge effects on the trays. The results are as shown in the following Table.

CONCLUSION
[0124] It was evident that the PPFM strains NLS0017, NLS0020, NLS21,NLS0037, NLS0038, NLS42, NLS46, NLS62, NLS64, NLS0065, NLS0066,NLS0068 and NLS0089 showed a reproducible and statistically significant increase in the wet weight of lettuce seedlings after the seed treatment. Also, together with an increase in shoot biomass, a corresponding increase in root development was observed. EXAMPLE 7. FLANDRIA SEED TESTS
Flandria lettuce seed was treated with the indicated PPFM isolates essentially as described in Example 5 to yield the following results.



[0126] It was evident that the PPFM strains NLS0017, NLS0037, NLS0066,NLS0020, NLS0042, NLS0065, NLS0089, NLS0046, NLS0021, NLS0069, NLS0068, NLS0064, NLS0062 and NLS0038 can enable an increased lettuce biomass in relation to treatments . EXAMPLE 8. IDENTIFICATION OF ORTHOLOGICAL GENES PRESENT IN METHYLOBACTERIUM SPECIES THAT MAY IMPROVE LETTUCE PRODUCTION
[0127] The PPFM strains listed in Table 1 were grown in solid agar media comprising Ammonium Mineral Salts (AMS) plus glycerol and peptone at 30°C for 5 days, essentially as described in the Patent Application Publication assigned to same dealership in US20130324407 and incorporated herein by reference in its entirety. Genomic DNA was extracted using MO-BIO Ultra Clean Microbial DNA Isolation Kit (Carlsbad, CA) and 1 μg of high quality DNA was used for Illumina Nextera XT library preparation followed by paired end sequencing by Illumina 2x100 on a HiSeq2000 system. The raw Illumina genomic sequence data was subjected to adapter- and quality-based trimming for quality control. Full Genome Shotgun Sequence Montage was achieved by assembling past quality data using the SPADES assembler again (33). For gene annotation and finding, reference training data were leveraged from TIGRFAM (9), Pfam, COG (10) and UniReflOO (11). rRNAs were identified with RNAmmer (5), protein coding genes were identified with Glimmer (3) and Maker (6), and tRNAs were identified with tRNAscan-SE (4). Gene functions were assigned with blastx (7), blastp (7), HMMER (8) and InterProScan against comprehensive protein databases described above (Reference data). The detection of polymorphisms (SNP or other DNA variations that occur as a result of insertions, deletions and substitutions (Indels)) in the Methylobacterium species isolates was performed with BWA (12) and the Samtools set (on the internet at samtools.sourcefrge .net/) and the Genome Analysis Toolkit (GATK, on the worldwide web site "broadinstitute.org/gatk/"), the structural variation was identified with BreakDancer (on the internet at breakdancer.sourceforge.net/) and CoGE (on the internet at genomevolution.org/Co Ge/). The genes encoding open reading frames were predicted from the assembled complete genomic sequences of NLS0017, NLS0020, NLS0037, NLS0042, NLS0065, NLS0066, NLS0135, NLS0071, NLS0109 andNLS0142 essentially as described above. The genome ortholog genes were clustered in and between using OrthoMCL (available on the world wide web site "orthomcl.org/orthomcl/"). The supposed functional annotations were assigned to gene products using BLASTP (available on the "blast.ncbi.nlm.nih.gov/Blast.cgi" website) against the UniProt database (available on the website of worldwide network "uniprot.org/"). Genes present in individual genomes of NLS0017, NLS0020, NLS0037, NLS0042, NLS0065 and NLS0066 that can improve lettuce production (as shown in Example 7), but absent in the entire genome set of NLS0135, NLS0071, NLS0109 and NLS0142 that do not improve lettuce production (as shown in Example 7), have been identified in OrthoMCL clumps using custom software. Encoded proteins found in Methylobacterium NLS0017, NLS0020, NLS0037, NLS0042, NLS0065 and NLS0066 that can improve lettuce production are provided in the sequencing listing as SEQ ID NO: 1-5125. The nucleic acid sequences encoding the proteins of SEQ ID NO: 1-5125 are SEQ ID NO: 5126-10250, respectively. Proteins encoded by genes present in NLS0017 but absent from NLS0135, NLS0071, NLS0109 and NLS0142 are provided as SEQ ID NO: 1-108 6. Proteins encoded by genes present in NLS0020 but absent from NLS0135, NLS0071,NLS0109 and NLS0142, are provided as SEQ ID NO: 1087-2176. Proteins encoded by genes present in NLS0037, but absent from NLS0135, NLS0071, NLS0109 and NLS0142, are provided as SEQ ID NO: 2177-2461. Proteins encoded by genes present in NLS0042, but absent from NLS0135, NLS0071, NLS0109 and NLS0142, are provided as SEQ ID NO: 2462-3347. Proteins encoded by genes present in NLS0065, but absent from NLS0135, NLS0071, NLS0109 and NLS0142, are provided as SEQ ID NO: 3348-3949. Proteins encoded by genes present in NLS0066, but absent from NLS0135, NLS0071, NLS0109 and NLS0142, are provided as SEQ ID NO: 3950-5125. Ortholog gene groups that represent genes encoding proteins found in the genomes of at least two individual genomes of NLS0017, NLS0020, NLS0037, NLS0042, NLS0065 and/or NLS0066 that can improve lettuce production (as shown in Example 7), but that are absent in the entire set of genomes of NLS0135, NLS0071, NLS0109, and NLS0142 that do not improve lettuce production are given in Table 7. In Table 7, ortholog gene groups are given in each row, where the sequence longer and the unique associated Seq ID Number are designated as a reference sequence to represent the orthologous cluster (Column 3 of Table 7). The ortholog group identification number is given in column 1 of Table 7, the closest gene identity based on database comparisons is given in column 2 of Table 7 and the reference sequence for each ortholog cluster is given in column 3 of Table 7. Examples of orthologous sequences found in NLS0017, NLS0020, NLS0037,NLS0042, NLS0065 and NLS0066 are provided as SEQ ID NO: in Table 7, in columns 4, 5, 6, 7, 8 and 9, respectively .TABLE 7. ORTHOLOGICAL GENE GROUPS








































































REFERENCES FOR EXAMPLE 81. Miller JR, Koren S, Sutton G (2010) Assembly algorithms for next-generation sequencing data. Genomics 95: 315 to 327.2. Zerbino DR, Birney E (2008)Velvet: algorithms for again short read assembly using de Bruijn graphs. Genome Res. 18: 821 to 829.3. Delcher AL, Bratke KA, Powers EC, Salzberg SL (2007)Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673 to 679.4. Lowe TM, Eddy SR (1997) tRNAscan-SE: a program for improved detection of RNA gene transfer in genomic sequence. Nucleic Acids Res 25: 955 to 964.5. Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T, et al. (2007) RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 35: 3100 to 3108.6. Cantarel B, Korf I, Robb S, et al. (2008) MAKER: An easy-to-use annotation pipeline designed for emerging model organism genomes. Genome Research 18: 188 to 196.7. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389 to 3402.8. Eddy SR (2009) A new generation of homology search tools based on probabilistic inference. Genome Inform 23: 205 to 211.9. Haft DH, Selengut JD, White O (2003) The TIGRFAMs database of protein families. Nucleic Acids Res 31:371 to 373.10. Tatusov RL, Fedorova ND, Jackson JD, Jacobs AR, Kiryutin B, et al. (2003) The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4: 41.11. Suzek BE, Huang H, McGarvey P, Mazumder R, Wu CH (2007) UniRef: comprehensive and non-redundant UniProt reference clusters. Bioinformatics 23: 1282 to 1288.12. Li H. and Durbin R. (2009) Fast and accurate short read alignment with Burrows-Wheeler Transform. Bioinformatics, 25: 1754 to 60
[0128] The inclusion of various references in this document is not to be construed as any admission by the Depositors that the references constitute prior art. Depositors expressly reserve their right to challenge any claims of non-patentability of inventions disclosed herein by references included herein.
[0129] Having illustrated and described the principles of the present invention, it should be evident to persons skilled in the art that an invention can be modified in arrangement and detail without departing from such principles.
[0130] Although the materials and methods of this invention have been described in terms of various modalities and illustrative examples, it will be evident to those skilled in the art that variations can be applied to the materials and methods described in this document without departing from the concept, the spirit and scope of the invention. All substitutes and similar modifications evident to those skilled in the art are intended to be within the spirit, scope and concept of the invention as defined by the appended claims.

权利要求:
Claims (15)
[0001]
1. Composition characterized by the fact that it comprises: (a) an isolated Methylobacterium selected from the group consisting of NLS0089 (NRRL B-50933); and (b) an agriculturally acceptable excipient, an agriculturally acceptable adjuvant or a combination thereof.
[0002]
2. Composition according to claim 1, characterized in that the isolated Methylobacterium is at a titer of at least about 1x106 CFU/g to at least about 1x1014 CFU/g for a solid composition and in a titration of about 1x106 CFU/ml to about 1X1011 CFU/ml for a liquid composition.
[0003]
3. Composition characterized in that it comprises: (a) (i) a solid substance with adherent Methylobacterium cultivated on it, or (ii) an emulsion with Methylobacterium cultivated on it; wherein the Methylobacterium is Methylobacterium isolated NLS0089 (NRRL B-50933); and (b) an agriculturally acceptable excipient, an agriculturally acceptable adjuvant or a combination thereof.
[0004]
4. Composition according to claim 3, characterized in that the solid substance with adherent Methylobacterium cultivated on it has a Methylobacterium titer of at least about 5x108 CFU/g to at least about 1x1014 CFU/g.
[0005]
5. Composition according to claim 3, characterized in that the composition further comprises an insecticide, a fungicide, a nematicide or a bactericide.
[0006]
6. Composition, according to claim 3, characterized in that the composition further comprises a microorganism, except Methylobacterium, in which said microorganism is biopesticidal or provides some other benefit when applied to a plant or plant part.
[0007]
7. Composition according to claim 3, characterized in that said agriculturally acceptable adjuvant is wet, an adhesive, a penetrant, an extender or a wetting agent that improves the effectiveness of the product or the ease of application of the product.
[0008]
8. Composition according to claim 7, characterized in that said adhesive comprises a polymer or copolymer of polyvinyl acetate, polymer or copolymer of polyvinylpyrrolidone-vinyl acetate, polymer or copolymer of polyvinyl alcohol, latex polymer, alginate, acrylic copolymer or acrylamide polymer or copolymer.
[0009]
9. Composition according to claim 4, characterized in that said adjuvant comprises a surfactant, dispersant, anti-caking agent, foam control agent or a dye.
[0010]
10. Composition according to any one of claims 1 to 9, characterized in that the composition is adapted to be used in the treatment of a plant, a plant part, or a propagule of a plant.
[0011]
11. Composition according to claim 10, characterized in that the propagule is a seed.
[0012]
12. Composition according to claim 1 or 3, characterized in that the composition is an essentially dry product that has a water content of 5% or less.
[0013]
13. Use of a Methylobacterium isolate NLS0089 (NRRL B-50933) characterized in that it is for the preparation of a composition for the treatment of a plant, part of a plant or propagule of a plant.
[0014]
14. Use according to claim 13, characterized in that the plant part is a leaf, a stem, a flower, a root, a tuber or a coleoptile.
[0015]
15. Use according to claim 13, characterized in that the plant is lettuce, corn, soybeans, tomatoes or a fruit plant.

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法律状态:
2019-07-23| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2019-12-24| B06G| Technical and formal requirements: other requirements [chapter 6.7 patent gazette]|Free format text: FOI SOLICITADA A ALTERACAO DO NOME DA INVENTORA ?RACHEL DIDONATO? DISPOSTA NA ENTRADA NA FASE NACIONAL PARA ?RACHEL DIDONATO FLORO?. NO ENTANTO, NAO FOI APRESENTADO NENHUM DOCUMENTO COMPROBATORIO DA ALTERACAO DO NOME. ENVIE DOCUMENTOS COMPROBATORIOS PARA A ALTERACAO DO NOME SOLICITADA SER EFETIVADA. |

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2021-02-02| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-03-30| B09W| Decision of grant: rectification|Free format text: REFERENCIA: RPI 2613 DE 02.02.2021 - CODIGO 9.1 |

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2021-06-22| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 04/12/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361911516P| true| 2013-12-04|2013-12-04|

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US61/911,516|2013-12-04|

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US201461954840P| true| 2014-03-18|2014-03-18|

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US61/954,840|2014-03-18|

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PCT/US2014/068558|WO2015085063A1|2013-12-04|2014-12-04|Compositions and methods for improving lettuce production|

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