Methods and Compositions for Controlling Root Lesion Nematodes

Abstract

The present disclosure provides compositions comprising Root-Lesion Nematode (RLN)-inhibitory Methylobacterium sp., as well as cell-free culture supernatants, fractions, and concentrates obtained from the Root-Lesion Nematode (RLN)-inhibitory Methylobacterium sp. Also provided are related methods for controlling RLN infections of plants, and methods of making the compositions.

What is claimed is: 
     
         1 . A method for reducing  Pratylenchus  sp. damage to a plant that comprises
 (a) applying a composition comprising a  Methylobacterium  selected from the group consisting of NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, NLS0089, derivatives thereof, and  Methylobacterium  related thereto and an agriculturally acceptable excipient and/or an agriculturally acceptable adjuvant to a plant part to obtain a treated plant part; and   (b) growing the plant from said treated plant part in the presence of  Pratylenchus  sp., wherein  Pratylenchus  sp. damage to the plant is reduced in comparison to a control plant from a control plant part that is not treated with the  Methylobacterium  and that is grown in the presence of  Pratylenchus  sp.   
     
     
         2 . The method of  claim 1 , wherein the  Methylobacterium  is present on said treated plant part in an amount of at least about 1×10 3  colony forming units (CFU) of said  Methylobacterium  per treated plant part. 
     
     
         3 . The method of  claim 1 , wherein the NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089 derivative is obtained by mutagenizing or transforming  Methylobacterium  NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089, respectively. 
     
     
         4 . The method of  claim 1 , wherein the NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089-related  Methylobacterium  is characterized by having a gene encoding a 16S RNA that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, respectively. 
     
     
         5 . The method of  claim 1 , wherein the  Methylobacterium  is heterologous to the plant part. 
     
     
         6 . The method of  claim 1 , wherein the  Methylobacterium  is: (i) NLS0021 and the plant part is not a lettuce plant part; (ii) NLS0038 and the plant part is not a tomato plant part; (iii) NLS0042 or NLS0934 and the plant part is not a soybean plant part; (iv) NLS0062 or NLS0069 and the plant part is not a corn plant part; or (v) NLS0089 and the plant part is not a broccoli plant part. 
     
     
         7 . The method of  claim 1 , wherein the  Pratylenchus  sp. damage is selected from the group consisting of a reduction in plant growth, yield, water-deficit tolerance, chlorosis, produce quality, and combinations thereof. 
     
     
         8 . The method of  claim 1 , wherein the reduction in damage is evidenced by an increase in shoot weight, root weight, or the combination thereof in the plant in comparison to the control plant. 
     
     
         9 . The method of  claim 1 , wherein the plant part is a seed, leaf, tuber, or root. 
     
     
         10 . The method of any one of  claims 1  to  9 , wherein the  Pratylenchus  sp. is selected from the group consisting of  Pratylenchus brachyurus, Pratylenchus coffeae, P. neglectus, Pratylenchus penetrans, Pratylenchus scribneri, Pratylenchus thornei, Pratylenchus vulnus , and  Pratylenchus zeae.    
     
     
         11 . The method of any one of  claims 1  to  9 , wherein the plant part is selected from the group consisting of a  Brassica  sp. corn, wheat, rye, rice, alfalfa, rye, sorghum, millet, soybean, tobacco, potato, peanut, carrot, cotton, coffee, coconut, pineapple, sugar beet, strawberry, oat, barley, tomato, lettuce, pepper, pea, onion, green bean, and cucurbit plant part. 
     
     
         12 . A plant part that is at least partially coated with a composition comprising a  Methylobacterium  selected from the group consisting of NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, NLS0089, derivatives thereof, and  Methylobacterium  related thereto and an agriculturally acceptable excipient and/or an agriculturally acceptable adjuvant, wherein said composition is provided on said plant part in an amount that reduces  Pratylenchus  sp. damage to a plant grown from the plant part in comparison to a control plant grown from a control plant part that is not treated with the  Methylobacterium.    
     
     
         13 . The plant part of  claim 12 , wherein the amount of  Methylobacterium  present on said plant part is at least about 1×10 3  colony forming units (CFU) of said  Methylobacterium  per plant part. 
     
     
         14 . The plant part of  claim 12 , wherein the plant part is a seed, leaf, stem, root, or tuber. 
     
     
         15 . The plant part of  claim 12 , wherein the NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089 derivative is obtained by mutagenizing or transforming  Methylobacterium  NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089, respectively. 
     
     
         16 . The plant part of  claim 12 , wherein the NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089-related  Methylobacterium  has a gene encoding a 16S RNA that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, respectively. 
     
     
         17 . The plant part of any one of  claims 12  to  16 , wherein the  Methylobacterium  is heterologous to the seed, tuber, or seedling. 
     
     
         18 . The plant part of  claim 17 , wherein the  Methylobacterium  is: (i) NLS0021 and the plant part is not a lettuce plant part; (ii) NLS0038 and the plant part is not a tomato plant part; (iii) NLS0042 or NLS0934 and the plant part is not a soybean plant part; (iv) NLS0062 or NLS0069 and the plant part is not a corn plant part; or (v) NLS0089 and the plant part is not a broccoli plant part. 
     
     
         19 . The plant part of any one of  claims 12  to  16 , wherein the  Pratylenchus  sp. damage is selected from the group consisting of a reduction in plant growth, yield, water-deficit tolerance, chlorosis, produce quality, and combinations thereof. 
     
     
         20 . The plant part of  claim 19 , wherein the reduction in damage is evidenced by an increase in shoot weight, root weight, or the combination thereof in the plant in comparison to the control plant. 
     
     
         21 . The plant part of any one of  claims 12  to  16 , wherein the  Pratylenchus  sp. is selected from the group consisting of  Pratylenchus brachyurus, Pratylenchus coffeae, P. neglectus, Pratylenchus penetrans, Pratylenchus scribneri, Pratylenchus thornei, Pratylenchus vulnus , and  Pratylenchus zeae.    
     
     
         22 . The plant part of any one of  claims 12  to  16 , wherein the plant part is selected from the group consisting of a  Brassica  sp. corn, wheat, rye, rice, alfalfa, rice, rye, sorghum, millet, soybean, tobacco, potato, peanut, carrot, cotton, coffee, coconut, pineapple, sugar beet, strawberry, oat, barley, tomato, lettuce, pepper, pea, onion, green bean, and cucurbit plant part. 
     
     
         23 . A method for obtaining a  Pratylenchus -inhibitory cell-free culture supernatant comprising growing a  Methylobacterium  selected from the group consisting of NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, NLS0089, derivatives thereof, and  Methylobacterium  related thereto in a culture comprising a liquid or an emulsion and separating the  Methylobacterium  from the culture supernatant, thereby obtaining a  Pratylenchus -inhibitory cell-free culture supernatant. 
     
     
         24 . The method of  claim 23 , further comprising the step of concentrating the cell-free supernatant. 
     
     
         25 . The method of  claim 23 , further comprising the step of fractionating to cell-free supernatant. 
     
     
         26 . The method of  claim 23 , further comprising the step of concentrating the cell-free supernatant. 
     
     
         27 . The method of any one of  claims 23 - 26 , wherein the culture comprising a liquid or an emulsion further comprises a solid substance. 
     
     
         28 . A  Pratylenchus -inhibitory cell-free culture supernatant, fraction thereof, or concentrate therefrom obtained by the method of any one of  claims 23 - 27 . 
     
     
         29 . A method for controlling Root Lesion Nematode (RLN) damage to a plant comprising:
 (i) applying a composition comprising an RLN-inhibitory  Methylobacterium  sp. isolate selected from the group consisting of  Methylobacterium  NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, NLS0089, derivatives thereof, and  Methylobacterium  related thereto to soil where a plant is growing or will be grown; and,   (ii) growing a plant or a plant from seed in soil subjected to the application of the composition and in the presence of RLN, wherein RLN damage sustained by the plant grown in the presence of the RLN is reduced in comparison to a control plant grown in the presence of the RLN.   
     
     
         30 . The method of  claim 29 , wherein the composition comprises a solid substance with adherent RLN-inhibitory  Methylobacterium  grown thereon at a titer of about 5×10 8 , 1×10 9 , or 1×10 10  colony-forming units per gram to about 5×10 13  colony-forming units of  Methylobacterium  per gram, wherein the adherent RLN-inhibitory  Methylobacterium  is selected from the group consisting of  Methylobacterium  NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, NLS0089, derivatives thereof, and  Methylobacterium  related thereto. 
     
     
         31 . The method of  claim 29 , wherein the composition comprises the RLN-inhibitory  Methylobacterium  sp. at a titer of about 1×10 4  or 1×10 5  colony-forming units per ml to about 1×10 9 , 1×10 10 , 1×10 11 , 5×10 11 , or 1×10 12  colony-forming units of  Methylobacterium  per mL of a liquid or emulsion. 
     
     
         32 . The method of  claim 29 , wherein the RLN-inhibitory  Methylobacterium  sp. is a  Methylobacterium  isolate selected from the group consisting of  Methylobacterium  NLS0021, NLS0038, NLS0069, derivatives thereof, and  Methylobacterium  related thereto. 
     
     
         33 . The method of any one of  claims 29 - 32 , wherein the composition is applied to the soil by broadcasting the composition, by drenching the soil with the composition, or by depositing the composition in furrow. 
     
     
         34 . The method of  claim 33 , wherein composition is deposited in furrow and wherein the depositing in furrow is performed prior to placing seed in the furrow, at the same time as placing seed in the furrow, or after placing seed in the furrow. 
     
     
         35 . The method of any one of  claims 29 - 32 , wherein the composition further comprises a nematicide that provides for inhibition of RLN growth, motility, and/or reproduction and/or reductions in RLN-mediated plant damage. 
     
     
         36 . The method of  claim 35 , wherein the nematicide is selected from the group consisting of an organophosphate, biological, and a carbamate nematicide. 
     
     
         37 . A method for reducing the populations of Root Lesion Nematodes (RLN) in soil comprising:
 (a) applying a composition comprising a  Methylobacterium  selected from the group consisting of NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, NLS0089, derivatives thereof, and  Methylobacterium  related thereto and an agriculturally acceptable excipient and/or an agriculturally acceptable adjuvant to a plant part to obtain a treated plant part; and   (b) growing the plant from said treated plant part in the presence of RLN, wherein RLN populations are reduced in soil where the plant was grown in comparison to soil where a control plant from a control plant part that is not treated with the  Methylobacterium  was grown.   
     
     
         38 . The method of  claim 37 , wherein the NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089 derivative is obtained by mutagenizing or transforming  Methylobacterium  NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089, respectively. 
     
     
         39 . The method of  claim 37 , wherein the NLS0021, NLS0038, NLS0042, NLS0934, NLS0062, NLS0069, or NLS0089-related  Methylobacterium  is characterized by having a gene encoding a 16S RNA that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, 99.9%, or 100% sequence identity across the entire length of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, respectively. 
     
     
         40 . The method of  claim 37 , wherein the  Methylobacterium  is heterologous to the plant part. 
     
     
         41 . The method of  claim 37 , wherein the  Methylobacterium  is: (i) NLS0021 and the plant part is not a lettuce plant part; (ii) NLS0038 and the plant part is not a tomato plant part; (iii) NLS0042 or NLS0934 and the plant part is not a soybean plant part; (iv) NLS0062 or NLS0069 and the plant part is not a corn plant part; or (v) NLS0089 and the plant part is not a broccoli plant part. 
     
     
         42 . The method of  claim 37 , wherein the plant part is selected from the group consisting of a  Brassica  sp. corn, wheat, rye, rice, alfalfa, sorghum, millet, soybean, tobacco, potato, peanut, carrot, cotton, coffee, coconut, pineapple, sugar beet, strawberry, oat, barley, tomato, lettuce, pepper, pea, onion, green bean, and cucurbit plant part. 
     
     
         43 . The method of any one of  claims 37 - 42 , wherein RLN populations are reduced in soil at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 85%, or at least about 95% in comparison to soil where a control plant from a control plant part that is not treated with the  Methylobacterium  was grown.