Microbes and methods for producing the same
The disclosure is generally directed to methods for screening, identifying, and producing microorganisms capable of imparting beneficial properties to plants. In some aspects, improved plant-associated soil microorganisms are generated by experimental evolution using a plant root exudate or root exudate compound.
1. A method for producing plant-associated soil microbial (PASM) cells, comprising:
(a) growing a genetically uniform population of PASM cells in or on a first microbial growth medium comprising a plant root exudate compound;
(b) harvesting at least some of the resulting PASM cells and growing the harvested PASM cells in or on a second microbial growth medium comprising the plant root exudate compound;
(c) optionally repeating step (b) at least one time; and
(d) selecting at least one PASM cell that is different compared to the genetically uniform population based on microbial traits.
2. The method of claim 1 , wherein the PASM cells are grown to log phase during at least one of the growth phases required by steps (a) or (b).
3. The method of claim 1 , wherein the plant root exudate compound is an antimicrobial compound that is antimicrobial with respect to the genetically uniform population of PASM cells grown in step (a).
4. The method of claim 1 , wherein the plant root exudate compound is one or more of the following:
(i) a phenol, a benzoxazinone, a flavonoid or isoflavonoid, a tannin, a coumarin, a terpenoid, an alkaloid, a t-cinnamic acid, a ferulic acid, a p-coumaric acid, a vanillic acid, a syringic acid, a 4-hydroxyphenylacetic acid, an indoleacetic acid, a benzoic acid, and a rosmarinic acid; and
(ii) a plant-derived monosaccharide, amino acid, peptide, protein, carbohydrate, sugar alcohol and organic acid.
5. The method of claim 4 , wherein the plant root exudate compound is an isoflavonoid selected from the group consisting of coumestrol, genistein, glycitein and daidzein.
6. The method of claim 1 , wherein the at least one PASM cell is selected on the basis of one or more of the following microbial traits:
(i) an increased growth rate;
(ii) an increased cell length and/or cell size;
(iii) an increased biomass; and
(iv) enhanced ability to form biofilms
compared to the genetically uniform population of PASM cells grown in step (a).
7. The method of claim 1 , wherein the at least one PASM cell is selected on the basis of one or more of the following:
(i) an increased resistance or novel immunity to the root exudate compound;
(ii) an increased resistance or novel immunity to the root exudate compound, wherein the root exudate compound is antimicrobial; and
(iii) an increased or novel ability to metabolize the root exudate compound;
compared to the genetically uniform population of PASM cells grown in step (a).
8. The method of claim 1 , wherein the second microbial growth medium is a liquid medium and the at least one PASM cell is selected on the basis of the optical density of the PASM cells grown in the second microbial growth medium.
9. The method of claim 1 , wherein the PASM cells are bacterial cells or fungal cells.
10. The method of claim 1 , further comprising fermenting the selected at least one PASM cell.
11. The method of claim 1 , further comprising applying the selected at least one PASM cell to a plant, a plant part or a locus surrounding the plant.
12. The method of claim 1 , wherein the plant root exudate compound is from a soybean plant.
13. A method for producing PASM cells, comprising:
(i) growing a genetically uniform population of PASM cells in a chemostat in a microbial growth medium comprising a plant root exudate compound; and
(ii) selecting at least one PASM cell that is different compared to the genetically uniform population based on microbial traits.
14. The method of claim 13 , wherein the plant root exudate compound is an antimicrobial compound that is antimicrobial with respect to the genetically uniform population of PASM cells grown in step (a).
15. The method of claim 13 , wherein the plant root exudate compound is one or more of the following:
(i) a phenol, a benzoxazinone, a flavonoid or isoflavonoid, a tannin, a coumarin, a terpenoid, an alkaloid, a t-cinnamic acid, a ferulic acid, a p-coumaric acid, a vanillic acid, a syringic acid, a 4-hydroxyphenylacetic acid, an indoleacetic acid, a benzoic acid, and a rosmarinic acid; and
(ii) a plant-derived monosaccharide, amino acid, peptide, protein, carbohydrate, sugar alcohol and organic acid.
16. The method of claim 15 , wherein the one or more plant root exudate compound is an isoflavonoid selected from the group consisting of coumestrol, genistein, glycitein and daidzein.
17. The method of claim 15 , wherein the at least one PASM cell is selected on the basis of increased tolerance to the isoflavonoid.
18. The method of claim 13 , further comprising sampling the PASM cells from an outflow of the chemostat to monitor genetic and/or phenotypic changes in the PASM cells.
19. The method of claim 13 , wherein the concentration of the root exudate compound in the medium is increased during the growth of the PASM cells to increase selective pressure.