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 medium comprising a soil inhibitor;
(b) harvesting at least some of the resulting PASM cells and growing the harvested PASM cells in or on a second medium comprising the soil inhibitor;
(c) repeating step (b) at least one time; and
(d) selecting at least one PASM cell that is different compared to the genetically-uniform population.
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 soil inhibitor is an antimicrobial compound that is antimicrobial with respect to the genetically-uniform population of PASM cells grown in step (a) and that is not plant-derived.
4 . The method of claim 1 , wherein the soil inhibitor is selected from the group consisting of a microbially-derived soil inhibitor, a nematode-derived soil inhibitor, a fertilizer, nitrogen or a pesticide.
5 . The method of claim 4 , wherein the soil inhibitor is 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, hydrogen cyanide, or pyoluteorin.
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 soil inhibitor;
(ii) an increased resistance or novel immunity to the soil inhibitor, wherein the soil inhibitor is antimicrobial; and/or
(iii) an increased or novel ability to metabolize the soil inhibitor;
compared to the genetically-uniform population of PASM cells grown in step (a).
8 . The method of claim 1 , wherein the second 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 medium.
9 . The method of claim 1 , wherein the PASM cells are bacterial cells or fungal cells.
10 . The method of claim 9 , wherein the PASM cells are selected from the group consisting of Proteobacteria, Firmicutes, Actinobacteria, and Ascomycota.
11 . The method claim 10 , wherein the PASM cells are Pseudomonas, Enterobacter, Stenotrophomonas, Burkholderia, Rhizobium, Herbaspirillum, Pantoea, Serratia, Rahnella, Azospirillum, Azorhizobium, Azotobacter, Duganella, Delftia, Bradyrhizobiun, Sinorhizobium Halomonas, Bacillus, PaeniBacillus, LactoBacillus, Mycoplasma, Acetobacterium, Streptomyces, Rhodococcus, Microbacterium, and Curtobacterium, Trichoderma, Ampelomyces, Coniothyrium, Paecoelomyces, Penicillium, Cladosporium, Hypocrea, Beauveria, Metarhizium, Verticullium, Cordyceps, Pichia, Candida, Coprinus, Corticium, Agaricus, Pythium, Mucor, or Mortierella.
12 . The method of claim 10 , wherein the PASM cells are Firmicutes.
13 . The method of claim 12 , wherein the Firmicutes are Bacillus or PaeniBacillus.
14 . The method of claim 1 , further comprising fermenting the selected at least one PASM cell.
15 . The method of claim 1 , further comprising applying the selected at least one PASM cell to a plant.
16 . A method for producing PASM cells, comprising:
(i) growing a genetically-uniform population of PASM cells in a chemostat in a medium comprising a soil inhibitor; and
(ii) selecting at least one PASM cell that is different compared to the genetically-uniform population.
17 . The method of claim 16 , wherein the soil inhibitor is an antimicrobial compound that is antimicrobial with respect to the genetically-uniform population of PASM cells grown in step (a) and is not plant-derived.
18 . The method of claim 17 , wherein the soil inhibitor is selected from the group consisting of a microbially-derived soil inhibitor, a nematode-derived soil inhibitor, a fertilizer, nitrogen or a pesticide.
19 . The method of claim 18 , wherein the soil inhibitor is 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, hydrogen cyanide, or pyoluteorin.
20 . The method of claim 16 , wherein the at least one PASM cell is selected on the basis of increased tolerance to a microbially-derived soil inhibitor.
21 . The method claim 16 , further comprising sampling the PASM cells from an outflow of the chemostat to monitor genetic and/or phenotypic changes in the PASM cells.
22 . The method claim 16 , wherein the concentration of the soil inhibitor in the medium is increased during the growth of the PASM cells to increase selective pressure.