Accelerated directed evolution of microbial consortia for the development of desirable plant phenotypic traits

1. An iterative plant phenotypic trait based screening method for selecting one or more microorganisms capable of imparting at least one beneficial phenotypic trait to a plant, comprising:

a) subjecting a plurality of plants to a growth medium in the presence of a first set of one or more microorganisms;

b) growing the plurality of plants in said growth medium;

c) selecting one or more plants from said plurality, following step b), based upon a beneficial plant phenotypic trait selection criteria, wherein said selected one or more plants exhibits the at least one or more beneficial phenotypic trait, as compared to other plants of said plurality;

d) acquiring a second set of one or more microorganisms from said one or more plants, or from said one or more plants and the growth medium containing said one or more plants, selected in step c);

e) repeating steps a) to d) one or more times, wherein the second set of one or more microorganisms acquired in step d) is used as the first set of microorganisms in step a) of any successive repeat; and

f) selecting one or more microorganisms that is associated with imparting a beneficial phenotypic trait to a plant.

2. The method according to claim 1 , wherein the selecting one or more microorganisms of step f) comprises:

isolating one or more microorganisms associated with imparting a beneficial phenotypic trait to a plant;

utilizing a molecular technique to characterize the one or more microorganisms isolated; and

selecting one or more characterized microorganisms that is associated with imparting a beneficial phenotypic trait to a plant.

3. The method according to claim 2 , wherein at least two microorganisms associated with imparting a beneficial phenotypic trait to a plant are isolated, characterized, selected, and combined into a microbial consortium.

4. The method according to claim 1 , wherein two or more microorganisms are acquired in step d), and further comprising:

i) separating the two or more microorganisms into individual isolates;

ii) selecting two or more individual isolates; and

iii) combining the selected two or more isolates,

wherein the combined isolates are used as the first set of one or more microorganisms in step a) of any successive repeat of the method.

5. The method according to claim 1 , wherein the one or more plants selected from the plurality of plants is at least one selected from the group consisting of: a plant part, seed, cutting, propagule, and combination thereof.

6. The method according to claim 1 , wherein the second set of one or more microorganisms are acquired from said one or more plants in step d).

7. The method according to claim 1 , wherein the second set of one or more microorganisms are acquired from the root, stem, and/or foliar tissue from said one or more plants in step d).

8. The method according to claim 1 , wherein the second set of one or more microorganisms are acquired from the plant rhizosphere in step d).

9. The method according to claim 1 , wherein the second set of one or more microorganisms acquired in step d) includes a hard to culture microorganism.

10. The method according to claim 1 , wherein the plurality of plants is ryegrass and the selecting of step c) is based upon selecting the ryegrass plants with the largest biomass.

11. The method according to claim 1 , wherein the plurality of plants is ryegrass and the selecting of step c) is based upon selecting the ryegrass plants with the largest biomass, and wherein the one or more microorganisms comprise a member selected from the group consisting of: Microbacterium ginsengiterrae, Bacillus cereus, Microbacterium oxydans, Rhizobium pusense, Curtobacterium ginsengisoli Penicillium daleae, Brevundimonas vesicularis, Aeromicrobium ponti, Microbacterium hydrocarbonoxydans, Sphingopyxis chilensis, Arthrobacter keyser, Penicillium melinii Rhizobium grahamii, Brevundimonas vesicularis, Rhizobium pusense, Curtobacterium ginsengisoli, Herbaspirillum rubrisubalbicans, Rhizobium etli, Exiguobacterium indicum, Mesorhizobium amorphae, Brevundimonas vesicularis, Arthrobacter keyser , and combinations thereof.

12. The method according to claim 1 , wherein the plurality of plants is basil and the selecting of step c) is based upon selecting the basil plants with the greatest median sugar content.

13. The method according to claim 1 , wherein the plurality of plants is basil and the selecting of step c) is based upon selecting the basil plants with the greatest median sugar content, and wherein the one or more microorganisms comprise a member selected from the group consisting of: Sphingomonas mali, Flavobacterium micromati, Penicillium sp., Sphingobium chlorophenolicum, Massilia niastensis, Flavobacterium limicola, Rhizobium alamii, Sphingopyxis sp., Pelomonas aquatica, Azospirillum lipoferum, Mesorhizobium amorphae, Asticcacaulis taihuensis, Ralstonia solanacearum, Microbacterium foliorum, Trichoderma, Burkholderia megapolitana, Mesorhizobium amorphae, Umbelopsis sp., Aquabacterium fontiphilum, Rhodanobacter terse, Sphingomonas mali, Sphingobium xenophagum, Pseudomonas moraviensis, Massilia niastensis, Flavobacterium limicola, Umbelopsis sp., and combinations thereof.

14. The method according to claim 1 , wherein the plurality of plants is maize and the selecting of step c) is based upon selecting the maize plants with the largest biomass.

15. The method according to claim 1 , wherein the plurality of plants is maize and the selecting of step c) is based upon selecting the maize plants with the largest biomass, and wherein the one or more microorganisms comprise a member selected from the group consisting of: Herbaspirillum frisingense, Acinetobacter sp., Xanthomonas translucens, Pseudomonas marginalis, Herbiconiux ginsengi, Burkholderia cepacia, Microbacterium oxydans, Pseudomonas moraviensis, Azotobacter chroococcum, Pseudomonas frederiksbergensis, Sphingomonas rosa, Rhizobium endophyticum, Bacillus thioparans, Terriglobus roseus, Novosphingobium rosa Azospirillum lipoferum, Streptomyces thermocarboxydus, Herbaspirillum frisingense , and combinations thereof.

16. An iterative plant phenotypic trait based screening method of creating a microbial consortium capable of promoting at least one beneficial plant phenotypic trait, comprising:

a) subjecting at least one plant to a growth medium, in the presence of a first plurality of microorganisms, and growing said plant in the growth medium;

b) selecting at least one plant following step a) based upon a beneficial plant phenotypic trait selection criteria, wherein said selected plant exhibits the beneficial phenotypic trait, as compared to other plants of said plurality;

c) acquiring a second plurality of microorganisms from said at least one plant selected in step b);

d) repeating steps a) to c) one or more times, wherein the second plurality of microorganisms acquired in step c) is used as the first plurality of microorganisms in step a) of any successive repeat;

e) isolating at least two microorganisms from said plurality of microorganisms that are associated with promoting at least one beneficial plant phenotypic trait;

f) utilizing a molecular technique to characterize the at least two isolated microorganisms;

g) selecting at least two microorganisms from the characterized microorganisms; and

h) combining the at least two selected microorganisms into a microbial consortium.

17. The method of claim 16 , wherein the characterization of step f), comprises: determining the relative abundance of the at least two microorganisms that are associated with promoting at least one beneficial plant phenotypic trait.

18. The method of claim 16 , wherein the characterization of step f), comprises: determining the relative abundance of the at least two microorganisms that are associated with promoting at least one beneficial plant phenotypic trait; and

wherein the selecting of step g), comprises: choosing at least two microorganisms from the characterized microorganisms, based on an increase in their relative abundance compared to their abundance from a previous iteration of steps a)-f).

19. The method of claim 18 , wherein the at least two microorganisms chosen comprise microorganisms whose relative abundance increased significantly relative to other microorganisms.

20. The method according to claim 1 , wherein said first set of one or more microorganisms in step a) are obtained by: subjecting a plurality of plants to a growth medium in the presence of one or more microorganisms, growing the plurality of plants in said growth medium, and acquiring said first set of one or more microorganisms from the plurality of plants or growth medium containing said plurality.

21. The method according to claim 1 , wherein the second set of one or more microorganisms are acquired from the stem from said one or more plants in step d).

22. The method according to claim 1 , wherein the second set of one or more microorganisms are acquired from the foliar tissue from said one or more plants in step d).

23. The method according to claim 1 , wherein the second set of one or more microorganisms are acquired from the root and stem from said one or more plants in step d).

24. The method according to claim 1 , wherein the second set of one or more microorganisms are acquired from the root and foliar tissue from said one or more plants in step d).

25. The method according to claim 1 , wherein the second set of one or more microorganisms are acquired from the root, stem, and foliar tissue from said one or more plants in step d).

26. An iterative plant phenotypic trait based screening method for selecting one or more microorganisms capable of imparting at least one beneficial phenotypic trait to a plant, comprising:

a) subjecting a plurality of plants to a growth medium in the presence of a first set of one or more microorganisms;

b) growing the plurality of plants in said growth medium;

c) selecting one or more plants from said plurality, following step b), based upon a beneficial plant phenotypic trait selection criteria, wherein said selected one or more plants exhibits the at least one or more beneficial phenotypic trait, as compared to other plants of said plurality;

d) acquiring a second set of one or more microorganisms from said one or more plants selected in step c);

e) repeating steps a) to d) one or more times, wherein the second set of one or more microorganisms acquired in step d) is used as the first set of microorganisms in step a) of any successive repeat; and

f) selecting one or more microorganisms that is associated with imparting a beneficial phenotypic trait to a plant.

27. The method according to claim 26 , wherein the plurality of plants is selected from the group consisting of ryegrass, basil, and maize.