Gnotobiotic rhizobacterial isolation plant systems and methods of use thereof
The invention provides methods of enriching and isolating novel microbial organisms, such as rhizobacteria, that promote the growth of plants by recreating the rhizosphere in a laboratory setting. The methods of the invention can be used to produce and characterize beneficial microbes grown at the root-soil interface and provide a technique for selectively enriching bacteria that live in close association with plants and foster the growth thereof.
1. A method of isolating one or more plant growth-promoting rhizobacteria (PGPR) from an environmental sample, the method comprising:
a) contacting an inoculum comprising the environmental sample with a surface-sterilized seed, wherein the environmental sample is obtained from the rhizosphere, rhizoplane, or endosphere of a plant, and wherein the environmental sample comprises diverse PGPR;
b) propagating the surface-sterilized seed in a gnotobiotic rhizobacterial isolation plant system (GRIPS), wherein the GRIPS produces a simplified model plant microbiome having controllable microbial diversity; and
c) isolating PGPR from the GRIPS.
2. The method of claim 1 , wherein the GRIPS comprises a growth medium that has been sterilized by autoclaving, fumigation, or exposure to gamma radiation.
3. The method of claim 2 , wherein the growth medium comprises soil.
4. The method of claim 3 , wherein the soil is field soil or artificial soil.
5. The method of claim 3 , wherein the growth medium further comprises sand.
6. The method of claim 2 , wherein the growth medium is maintained in round-bottom or cylindrical glass tube.
7. The method of claim 1 , wherein the is PGPR are selected from the group consisting of unclassified Bacilli, Naxibacter, Bacillus , unclassified Geobacilli, Tumebacillus, Paenibacillus, Chitinophaga, Methylobacterium , and Sphingopyxis.
8. The method of claim 7 , wherein the bacterium is a Bacillus.
9. The method of claim 1 , wherein the surface of the seed has been sterilized by contacting the surface with ethanol.
10. The method of claim 1 , wherein the inoculum comprises a serial dilution of the environmental sample sufficient to permit inoculation of the seed with the PGPR.
11. The method of claim 1 , wherein the inoculum comprises soil and water.
12. The method of claim 1 , wherein the inoculum is obtained by mixing soil comprising a homogeneous or heterogeneous population of rhizobacteria and water to form a suspension.
13. The method of claim 12 , the method further comprising diluting the suspension.
14. The method of claim 13 , wherein the suspension is diluted by a factor of from 10 to 100,000.
15. The method of claim 1 , the method further comprising exposing the inoculum to heat or ethanol.
16. The method of claim 2 , the method further comprising adding water to the growth medium during the incubating.
17. The method of claim 2 , the method further comprising contacting the growth medium with one or more antibiotics during the incubating.
18. The method of claim 2 , the method further comprising contacting the growth medium with one or more selected from the group consisting of chitin, 1-aminocyclopropane-1-carboxylic acid, and insoluble phosphate.
19. The method of claim 1 , the method further comprising maintaining the seedling within the GRIPS for about 2 weeks following germination of the seedling.
20. The method of claim 19 , wherein the seedling is exposed to alternating light/dark cycles while maintained within the GRIPS.
21. The method of claim 1 , wherein the one or more PGPR are isolated from the root of the seedling by:
a) mixing the root with water;
b) separating the mixture formed in step (a) by centrifugation; and
c) inoculating a culture medium with a pellet produced in step (b).
22. The method of claim 21 , the method further comprising maintaining the culture medium under conditions suitable for the growth of the one or more PGPR.