Description of Proposed Research
a. Project Justification and Rationale
Iron deficiency chlorosis (IDC) is a wide-spread problem strongly affecting soybean production in North Dakota. The characteristic yellowing of plant leaves suffering from IDC is caused by a lack of chlorophyll formation due to poor function of iron-requiring enzymes involved in chlorophyl biosynthesis. North Dakota soils normally contain more than enough iron for plant function, however much of the iron is not in soluble form needed by the plant. A reduction in iron solubility at high soil pHs caused by high levels of CaCO3 (lime) in the top-soil is the main cause of IDC; white the iron is there, it isn’t available to the plant. High lime soil is common in North Dakota, and IDC is exacerbated by salinity which is also becoming more and more common.
Despite a decades-long recognition of the problem, few effective solutions are available for farmers. One option involves applying iron fertilizer furrow at planting. But only red chelate fertilizers such as Soygreen (EDDHA) are effective, and these fertilizers are almost impractically expensive for most farmers at a cost of ~$18/acre at the recommended rate – which often needs to be exceeded to alleviate IDC. Some genes in soybeans that confer resistance have been identified and varieties that incorporate these genes can help, though traits to improve IDC tolerance are often not sufficiently incorporated into commercial varieties with other desirable traits such as weed control.
The ability of microbes to solubilize iron, making insoluble iron available to the plant, has long been recognized (Crowley et al. 1988) and represents a new opportunity to combat IDC. Bacteria such as pseudomonads can release siderophore compounds which are effective in solubilizing iron and can improve the iron nutrition of plants (Vansuyt et al. 2007). Such microbes could be cultured and deployed with rhizobia as inoculants to combat IDC. Further, by isolating these microbes from North Dakota soils, the likelihood they will be persistent and effective when growers applying them in our environmental conditions would be enhanced (a “tailored inoculant” approach).
Crowley, D.E., Reid, C.P. and Szaniszlo, P.J., 1988. Utilization of microbial siderophores in iron acquisition by oat. Plant Physiology, 87(3), pp.680-685.
Goos, R.J., Johnson, B., Jackson, G. and Hargrove, G., 2004. Greenhouse evaluation of controlled-release iron fertilizers for soybean. Journal of plant nutrition, 27(1), pp.43-55.
Lewis, R.W., Islam, A.A., Dilla-Ermita, C.J., Hulbert, S.H. and Sullivan, T.S., 2019. High-throughput Siderophore screening from environmental samples: plant tissues, bulk soils, and rhizosphere soils. JoVE (Journal of Visualized Experiments), (144), p.e59137.
Vansuyt, G., Robin, A., Briat, J.F., Curie, C. and Lemanceau, P., 2007. Iron acquisition from Fe-pyoverdine by Arabidopsis thaliana. Molecular Plant-Microbe Interactions, 20(4), pp.441-447.
b. Brief description of Proposed Research
In this study we aim to build on previous work to assess the potential of the soybean microbiome as a new tool to combat IDC. In FY22, in a study that analyzed four fields in Eastern ND with varying levels of IDC, we observed a significant correlation in the structure of the soybean root and rhizosphere microbiome with the IDC level of the soil. We hypothesized that unique groups of microbes that are enriched under IDC conditions could help alleviate IDC in soybeans when cultured and used as inoculants along with root nodule forming rhizobia. We have adapted a greenhouse assay for IDC from Jay Goos that we hope will be suitable for measuring growth potential of microbiome members, and a optimized plate screening assay that can identify siderophore producers (microbe-produced iron solubilizing molecules that function like Fe-chelating fertilizers). In FY24 we have made significant progress in the isolation of a culture collection of IDC-recruited microbes and are currently preparing to screen them for their ability to produce siderophores and reduce IDC in the greenhouse. With this study we aim to bring this research towards translation by identifying the most effective microbes in our collection at reducing IDC as well as improving symbiotic nitrogen fixation under IDC. We will do this with an IDC-induced greenhouse study by applying paired microbial inoculants of IDC-recruited microbes with nitrogen-fixing rhizobia. To ensure the products are able to effectively colonize the soybean microbiome, we will evaluate their persistence in the soybean microbiome throughout the plant lifecycle.
Objective 1) Test elite siderophore-producing IDC-recruited microbes for alleviation of IDC symptoms in a greenhouse assay. From siderophore production screening of our culture collection in FY24, we will select five IDC-recruited microbes with the highest siderophore production as candidates for direct evaluation of IDC reduction in a greenhouse assay. Each microbe will be tested independently using the Jay Goos IDC assay in the greenhouse that we have successfully adapted. Reduction of IDC will be measured based on increased chlorophyl content of soybean leaves inoculated with the microbes.
Objective 2) Evaluate improvement of root nodule symbiosis from co-inoculation with IDC-recruited microbes. Concurrently with the above assay, we will measure the recovery of symbiosis phenotypes. Our data has shown a significant inhibition of nodulation associated with IDC. Therefore, we will include the rhizobium inoculant Bradyrhizobium japonicum USDA110 as a mixed inoculum with potential IDC-reducing strains and screen for improves symbiosis characteristics (increased nodule number and size) along with IDC reduction in Objective 1.
Objective 3) Evaluate persistence of inoculated microbes in the soybean microbiome. It is critical that any microbial product that is to be applied in the field can survive and thrive in the soybean microbiome over time. To evaluate the ability of the candidate IDC_reducing microbes to persist in the soybean microbiome, we will perform amplicon sequencing of the soybean root and rhizosphere microbiome in the experiment above. To track our introduces strains, they will be labeled with a synthetic 16S molecule to allow specific tracking of the relative abundance of each of the introduced strains. The data regarding the persistence of introduced strains could guide the choice of strains for proposed field tests should more than one successfully reduce IDC and improve rhizobium symbiosis.
d. Potential barriers.
While we have successfully demonstrated the ability to induce IDC in the Jay Goos IDC assay, we have not yet proven that microbial inoculation can reduce IDC in this assay (this is ongoing work in FY24). If we are unable to measure reduction of IDC using this assay, we will adapt other assays from the literature using more sterile systems for objectives 1 and 2.
e. Communication and outreach.
The results will be made available to North Dakota Soybean growers by multiple mechanisms. We will communicate results through the ND Soybean Magazine, and we will volunteer to speak about the results at field days. We will also communicate our results on the social media platform twitter.
f. Timeline
July to September 2024: Pilot assays for IDC reduction and symbiosis improvement in the greenhouse, engineering of strains with synthetic 16S molecules to allow tracking in the microbiome.
October 2024 to February 2024: IDC reduction and symbiosis improvement trials with each of five individual microbes.
March 2024 to May 2024: Microbiome sequencing and data analysis