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 (Mamidi et al. 2014), 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), and antagonize pathogens (Zhang et al. 2018). 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).
References.
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.
Zeng, J., Xu, T., Cao, L., Tong, C., Zhang, X., Luo, D., Han, S., Pang, P., Fu, W., Yan, J. and Liu, X., 2018. The role of iron competition in the antagonistic action of the rice endophyte Streptomyces sporocinereus OsiSh-2 against the pathogen Magnaporthe oryzae. Microbial ecology, 76, pp.1021-1029.
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. In FY24, we cultured many of the microbial groups that we found to be responsive to responsive to IDC and optimized a pot assay to test their ability to reduce IDC in soybeans. Excitingly, we found that a cocktail of responsive microbial isolates significantly reduced IDC. Recently, in ongoing FY25 funding, we have now narrowed down IDC-reduction to four individual responsive taxa, each of which significantly reduces IDC when inoculated in pots. These include two members of Variovorax (ASV105 and ASV102), Cellvibrio (ASV64) and Pseudoxanthomonas (ASV27). In this study, we will evaluate the efficacy of each of these microbes when applied in field trials as co-inoculants with the rhizobium inocuant Bradyrhizobium japonicum USDA110.
Objective 1) Evaluate soybean IDC reduction and yield improvement following application of four IDC-reducing microbial inoculants. The central aim of this project is to test the four microbial isolates for their potential to reduce IDC in the field. To do so, we will perform field trials of 2 row plots that are 15 foot long and at 30 inch row spacings. Plots will be planted at 140,000 seeds/ac. The trials will be performed at an IDC inducing location (such as Colfax or Leonard) that is also used by the breeding program for IDC variety trials. Trials will also be conducted in two fields without a history of IDC. The trials will include two soybean lines; one IDC sensitive (ND17009-GT) and one IDC-resistant (A11). We will use six different treatments. These will include 1) no addition (negative control), 2) Variovorax ASV105, 3) Variovorax ASV102, 4) Cellvibrio ASV64, 5) Pseudoxanthomonas 6) Soygreen® fertilizer (positive control). Each treatment will also include Bradyrhizobium japonicum USDA110 liquid rhizobium inoculant. The treatments will be applied in six independent replicates, arranged in a randomized complete block design. Microbes will be grown and applied directly to the seeds as liquid inoculants before planting. The cones of the planter will be sterilized with ethanol between each treatment. Soygreen® will also be combined with seeds in envelopes before planting. At six weeks after planting, IDC development will be evaluated using the IDC rating scale used to assess IDC-resistance in the breeding program, as well as using an SPAD chlorophyll meter. After the crop matures, plots will be harvested by small-plot combine, and yield will be adjusted to 13% moisture. Changes in yield will be evaluated for each treatment.
Objective 2) Evaluate changes in nodulation in response to inoculation with IDC-reducing microbes. From previous work, we have observed a significant reduction in nodulation associated with IDC in soybeans. Therefore, we also wish to assess the synergy between IDC-reducing microbes and rhizobia by evaluating the nodulation of soybeans in the field trial above. To do so, 5 plants will be collected from each plot 6 weeks post-planting. The nodules will be collected and the nodule number and mass will be analyzed and compared amongst the treatments.
Objective 3) Evaluate changes in disease (root-rot) in response to inoculation with IDC-reducing microbes. Some research into iron-chelation has indicated that microbes with the capacity to chelate iron may also provide increased protection from pathogens. Therefore, we will also evaluate the effects of these describe microbes on the development of seedling diseases and root rots. Around the V1-V2 growth stage, stand counts will be performed to determine the germination rate. Plots will also be evaluated for root rot severity (%) by sampling and rating 10 seedlings from the front and 10 seedlings from the back of each plot. These will follow a standard root rot severity on a scale of 1-5 of increasing severity. Plots will also be evaluated for vigor at this growth stage on a 1-5 scale. In conjunction with Objectives 1 and 2, the five collected plants 6 weeks after planting will also be evaluated for root rot.
d. Potential barriers.
Since IDC can routinely change due to weather conditions, there is risk some site may not develop IDC symptoms as expected, therefore we will plant the trial in two to three locations to ensure at least one location shows IDC symptoms.
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
May to September 2024: Field trial planting, maintenance and data collection.
October 2025 to April 2026: Data analysis and manuscript preparation.