Iron deficiency chlorosis (IDC) is the symptom typically observed in soybean growing in high-pH soils with high carbonate concentrations where iron availability is reduced. Currently, the most important management option is the selection of ID-resistant cultivars. Several studies have detected QTL that confers resistance to IDC.
Other limitations on soybean production are due to soybean diseases that reduce yield. Several species of Fusarium are well recognized as soybean pathogens, such as F. virguliforme, the causal agent of sudden death syndrome (SDS), F. oxysporum causing Fusarium wilt or F. graminearum, a major necrotrophic pathogen causing severe root rot. F. graminearum also is the most frequently recovered species of Fusarium in fields in Iowa. Management of soil-borne diseases like Fusarium root rot depends mainly on genetic resistance or seed treatments during emergence and the seedling stages. Soybean resistance to F. graminearum was described in the soybean cultivar Conrad, and putative Quantitative Trait Loci (QTL) associated with resistance to F. graminearum were.
Although soybean plants with IDC symptoms often display Fusarium root rot symptoms, currently the basis of this association is not clear. In general, research programs focus on identifying resistance to a particular stress and do not test susceptibility to other biotic or abiotic stresses. Consequently, improved varieties may respond unpredictably when grown in field conditions.

The objective of this study is to characterize soybean genes that are differentially regulated by the host during F. graminearum infection in an iron deficiency environment in order to identify new potential resistant mechanisms and candidate genes involved in the defense response.
Our specific objectives are to:
1. Evaluate phenotypically IDC-resistant and susceptible soybean cultivars inoculated with F. graminearum under iron deficiency conditions in a hydroponic system.
2. To elucidate the comprehensive gene expression in response to the pathogen and iron deficiency simultaneously (RNA seq analysis).
3. Identify, compare, and analyze differentially expressed genes.

To our knowledge, transcriptional changes in soybean roots that have been infected by F. graminearum growing in an environment with low iron availability have not been studied.
The finding generated will be useful for determining environmental conditions and stress factors on the epidemiology of soilborne pathogens that affect seedlings and taking steps toward identifying effective management. In addition, these results may be useful in developing new methods of broadening the resistance of soybean to F. graminearum and iron deficiency.

Updated October 1, 2024:
To characterize genetically the soybean response to abiotic stress (iron deficiency) and biotic stress (F. graminearum-FG infection), first, we worked on the optimization of a screening method to evaluate simultaneous stress effects in a hydroponic system. We standardize the response of soybean near-isogenic lines Clark (PI548553, iron efficient) and IsoClark (PI547430, iron inefficient) under combined iron deficiency+ Fg inoculum conditions. Three weeks after planting, the growth parameters and chlorophyll content were measured. Detailed root morphological and architectural measurements will be analyzed by WinRHIZO root-scanning software.
The RNA extraction was realized from root and leaf tissues using Qiagen RNeasy and Min-Elute kits. After running the quality checks on the samples, they will be submitted to the Iowa State University DNA Facility for library construction and sequencing (RNA seq). The bioinformatic analysis will allow us to identify, compare, and cluster differentially expressed genes between roots and leaves within each genotype for each treatment.

A 3-month no cost extension was granted on the project to complete sample processing.

Updated May 6, 2025:

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In the north central U.S., the calcareous soil favors the development of Iron deficiency chlorosis (IDC) in soybeans. An additional problem is that soybean plants with IDC symptoms often display Fusarium root rot (FRR) symptoms, caused by Fusarium graminearum, a major necrotrophic fungus and the most frequently recovered species of Fusarium in fields in Iowa. The combined impact of IDC and FRR results in yield loss, leading to substantial economic losses for soybean growers. Currently, the basis of the IDC-FRR association is not clear, and management becomes more complicated when they coexist. In general, research programs focus on identifying resistance to a particular stress and not multiple stress conditions. Consequently, improved varieties may respond unpredictably when grown in field conditions. To investigate soybean responses to both stresses, IDC and FRR, soybean lines Clark (iron efficient) and IsoClark (iron inefficient) were compared. F. graminearum inoculated and non-inoculated seeds were germinated on germination paper for 7 days. The seedlings were transferred to a hydroponic system and grown in iron-sufficient and iron-deficient nutrient solutions for 2 weeks. F. graminearum infestation and iron deficiency consistently impacted growth parameters, root morphological characteristics, and content of chlorophyll. Interestingly, the pathogen may strongly affect Clark (iron efficient cultivar) than IsoClark.

We also collected root and leaf tissue samples and we extracted the RNA for gene expression analysis (RNA seq analysis). Sequencing generated 1.9 billion reads, 1.7 billion of which (~84%) mapped uniquely to the soybean genome. The large quantity of sequence data generated by some of the samples meant the standard analysis pipeline failed at a critical juncture due to lack of computational power. Accordingly, we developed a novel method to handle the data that was much less computationally intensive and will be incredibly helpful in saving time and computational power for future experiments. Overall, we were able to determine that 33659 genes are expressed in leaves and 35028 genes are expressed in roots. We identified soybean genes in Clark and IsoClark responding to single stresses (iron deficiency and fungal infection) and combinatorial stresses (Fusarium infection + iron deficiency). The response to iron deficiency in Clark and IsoClark is well characterized by previous publications, and we will compare the genes identified in this study to previous studies to confirm that iron stress was induced. However, it is necessary to evaluate the impact of F. graminearum on IDC-tolerant/susceptible soybeans. We identified 9315 genes differentially expressed due to Fusarium infection in Clark leaves and 4765 genes in IsoClark leaves. Similarly, we identified 8195 genes differentially expressed due to the pathogen in Clark roots and 6407 genes differentially expressed in IsoClark roots.

To investigate how soybeans respond to iron deficiency when previously inoculated with F. graminearum, we compared genes differentially expressed in plants inoculated with the fungus in sufficient and deficient iron conditions. These analyses identified 1425 genes in leaves and 1499 genes in roots of Clark and IsoClark combined. However, visualization of these genes showed that the majority of the genes were expressed in the same direction under both stresses, indicating a similar or possible additive response to combined stresses. We are particularly interested in genes with opposing expression patterns under the two conditions. Initial investigations have identified 781 genes differentially expressed of interest in leaves and 190 in roots (depicted by * on the heat maps). Further investigation and characterization of these genes will continue as we prepare a manuscript for publication, i.e., to assign the biological roles to the identified gene clusters related to defense, iron homeostasis, cell cycle, gene silencing, and photosynthesis.

CONCLUSIONS
What new knowledge was developed/discovered in the research supported by the ISRC?
• We have developed a novel bioinformatics pipeline to manipulate the large file sizes generated in this experiment. This is going to greatly improve the speed at which future analysis takes place (~3 hrs for one step down to 1 min).
• We have identified genes differentially expressed due to Fusarium infection in Clark (9315 genes) and IsoClark (4765 genes). These genes can be mined to better understand the infection and plant response to Fusarium infection.
• We have identified 781 genes in leaves and 190 in roots with expression patterns that change direction under iron deficiency stress and Fusarium infection. These genes indicate an interaction between iron deficiency and Fusarium infection that has not been previously reported.

What are practical applications of the research? How long?
• The genes identified by this research immediately become high-priority genes to characterize and investigate in future iron and Fusarium studies. While tissue for this study was collected after extended stress, studies in iron deficiency have shown similar genes to be of high interest in understanding the soybean iron stress response.

How might this new knowledge/discovery affect the success of positive impact for industry and/or farmers?
• The knowledge provides the first evidence that there is an interaction between Fusarium infection and iron deficiency. Further, these results provide insight into how soybean responds to Fusarium infection. Understanding the soybean responses to these stresses is crucial for more effective management.
• In addition, these results may be useful in developing new methods of broadening the resistance of soybean to F. graminearum and iron deficiency.

SUPPORTING ATTACHMENTS CAN BE FOUND IN THE ATTACHED FILE

Results may be useful in developing new methods of broadening the resistance of soybean to F. graminearum and iron deficiency.