Updated June 30, 2024:
Research Project Title: Potential for combatting iron deficiency chlorosis with the soybean microbiome FY24.
Principle Investigator: Barney Geddes
Research Overview:
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.
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 (see FY22 report). We hypothesize 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 already optimized a greenhouse assay that will be suitable for measuring growth potential of microbiome members, and a plate screening assay that can identify siderophore producers (microbe-produced iron solubilizing molecules that function like Fe-chelating fertilizers). With this study we aim to utilize these resources to attempt to identify individual microbes or groups of microbes, cultures from the IDC soybean microbiome that could have a beneficial effect to soybean plants grown under IDC conditions.
Objectives:
Objective 1) Culture a community of North Dakota microbes from the soybean microbiome.
Objective 2) Siderophore production screen from members of soybean microbial community.
Objective 3) Evaluate reduction of IDC from microbial inoculants with an optimized “Goos” Greenhouse assay.
Materials and Methods:
Objective 1) To address objective 1, we adapted a high throughput culturomics pipeline developed by J. Zhang et al., 2021, to isolate the members of the soybean microbiome. We cultured bacteria from freshly harvested soybean roots using dilution-enrichment culturing techniques. We used a concentration of diluted root homogenates that allowed for approximately 40% of the wells to exhibit bacterial growth originating from a single bacterial cell. Wells with bacterial growth were split for 16S rRNA sequencing and high-quality glycerol stocks. We created our own bioinformatic pipeline to identify matched amplicon sequence variants to those tracked in microbiome data from the field. According to the analysis, the wells with a purity greater than 95% were selected from the preserved glycerol stocks and streaked repeatedly on agar plates. Finally, these isolates were validated by full-length 16S rRNA gene Sanger sequencing before being stocked and stored at the temperature of -80°C.
Objective 2) We have optimized a Chrome Azurol S colorimetric assay (Louden et al., 2011) to identify the siderophore-producing microbes based on orange color formation in the CAS medium. This assay utilizes Chrome Azurol S (CAS) substrate and hexadecyltrimethylammonium bromide (HDTMA) as indicators. The CAS/HDTMA agar media is prepared by mixing it with FeCl3, which serves as a blue dye. We assessed the siderophore-producing ability of each of the 64 soybean bacterial isolates cultured from the high-IDC (Leonard) and no-IDC (Casselton) sites. We cultured these individual microbes on TSOY agar media from their respective freezer glycerol stocks. Following a 72-hour incubation at 28°C, the culture plates were overlayed with CAS/HDTMA-agar media. Siderophore molecules secreted by candidate microbes chelated iron from the CAS/HDTMA complex after 4-6 hours incubation period and transformed color from blue to orange, indicating the potential presence of siderophore-producing microbes.
Objective 3) We optimized a greenhouse assay originally developed by Dr. Jay Goos to evaluate the capacity of our taxonomically diverse SynCom members to promote plant growth under iron-deficient conditions by enhancing access to unavailable iron. The experiment was conducted using a sterile IDC soil/sand potting mixture where 10 mM Nitrate/5 mM Bicarbonate was supplemented to induce iron-deficient conditions as found in calcareous soils. For iron-rich medium, soil/sand mixture was treated with the 10 mM Nitrate/5 mM Bicarbonate and iron-chelating fertilizer Soygreen, an efficient and highly soluble iron source. Two pregerminated soybean seedlings were planted per pot under available and unavailable iron sources with SynCom inoculation and a control group without SynCom inoculation. We included 20 replicated pots per condition. The plants were cultivated on iron-rich or iron-deficient medium for three to four weeks while exposing them to live or no SynCom members.
Research Results and Outcomes:
Objective 1) As a result of our cultivation efforts we successfully cultivated 64 unique isolates from the no-IDC and high-IDC sites (Table 1). This enabled us to selectively choose microbes from our culture collection that demonstrated high levels of colonization of the soybean root and rhizosphere and displayed sequence identity match with the field microbiome data. This broader culture collection serves as a valuable community resource, enabling us to create a well-defined synthetic bacterial community (SynCom) to explore the potential of enriched or depleted microbes during IDC to produce siderophores and/or alleviate IDC stress in soybeans
Table 1. Microbes in the process of culturing for Objective 1.
ASV number Genus Location Year Siderophore production
9 Variovorax robiniae Casselton (No IDC) 2022 -
10 Pseudomonas silesiensis Casselton (No IDC) 2022 +
13 Pseudomonas cerasi Casselton (No IDC) 2022 -
15 Pseudomonas koreensis Casselton (No IDC) 2022 +
24 Pseudarthrobacter sulfonivorans Casselton (No IDC) 2022 -
34 Phyllobacterium ifriqiyense Casselton (No IDC) 2022 -
37 Pseudomonas oryzihabitans Casselton (No IDC) 2022 +
55 Lysobacter antibioticus Casselton (No IDC) 2022 -
61 Pantoea agglomerans Casselton (No IDC) 2022 -
68 Chryseobacterium gregarium Casselton (No IDC) 2022 -
71 Paeniglutamicibacter sulfureus Casselton (No IDC) 2022 -
75 Paenarthrobacter nitroguajacolicus Casselton (No IDC) 2022 -
84 Cellulomonas cellasea Casselton (No IDC) 2022 -
87 Variovorax paradoxus Casselton (No IDC) 2022 -
88 Aeromicrobium ginsengisoli Casselton (No IDC) 2022 -
89 Aeromicrobium ginsengisoli Casselton (No IDC) 2022 -
102 Variovorax paradoxus Casselton (No IDC) 2022 -
105 Variovorax paradoxus Casselton (No IDC) 2022 +
110 Variovorax ureilyticus Casselton (No IDC) 2022 -
120 Massilia agri Casselton (No IDC) 2022 -
126 Rhodococcus qingshengii Casselton (No IDC) 2022 -
129 Curtobacterium pusillum Casselton (No IDC) 2022 -
130 Pseudorhodoferax soli strain TBEA3 Casselton (No IDC) 2022 -
131 Hydrogenophaga intermedia Casselton (No IDC) 2022 -
132 Polaromonas eurypsychrophila Casselton (No IDC) 2022 -
136 Bacillus proteolyticus Casselton (No IDC) 2022 -
23a Pseudomonas brassicacearum Leonard (High IDC) 2022 -
28a Ensifer adhaerens Leonard (High IDC) 2022 -
43 Pseudoxanthomonas japonensis Leonard (High IDC) 2022 -
64 Cellvibrio ostraviensis Leonard (High IDC) 2022 -
72 Pseudomonas brassicacearum Leonard (High IDC) 2022 +
3 Ferrovibrio Leonard (High IDC) 2023 -
4 Ferrovibrio Leonard (High IDC) 2023 -
6 Pseudomonas Leonard (High IDC) 2023 +
7 Bosea Leonard (High IDC) 2023 -
10 Asticcacaulis Leonard (High IDC) 2023 -
11 Ensifer Leonard (High IDC) 2023 -
13 Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium Leonard (High IDC) 2023 -
16 Pseudoxanthomonas Leonard (High IDC) 2023 -
20 Pseudoxanthomonas Leonard (High IDC) 2023 -
22 Pseudoxanthomonas Leonard (High IDC) 2023 -
23 Lysobacter Leonard (High IDC) 2023 -
24 Lysobacter Leonard (High IDC) 2023 -
25 Lysobacter Leonard (High IDC) 2023 -
27 Sphingopyxis Leonard (High IDC) 2023 -
28 Novosphingobium Leonard (High IDC) 2023 -
33 Chitinophaga Leonard (High IDC) 2023 -
35 Taibaiella Leonard (High IDC) 2023 -
36 Chryseobacterium Leonard (High IDC) 2023 +
41 Dyadobacter Leonard (High IDC) 2023 -
42 Microbacterium Leonard (High IDC) 2023 -
43 Pseudarthrobacter Leonard (High IDC) 2023 -
44 Microbacterium Leonard (High IDC) 2023 -
54 Aeromicrobium Leonard (High IDC) 2023 -
60 Methylibium Leonard (High IDC) 2023 -
61 Variovorax Leonard (High IDC) 2023 -
62 Variovorax Leonard (High IDC) 2023 +
65 Ramlibacter Leonard (High IDC) 2023 -
66 Variovorax Leonard (High IDC) 2023 -
68 Xylophilus Leonard (High IDC) 2023 -
69 Limnohabitans Leonard (High IDC) 2023 -
70 Paucibacter Leonard (High IDC) 2023 -
71 Acidovorax Leonard (High IDC) 2023 -
85 Pseudorhodoferax Leonard (High IDC) 2023 -
Objective 2) Using the optimized CAS assay we evaluated siderophore production in each of the cultured ND soybean microbes. The resulting data is summarized as a column in Table 1 and example photos in Figure 1. Overall, 8/64 microbes showed potential for siderophore production.
Figure 1. CAS assay results for soybean isolates. A positive result is visualized as an orange halo surounding the spot of microbial inoculant.
Objective 3) We successfully optimized an assay for evaluating IDC reduction by microbial inoculants. The resulting experiment involved using a sterile IDC soil/sand potting mixture where 10 mM Nitrate/5 mM Bicarbonate was supplemented to induce iron-deficient conditions as found in calcareous soils. Microbial inoculants were added to pre-germinated soybean roots before planting, and an iron-chelating fertilizer Soygreen was added as a positive control to uninoculated pots. To test the assay, we created a microbial consortium that included 11 members from our soybean isolate collection that either showed significant enrichment or depletion in response to IDC based on previous years microbial community data. The experiment successfully induced IDC in soybeans that were untreated (Figure 3 and 6)). Remarkably, the microbial treatment showed significant alleviation of IDC symptems to levels (Figure 4 and 6) comparable to adding the soygreen fertilizer (Figure 5 and 6).
Figure 3. Images of IDC symptoms induced in the greenhouse by the newly optimized assay.
Figure 4. Images of microbe-treated soybeans grown in the same IDC symptom producing conditions as Figure 3.
Figure 5. Images of Soygreen-treated soybeans grown in same IDC symptom producing conditions as Figure 3.
Figure 6. Recovery of shoot dry weight from microbial treatment (SynCom) and Soygreen treatment in newly developed assay.
Discussion:
Overall, we have developed and optimized several elements of a comprehensive pipeline for isolation and characterization of microbes with the potential to reduce iron deficiency chlorosis in soybean. Our preliminary data from testing the system indicate that there is significant potential for microbial inoculants to reduce IDC and we have effectively developed an experimental procedure that can screen for and measure reduction of IDC from microbial treatments. Going forward, utilizing this system to detect and characterize individual microbes with the potential to reduce IDC will prove a powerful approach to provide sustainable management solutions to this
Conclusion/Benefit to Soybean Farmers:
IDC remains an important agronomic issue without perfect solutions. The microbiome offers a solution that can supplement genetics for IDC resistance which would be more cost effective than iron fertilizers and could be applied concurrently with rhizobium inoculants. In this project we successfully developed a pipeline for the isolation of potential IDC-reducing microbes and characterization of their potential for IDC reduction. Early results indicate microbial applications have the capacity to reduce IDC at a similar effectiveness to Soygreen fertilizer, the current best option.
References:
1. Louden, B. C., Haarmann, D., & Lynne, A. M. (2011). Use of blue agar CAS assay for siderophore detection. Journal of Microbiology & Biology Education, 12(1), 51–53.
2. Zhang, J., Liu, Y.-X., Guo, X., Qin, Y., Garrido-Oter, R., Schulze-Lefert, P., & Bai, Y. (2021). High-throughput cultivation and identification of bacteria from the plant root microbiota. Nature Protocols, 16(2), 988–1012.
View uploaded report 
Research Project Title: Potential for combatting iron deficiency chlorosis with the soybean microbiome FY24.
Principle Investigator: Barney Geddes
Why the Research is Important to North Dakota Soybean Farmers:
Iron Deficiency Chlorosis (IDC) is a significant problem in North Dakota that negatively impacts soybean production. IDC causes the leaves of soybean plants to turn yellow due to a lack of chlorophyll, which is essential for plant health and growth. This happens because, although North Dakota soils have enough iron, it is often in a form that soybeans cannot absorb. High soil pH and salinity, common in North Dakota, exacerbate this issue. Finding an environmentally and economically sustainable solution to IDC is crucial for improving soybean yields and ensuring the profitability of soybean farming in the region.
Research Conducted:
The research aimed to explore the potential of the soybean microbiome (the community of microbes around soybean roots) to alleviate IDC. We did this through research in three key areas that together build a pipeline for harnessing the microbiome to reduce IDC.
1. Microbe Culturing: We isolated and cultured microbes from soybean roots harvested from fields with varying levels of IDC.
2. Siderophore Screening: We tested these microbes for their ability to produce siderophores, compounds that can solubilize iron and make it available to plants.
3. Greenhouse Assay: We optimized a greenhouse assay was used to evaluate how these microbial inoculants affected soybean growth under iron-deficient conditions.
Findings of the Research:
1. Microbial Isolation: 64 unique microbial isolates were obtained from soybeans grown in both high-IDC and no-IDC sites.
2. Siderophore Production: Eight of these microbes showed potential for producing siderophores.
3. Greenhouse Results: The greenhouse tests showed that it is practical to screen for IDC-reduction affects by the microbiome. We found that applying certain microbial inoculants significantly reduced IDC symptoms in soybeans, similar to the effects of using the iron fertilizer Soygreen (Figure 1).
Benefits and Recommendations to North Dakota Soybean Farmers and the Industry:
The research paves the way for developing microbial inoculants to reduce IDC in soybeans, potentially offering a cost-effective and sustainable solution. For North Dakota soybean farmers, this means:
1. Reduced Costs: Microbial inoculants could be a cheaper option compared to iron fertilizers.
2. Ease of Application: These inoculants can be applied along with rhizobium inoculants, simplifying the process for farmers.
3. Sustainable Solution: Utilizing the natural soil microbiome is an environmentally friendly approach to managing IDC.