Using native rhizobia to create a drought-resilient field pea production system

To date, we do not have conclusive results. Currently, we have 91 rhizobia candidates. It is unclear how many of these samples are unique. Genetic sequencing will be utilized to determine how many species we actually have. However, we have begun the screening process, which involved developing a workflow and a series of criteria from which to evaluate each candidate species. Below are the representative results of rhizobia growth at varying levels of salinity.

Figure 1. Results of OD600 measurements of bacterial isolates USDA 110, S18, and S16 after 3 days of growth. Treatments used were 0.0017M NaCl (Control), 0.3 M NaCl, and 0.5M NaCl.

Figure 2. Results of OD600 measurements of bacterial isolates USDA S4, S17, and S21 after 3 days of growth. Treatments used were 0.0017M NaCl (Control), 0.3 M NaCl, and 0.5M NaCl.

Due to having little difference in the ranges of values between 0.3M and 0.5M NaCl, the “Medium” scale, we adjusted experiment to use “High” salinity treatment range to attempt to see more of a variation for field testing.

Figure 3. Results of OD600 measurements of bacterial isolates USDA 110, S18, and S16 after 3 days of growth. Treatments used were 0.0017M NaCl (Control), 0.7 M NaCl, and 0.5M NaCl.

Due to having a lower level of salt stress in the 0.7M NaCl range, the samples isolated from these glycerol stocks were sent for further study in the field trial experiment. Additionally, S3 isolate was added to the high salinity trial test after becoming contaminated during its medium salinity test. The S3 results for this test show salt stress at approximately 1M NaCl, but not 0.7M NaCl. Therefore, it was sent out to be used for field trial measurement in addition to the other listed samples. These results demonstrate that we see a broad range of response to abiotic stress and that we can isolate rhizobia with abiotic stress tolerance. The second phase was to take potential candidates to the greenhouse to impose stress in concert with a growing pea plant. 

Greenhouse Trials

Isolates from the high salinity and N-plate fixing plates were grown in VR broth media and shaken (245rpm, 28 degrees Celsius) and diluted until they reached OD₆₀₀=0.08. Treatments were subdivided thusly in equal concentrations_, along with high nitrogen and low nitrogen solution control treatments.

Table 1. Treatment scheme for mass inoculations. Salinity Treatment =  Week 1-2 ddH20. Week 3, 0.03M. Week 4, 0.06MNaCl, and 0.09M NaCl). Drought Treatment = Week 1, 100% H20. Week2, 100%, Week 3, 75% H20, Week 4, 50% H20, Week 5 25% H20.

Figure 4. Nodules observed from Inoculant 3.

Unfortunately, nodulation was only observed for Inoculant 3 (I.3), and there were correlations observed between I.3 and the other inoculants and the High Nitrogen control.3 had a higher average dry weight, as well as increased shoot length. Since further sequencing data points to plant growth promoting bacteria being a large source of isolates, this may be the attributed phenotypic properties observed. During harvesting on Week 6, only plants from I.3 contained observable nodulation. Their phenotype was observed to be very dark red/black nodules. These studies will be repeated to ascertain the reason for not getting nodulation after culturing the bacteria.

Field Observations

Lastly, the end goal of this project is to produce rhizobia that can be used in the field by farmers. Unfortunately, Covid created a number of obstacles in establishing field plots in 2020. We were unable to work with producers to establish our drought plots. However, we were able to at least set out a single site with several cultured rhizobia. The goal was to simply examine both N fixation and yield from two separate field pea varieties when inoculated with our native rhizobia species as compared to commercial granular and liquid inoculants. 

Figure 5. N derived from the atmosphere as a percent of total N in the plant across two field pea varieties (Carver and Profit)

As Figure 5 shows, the 3 native rhizobia inoculants performed similarly to the commercial granular and liquid inoculants. There was some variation between varieties, however, in general, all rhizobia fixed between 60-70% of N from the atmosphere. Likewise, total N fixed ranged from 60-80 lbs of N/acre, which was favorable to commercial inoculants. Hence, we these results are not conclusive, they do offer positive results. These tests will continue in 2021 with the addition of sites under saline and drought stress.

Figure 6. Total N (lbs/ac) fixed from the atmosphere across two field pea varieties (Carver and Profit)

2021 Results (lab results continued from 2020)

Objective 1: Rhizobia isolation.

Due to significant disruption from the Covid pandemic, much of our rhizobia screening was delayed until into 2020 and 2021. Due to the goal of isolating rhizobia that was resistant to high salinity environments, initially in this experiment’s conception, we chose the use of high salinity media broth as a “filtering” step before plating, choosing isolates based on morphology determination, and extracting dna for sequencing. However, this led to a number of false positives discovered after sequenced several isolates. Non-rhizobia isolates mostly composed of Pseudomonas and Baccilus strains. Because of this, a more stringent parallel plating system was used to first isolate candidate colonies before dna isolation. This system led to significant success in reducing the levels of false positives. And only after confirmation were the isolates then subjected to salinity stress test for further down-stream analysis.

Salinity-filtering Isolation

            The following process was used for the initial phase of rhizobia isolation. Initially, samples were collected after incubation in 0.5M NaCl until post log phase, bacterial suspensions were plated onto vincent’s rich (VR) solid media supplemented with Congo Red (CR) (%1.2 Agar, 25 mg/L Congo Red). Positive isolates had favorable morphology (round, convex, glistening colonies) and color absorption from the CR. After 5 days of growth, samples with visible growth were replated on VR media. DNA was then extracted using DNeasy Ultraclean Microbial kit, as per instructions.

Parallel-plating Isolation

            After false-positives were tested, an alternative method of isolation method was established, based on existing protocols (Working with Rhizobia Handbook, 2016). Samples were grown in VR liquid broth, with the addition of vancomycin antibiotic (concentration 1 mg/L), to limit contamination from gram-positive bacteria. After 3 days of growth, visible turgid samples were then streaked on CR plating for 3 days. Afterwards, colonies were chosen with favorable morphology, and further downstream parallel plating was added to differentiate rhizobia from possible non-rhizobia contaminants. Bromethyl bromothymol blue (BTB) (VR agar, 5 mL/L of 0.5% w/v in 0.2M KOH) plating and glucose-peptone agar (5 g glucose, 10 g peptone, 10 g agar) (GPA) plating was used.  While colonies grow in media there are differentiations in phenotype on the plate based on pH-associated enzymatic reactions. While slow-growing rhizobia such as Bradyrhizobium, as well as contaminants such as Pseudomonas and Bacillus produce basic pH changes in the media during colony growth, fast-growing Rhizobium species produce an acidic reaction in the media. The addition of BTB dye to VR media produces a color change during pH shifts. Normally producing a green plate, BTB VR plates turn yellow when acidic and blue when basic. Additionally, GPA agar is used in medium where it is difficult for rhizobium to develop. BTB dye was added to the GPA agar to determine if contaminates are present. As can be seen, the test works as expected. The samples grew well on the CR plates as well as the BTB plates. In the latter, the colonies produced a yellow color that is described for an acidic reaction.

Figure 7. Plating scheme initially spreading colonies on Congo Red plates. Colonies without significant red contamination were streaked to BTB and GPA plates in quadrants searching for yellow coloring and no growth, respectively.

Figure 8. Scheme representation of parallel plating scheme. Bacteria isolates are cultured in VR broth supplemented with vancomycin gram-positive inhibitor. They are then spread on Congo Red plates, and round, convex, and glistening colonies (without significant red color absorption) are spread onto BTB and GPA plates concurrently. Samples that show yellow reactions in BTB plating, but no growth in GPA plating were made into a glycerol stock and used for sequencing.

Salinity Resistance of Suspected Rhizobia

After successful plating, we conducted additional salt tolerance testing for rhizobia with potential drought stress conditions. A previous study had categorized tolerance scales for tested growth of microorganisms in saline conditions based on three groups: sensitive (0-50 mM NaCl), tolerant (100-500 mM NaCl) and extremely tolerant (600-1800 mM NaCl) (Pereira, 2008). Due to filtering potential helpful rhizobia, we determined testing scales of 0.00017M (control), 0.25M NaCl, and 0.5M NaCl, settling on an upper “tolerant” scale. While extremely tolerant upper limits of up to 1M was initially used for earlier testing, this had the potential to cause excessive filtering of helpful rhizobia species from the collected nodule glycerol stock cultures. Future tests exclusively used this scale for testing. Below is the results for salinity testing from the available rhizobia isolates following earlier screening and culling of the total candidate pool. As seen in Figure 9, the initial rhizobia isolates selected for greenhouse and field studies proved to be tolerant to high salinity, amongst several others. Overall, bacterial growth rate is considered to be in salt stress if there is 75% inhibition. Additional tests in the future might continue this work by testing further salt stress to test the limits of the bacteria’s osmotic stress, as well as an examination in the differences between genes governing abiotic osmotic stress.

Figure 9. Salinity test using bacteria isolates grown in three different scales of salinity,, 0M NaCl, 0.25M NaCl, and 0.5M NaCl. Samples were grown until post log phase, and bacterial suspensions of OD₆₀₀ 0.13, at a 1:100 ratio to VR. Samples were incubated for 5 days and bacterial pellets were centrifuged and resuspended in ddH20.

Rhizobia Identification

            From the tests in Figure 9 (candidates on the right hand of the graph), we cultured rhizobia that were tested successfully for saline resistance in VR liquid media, and we extracted dna using DNeasy Ultraclean kits per specifications. We verified fidelity using Nanodrop 2000C. 16S primer pairs 27F and 519R were used for amplification. PCR protocol as follows (Degrees Celsius): Initial denaturation of 95 degrees for 4 minutes. 30x cycle of 95 degrees for 30 seconds, Annealing temp of 50 degrees for 45 seconds, and elongation phase of 72 degrees for 1 minute. And a final elongation phase of 72 degrees for 10 minutes. We verified pcr product using gel electrophoresis. After sequencing, samples were identified using BLAST (16S rDNA), and 5 candidate rhizobia strains were ultimately used for additional testing from collaborative greenhouse experiments.  We uploaded sequence data to Geneious software, and we created phylogenetic trees from overall rhizobia collection results using Muscle (Tamura-Nei, Neighbor-Joining) for dna alignment and then tree building. The tree can be seen below. The highlighted red portions are the samples used in the field. In the Tree, a Pseudomonas strain, extracted from the same sample site and provided as a positive control for phenotype tests, as well as a well-characterized Glycine max-compatible Bradyrhizobium strain, was used as comparison points.

Figure 10. Phylogenetic trees, presented as rooted and circular trees.

Greenhouse Testing

Initial salinity testing, and sequencing of rhizobia isolates were used for field testing included V23.2, V2762, V47, V54, and V85, narrowed down from the original rhizobia isolates sequenced due to their high saline tolerance. Trials were ran to test for their effectiveness for growth in greenhouse settings. Similar inoculation strategy was used for all tested isolates in the greenhouse. Samples were grown in primary cultures of VR broth with ampicillin antibiotics (20ng/ml), and then post log phase were used to reinoculated secondary cultures until post-log phase, to ensure much reliable growth levels. Pea seeds were sterilized by being washed using 10% chlorax solution and 70% ethanol for 2 minutes each. They were rinsed with distilled water 10 times, and then placed in distilled water for 3 hours to imbibe.

Sterilized vermiculite/perlite mixture (3:1 ratio) were added in 1.5’’ by 8.25’’ cone-tainer cells. Pea seeds were added 1.5’’ below the fill line. Samples were watered with alternating ddH2O and ¼ PNS nutrient solution. After 1 week of growth, the samples were inoculated using cultured rhizobia. Two treatments were not inoculated with rhizobia, adding instead a nitrogenous Hoagland solution during the tray’s 1/4^th PNS watering as a high nitrogen control. Another control had no rhizobia and no supplemental nitrogen source added. By comparing these controls, we can measure what value nitrogen sources supplemented by rhizobia.  After 2 weeks of growth, the watering sources were supplemented with 0.03M NaCl. This value is increased an additional 0.03 until a final saline concentration of 0.09 NaCl was achieved. This value was chosen to stress the plant for abiotic conditions and test if the inoculated rhizobia was able to provide a mutualistic benefit by helping the plant survive under higher salinity conditions compared to the controls. These results showed a range of effectiveness for rhizobia isolated from the Midwest soils, both from their ability to impart biomass from saline resistance, as well as their capacity for measured nitrogen production.

Our goals in part focused on isolating these rhizobia for their ability to compete with market inoculants. There are many commercial inoculants that are offered in the market, that use a “one-size-fits-all” approach to develop high performing inoculants. In this trial, a commercial peat inoculant (CP) was added in comparison to the low and high nitrogen control. Salinity treatment was used for all samples. As can be seen from below, there was a range of effects observed for both saline and control treatment results. This was accomplished by, similar to the field, inoculating the pea seed in the same method as with inoculation in the field, simultaneously with rhizobia inoculation.

In the figures 11 and 12 below, we see the results for the non-saline treatments of the inoculated plants. And while V85 and V54 show a much higher distribution and mean (approximately by 3x) than the other V series, they are not statistically different at this level using Tukey test. Though when measuring shoot/root ratios, Peat control and V47 were statistically different than the other samples.

For the saline treatment (Figure 13 and 14), there was no significant difference observed between the ethylene production of the nodules measured. However, the Dry Shoot/Dry Root ratio showed that V47 provided the highest increase to shoot biomass distribution, with V2762 as expectedly the lowest alongside the Peat Control. This data showed that after saline conditions treatment, our rhizobia had a beneficial effect that was significantly greater than a negative control. Visual images also were important for this experiment, showing significant senescense and yellowing in the V23.2 and V2762 rhizobia pots. While this information gave conflicting information, it was useful to help narrow down focus for further field analysis. V2762 had difficulty producing nodules compared to the other inoculants, while previous work with V23.2 showed it to be less effective as well. While in this experiment it produced a significant amount of nodules, this was not notably producing significant amount of ethylene. The notable senescence present in those two sample pots were also a contributing factor for use in their long-term growth in the field.

Further, nodule number under saline conditions varied significantly between rhizobia species, but these values do not necessarily correlate with biomass production. As Figures 15 and 16 show, V23.2 and V85 had relatively large numbers of nodules but middling shoot biomass, whereas V47 had very low nodule numbers but had the highest average biomass production.

Additional Rhizobia Isolation and Greenhouse Testing.

While screening for rhizobia samples initially for saline resistance, we potentially screened against possible rhizobia samples that might offer distinctive and beneficial phenotypes/mutualistic effects. There is an ever-growing body of research in not just single inoculant discoveries, but the examination of dual-inoculant studies that have shown increased growth benefits to legumes. By expanding our saline filtering step, we sought to explore the additional rhizobia strains that may have been initially filtered out, but could still have future capacity to provide mutualistic benefits in either single or dual-inoculant future experimental designs.

From our initial batch of testing, there were several strains listed with potential for very high nitrogenase enzyme production. Initial testing of these samples did not use salinity control, only as a biomass/ARA experiment. Downstream testing with these samples in mutualistic environments would be necessary to further match the overall goals of this project. If these samples are able to reproduce similar results under stress conditions, then there is potential for them to be used in further inoculant tests for field testing. Continuation of rhizobia study from isolated nodule samples is of potential significance for agricultural research.

Intrinsic Antibiotic Resistance

            Testing for intrinsic antibiotic resistance (IAR) is necessary for culturing and testing bacterial isolates. Initially, ampicillin testing using liquid broth culture growth was conducted due to Rhizobium species having already widely established ampicillin resistance. However, we widened our tests for antibiotic resistance to gain more information on resistance plasmids, as well as to have multiple antibiotic options in case of potential ampicillin resistance. We used testing kits composed of antibiotic discs infused with ampicillin (10mcmg), chloramphenicol (30mcmg), streptomycin (10mcmg), and tetracycline (30mcmg). After culturing bacteria isolate in VR broth and resuspending the centrifuged bacteria pellet, sample turbidity was reduced to 0.13 OD₆₀₀, and then 100ul of suspension was added to a VR solid plate. The suspension was spread using sterile glass beads. We separated the plates into quadrants, and antibiotic discs were aseptically affixed to the plate and stored in a 28 degrees Celsius incubator for 5 days. Afterwards, the halo-zone surrounding antibiotic discs, if any, were recorded (Figure 17,18). This provided a measure of the rhizobia’s susceptibility to tested antibiotics. The results of IAR for measured isolates that were used for downstream analysis is presented below. Notably, ampicillin remained the most consistent antibiotic for IAR. Samples were also widely resistant to chloramphenicol, but this resistance was inconsistent for several of the samples. Expectedly, all rhizobia samples had the least resistance to tetracycline, followed consistently by streptomycin.

Phenotype Measurements

            While salinity measurements provided a useful baseline measurement for stress resistance, there are many other sample tests that have been developed that are useful in establishing plant growth promoting effects. 

IAA Production

            Phytohormone secretion is one of the major ways in which soil bacteria are studied for their mutualistic benefits. Indole-3-Acetic Acid (IAA) is a very important phytohormone, a secondary metabolite of L-tryptophan that acts as a regulator of many biological processes for plant development while acting on organogenesis, trophic responses, and cellular responses such as cell expansion, division, differentiation, and gene regulation (Lebrazi et al., 2020). The production of IAA produced by colonizing bacteria in the plant rhizosphere proposed to act in conjunction with endogenous IAA in plant to stimulate cell proliferation, elongation, and enhancement of host’s uptake of minerals and nutrients from the soil (Shokri, 2010). The levels of IAA produced by a bacterium in the rhizosphere determines its effect on the host plant, with high levels inducing developmental abnormalities and stimulating formation of lateral and adventitious roots, while low levels promoting root elongation (Bal et al., 2013).

 We sought to examine our rhizobia isolates for IAA production using previously established protocols. We cultured rhizobia isolates, resuspended bacterial pellet in VR broth, and reduced to 0.13 OD₆₀₀.  Samples were used to inoculate VR broth supplemented by 1 g/L of L-tryptophan. After 5 days of growth, these samples were removed, and centrifuged at 10,000g for 1 minute. 1ml of the supernatant was used to combine with Salkowski’s reagent (50ml 35% perchloric acid + 1ml 0.5M FeCl₃.) at a 1:2 ratio. We set these samples in the dark and incubated them for 1 hour, as the chelating iron-based Salkowski reagent produced darker pink/purple colors with greater presence of IAA.  Alongside, we used IAA standards combined with Salkowski reagent to estimate IAA concentrations. Afterwards, the samples were added to a flat-bottomed clear pcr plate, and the absorbances were read using a spectrophotometer set to 540nm.

The overall results for IAA experiments are presented below. Three graphs were presented here to demonstrate different measurements. The first graph shows results from calculations using the standard curve to derive concentration of IAA from the fluorescence values. The second graph shows a normalization of the concentration data to adjust for “cell number” of bacteria present in the final sample before IAA collection. The latter may serve to provide an idea of “efficiency” of the size of a culture  Overall, these samples reveal lower levels of IAA produced by our field trial samples, especially V47 and V85. However, the V23.2 sample produced higher levels of IAA, and there was a wide range of IAA concentrations provided by our measured samples. Since IAA concentrations may be produced at different levels in under a variety of conditions, this is an exploratory snapshot that can allow us to dive further into other rhizobia of interest, as well as to compare how the experimental snapshot of this phenotype compares between sequencing data.

Phosphate Solubilization

            Organic phosphates are widely present in soils but unusable by agricultural plants. While synthetic fertilizers are the typical alternative to soil organic phosphorus, this is, similar to nitrogenous fertilizers, a costly and polluting strategy in which only a fraction of used phosphate is absorbed into the soil. Many bacteria, however, do have the ability to convert organic phosphates in soil to a usable form by the legume. This is a PGPR that is favored by microbiology industries. Therefore, we used this as an additional test for our isolates to further score their effectiveness and potential future benefits as an inoculant product.

            Using Pikovskaya’s agar we made a solid plate media contained with organic phosphates. We cultured isolates in VR, centrifuged at 10,000g for 1 minute, and resuspended pellet in sterile ddH2O. We reduced to 0.6 OD₆₀₀, and spot inoculated solid media plates of Pikovskaya’s media. After 7 days of growth, we measured halo-zones surrounding colonies. The figure below shows the overall results of the phosphate solubilization tests conducted over our rhizobia isolates. The tested strains showed very similar levels of solubilization throughout the trials, though there were several samples that showed no solubilization under these conditions. Notably S16R182 showed the highest index levels.

Isolation of non-rhizobia species

            This project led to the identification of several non-rhizobia species from the crushed nodule glycerol stocks. There was a large number identified due to the initial false positives produced from the early isolation steps of the experiment (Figure 23). There was no significant phenotype testing from these bacteria due to falling outside the purview of our goals. However, notably Pseudomonas sample S7R4 provided a useful tool as a positive control for our several phenotype tests, including IAA, and phosphate solubilization testing. It was notable for outperforming a well-characterized phosphate positive control strain Pseudomonas aeruginosa strain ATTC 2853 in phosphate solubilization tests.

            The use of non-rhizobia species, including Bacillus and Pseudomonas strains, as co-inoculants has shown to have beneficial cumulative effects. Non-rhizobial plant growth promoting bacteria has been shown to improve nodulation and grain yield of the legumes upon co-inoculation with crop specific rhizobia (Kohr, 2017). Therefore, these strains will be kept in storage and might provide further use for other researchers investigating co-inoculation effects. Below is a phylogenetic tree constructed from the non-rhizobia extracted isolates from the sample sites.

Field Studies - 2021

In 2021 we experienced drought conditions at both test sites. In western South Dakota (Sturgis) the earlier planting actually experienced more extreme drought conditions over the later planting date due to some late season rains. As figure 24 shows the late planting in general had higher nitrogen fixed from the atmosphere (NDFA) (as a percent of total N in the plant) and total nitrogen fixation. Overall, NDFA and total nitrogen fixation were roughly half of a typical year. Two native species - V47 and V85 - did comparably well against the commercial liquid and granular inoculants. Although the commercial granular inoculant had the highest N fixation, it was not statistically different from the two highest performing native rhizobia species. 

At our second research site in the middle of the state, the drought was more severe and impacted the second planting more so than the first planting date. Figure 25 shows both the NDFA and total N fixation measured at this site. From a methodological standpoint, the uninoculated control (Cont_UnIn) was lower that than all other treatments at both sites, which suggests that our methods are free from cross-contamination. There is still native rhizobia in the soil at each research site, so some level of nitrogen fixation is expected. The results were not entirely consistent the with the other research site. However, V85 still performed quite well against the commercial controls. Lastly, Figure 26 depicts seed yield at both research sites. Again, overall yield was roughly 50% of long-term averages. There was not a consistent trend between treatments. V85 had the highest yield under the more mild drought stress conditions  but did not fare as well under severe drought, despite having higher N fixation.

Overall, the 2021 lab and greenhouse studies increased our confidence in developing competitive rhizobia using native species. We have now developed a catalog of over 90 different rhizobia species and we have two very promising candidates from this pool. In 2022, we will continue our field studies for another year and expand to one additional salt-stressed field. Further we are in the process of obtaining the genetic sequences of several of our candidate species so that we can conduct an analysis of the different genes involved in conferring drought/salt tolerance. This work will allow us to streamline the rhizobia screening process with the possibility of identifying genetic markers for stress tolerance. 

2022 Results

Field Studies -

In 2022, we repeated field the same field studies as in 2021. Two sites were established in Pierre, South Dakota and Sturgis, South Dakota at two planting dates (1. On-time and 2. Late planting). Unfortunately, the Sturgis site received several significant hail events during the growing season. While data were collected for this site, we did not feel that they were reliable due to the potential for inhibited growth effects by the hail. Hence, the data presented here is for the Pierre site only.

Nitrogen Fixation

Contrary to 2021, precipitation in 2022 was much more conducive for plant growth. Both NDFA and total N fixation were correspondingly increased. On average, NDFA was 74% and total N fixation was 100 lb/ac for the on-time planting. For the late planting, average NDFA was reduced to 65% and total N fixation was 44 lbs/ac - a reduction of over 50%. Between treatments, the uninoculated control treatment had the lowest NDFA overall at 64% at the on-time planting and 58% at the late planting. As Figure 27 shows, the native rhizobia 23.2 had the highest NDFA at both planting times at 76% and 68%, respectively. Despite these differences, the inoculants were only statistically different (p<0.05) than the uninoculated control.

Similar to NDFA, total N fixation followed a similar trend although treatment differences were much greater at the on-time planting and were relatively similar at the late planting. In general, the native rhizobia fixed more nitrogen than either the uninoculated or liquid inoculant controls. V47 fixed the most nitrogen at 108 lbs/ac which compares quite favorably to the uninoculated control, which fixed 92 lbs/ac at the on-time planting (Figure 27). 

Figure 27. The left-hand graph depicts the percent of total N in the plant derived from the Atmosphere (NDFA %) and the figure on the right depicts the total N fixed in lbs/ac by rhizobia treatment.

Yield

Similar to N fixation, yields were significantly decreased at the later planting date. On average, yields were 49 bu/ac at the on-time planting and just 17 bu/ac at the late planting. Within planting dates, yields were not statistically different between rhizobia inoculants although V23.2 trended lower, which suggests that this particular rhizobia may consume higher amounts of carbon than other rhizobia species (Figure 28). Moreover, this figure highlights the importance of inoculants for conserving soil N. As this figure shows, the uninoculated control yields were comparable to the other rhizobia inoculants. Assuming similar total nitrogen in the plant, the uninoculated control uses significantly more soil-derived N, which would leave less residual N for subsequent crops.