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

Progress report for LNC18-405

Project Type: Research and Education
Funds awarded in 2018: $199,813.00
Projected End Date: 11/01/2022
Grant Recipient: South Dakota State University
Region: North Central
State: South Dakota
Project Coordinator:
Christopher Graham
South Dakota State University
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Project Information

Summary:

Legumes such as field peas have emerged as a viable option for farmers to diversify their cropping rotations. An added benefit of legumes in rotation is their ability to fix nitrogen through a symbiotic relationship with rhizobia bacteria. The nitrogen fixed by this interaction not only helps the legume crop, but also the subsequent crop as the fixed nitrogen is released to the soil as legumes decompose at the end of the season. This process allows farmers to be less reliant on expensive nitrogen fertilizers for both crops. However, as field peas continue to expand into the northern Plains, where dry, hot summers are common, it is evident that heat and drought stress are major limiting factors to both production and adoption by farmers. The problem of drought stress is two-fold in that it not only affects yield, but it also severely limits nitrogen fixation by rhizobia.

It is possible that native rhizobia, acclimated to the local climate and soils, are more effective at nitrogen fixation under stress conditions. This project intends to 1) examine the paired use of drought tolerant field peas and native rhizobia versus commercial rhizobia to maintain nitrogen fixation in field peas during drought stress, 2) identify the most effective inoculation methods for nitrogen fixation, and 3) create an open-source culture method that farmers can utilize on their own farms to be used as either a standalone inoculant or in combination with commercial inoculant.

Project Objectives:

Project Outcomes

1) Evaluate the use of native rhizobia to maintain nitrogen fixation in field peas during drought stress and improve crop performance

2) Identify the most drought tolerant FP varieties and the most effective inoculation methods for nitrogen fixation in field peas and ensure that farmers are employing these techniques

3) Educate farmers on the value of effective nitrogen fixation and the importance of legumes in diversified cropping rotations

4) Create an open-source culture method that farmers can utilize on their own farms to be used as either a standalone inoculant or in combination with commercial inoculants

Introduction:

Legumes such as field peas (FP) have emerged as a viable option for farmers to diversify rotations. In fact, SARE
has recently funded several projects examining the value of FP in intensified farming rotations, which replaces the
traditional wheat-fallow system. An added benefit of legumes is their ability to fix atmospheric nitrogen through
symbiotic relationships with ‘rhizobia’ bacteria. This fixed nitrogen helps the legume crop, and also the
subsequent crop as the nitrogen is released to the soil during plant decomposition. This process allows farmers to
be less reliant on expensive nitrogen fertilizers for both crops. To our knowledge, SARE has not funded similar
projects or projects related to rhizobia development.

However, as FP acreage expands across the northern Great Plains, where dry, hot summers are common, it is
evident that heat and drought stress are major limiting factors to production and adoption by farmers. The
problem of drought stress in FP is two-fold. It not only affects yield, but also limits nitrogen fixation by rhizobia.
Drought stress leads to a reduction in nodulation due to limited root hair growth which consequently reduces the
amount of N-fixation (Bordeleau, 1994). Studies have shown that even moderate drought conditions can lead to a
50% or greater reduction in N-fixation (Kirda, 1989). Consequently, farmers lose money on poor yields and lose
the soil health benefits of nitrogen fixation.

We currently conduct FP variety trials at five locations across the state. The past two growing seasons have been                                          defined by drought and this project stems from our experiences with these variety trials and producer concerns.
There has been some concern that inoculants are failing under drought stress, hence the need for more drought
tolerance in both plant and inoculant. Moreover, during our field tours, farmers remarked that if drought is severe,
a modestly productive legume would still be beneficial as a soil health builder even when not producing a viable
yield. This would be preferable to other crops such as failed wheat or corn. In other words, where a harvest is not
viable due to drought, field peas are a preferable option to wheat due to the low C:N, which degrades rapidly and
provides a valuable green manure.

Native rhizobia populations arise in the soil by two means, A) from compatible strains nodulating wild legumes or
B) commercial strains remaining in the soil from previous cropping seasons (Vessey and Chemining'wa, 2006).
The failure of commercial inoculants under drought conditions is not surprising. It is well established that when
microorganisms are introduced into a new environment they often fail to survive because the environment is too
different from their native growing conditions or they cannot compete well with the native community and thus fail
to become established (Mendes, 2013). This is particularly true for rhizobia where it is recognized that inoculation
efficacy can be improved by using strains that were isolated from environments similar to where they will be used
(Lupwayi, 2006). In spite of this, currently, most commercial rhizobia inoculants are sold as 'one size fits all.' The
same rhizobia strains used in South Dakota are also used in South Carolina.

We envision an integrated approach that matches drought tolerant FP varieties with compatible, drought tolerant
rhizobia adapted to local growing conditions so both nitrogen fixation and yield can be maintained during drought
and heat stress. In order to improve rhizobia survival and activity, we will evaluate the interaction between
commercial and native rhizobia to identify suitable crop variety-rhizobial strain pairs for improved performance
under drought and heat stress. A combination of commercial and native rhizobia may work in concert.
Commercial rhizobia could maximize nitrogen fixation during optimal conditions while native rhizobia will
supplement nitrogen fixation during stress conditions. While the examination of native rhizobia is not a new
concept, evaluating native and commercial inoculants together is. Typically, research on native rhizobia focuses
on the value of the native rhizobia to obviate the necessity of applying commercial inoculants (e.g. Chemining'wa
and Vessey, 2006). But most studies fail to culture the native rhizobia and apply it at similar rates that a
commerical inoculant is applied at. Therefore, while the native population may be effective at nodulating the target
legume, it is not present in sufficient quantities to compete with a commercial inoculant. Moreover, there is little if
any information in the literature that examines how different rhizobial strains might work symbiotically under
varying abiotic stressors.

The hypotheses for this research are: 1) native rhizobia are better adapted to their local environment and can
maintain nitrogen fixation more effectively than commercial rhizobia under stress conditions when supplied in
adequate concentrations. And 2) specific legume variety-rhizobial strain combinations will perform differently due
to compatibility and competitiveness.

Research

Hypothesis:

Native rhizobia cultured from drought-prone regions can outcompete commercial inoculants under drought conditions in field pea production.

Materials and methods:

In 2020, soil was gathered from 24 unique sites across Montana, North Dakota and South Dakota. The sites represent different (never cultivated) biomes including high-altitude, mountainous regions, forest, undisturbed prairie and shrub-steppe grassland. Uninoculated field peas were grown in each of these soils under lab conditions and harvested after 8 weeks to determine colonization by native rhizobia. Nearly all soils produced nodules. Of these nodules, 92 rhizobia samples were successfully cultured. These samples will then be screened for drought tolerance and candidate rhizobia will be tested for further greenhouse and field studies in 2020. This process was repeated during the 2021 calendar year.

Lab-Based work:

From the grown field peas that were successfully nodulated from the trap experiments, five representative nodules from plants of each sample site replicate were crushed in Vernon’s Rich (VR) media (For the first 10 glycerol stocks, however, single representative nodules of three listed sites were first used). After being strained through a 70micro meter nylon strainer, 750 microliters of filtrate was mixed with equal parts 50% glycerol solution and frozen in a -80 degree Celsius freezer. This created 91 total glycerol stocks of rhizobia “soup” cultures.

Bacteria colony isolates were acquired by culturing the glycerol stocks in 10ml of liquid VR medium in 50ml glass vials and placed in a shaker (28 degrees at 245 rpm). After successful growth, broth was streaked on 1.2% VR agar plates. Five representative colonies were picked and transferred into Nitrogen-Free media. After 1 week of growth, successful isolates were harvested and stored into glycerol stocks.

We are attempting to determine drought tolerance by both withholding sufficient moisture, but also through the addition of salinity which creates a similar stress on the rhizobia. To determine saline tolerance, isolates were screened in NaCl supplemented liquid VR cultures (3 replicates) and measured at OD600 after a 3-day period. USDA 110, a highly characterized rhizobia, was used as a control to determine reliability of experiment. Below are the results of the initial run (concentrations of 0.0017M, 0.3M, and 0.5M).

Field-based work:

Three native rhizobia species were selected to be field tested along with a commercial and liquid inoculant in 2020. Two varieties of field peas (Carver and Profit) were inoculated with each rhizobia species and planted in 5x30ft plots with 6 replications. Sub-plots were harvested at 50% flowering for measurement of N fixation - determined through the 15N natural abundance method. 

In 2021, further lab and field testing was conducted. At this point in the research we have now narrowed our rhizobia search down to just a few promising candidates. These candidates were selected based on previous lab and field-based measurements. The field trials took place at two locations in western and central South Dakota. The stimulate drought conditions, we utilized two planting dates at each location: the first date was considered a normal 'on-time' planting and the second was a late planting, which pushes the critical anthesis period into the warmer part, drier part of the summer growing season. 

Research results and discussion:

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.

 

Treatments

Inoculant

Salinity

Drought

H20

Inoculant 3 

6 Replicates

6 Replicates

6 Replicates

Inoculant 2 

6 Replicates

6 Replicates

6 Replicates

Inoculant 1

6 Replicates

6 Replicates

6 Replicates

High Nitrogen

6 Replicates

6 Replicates

6 Replicates

Low Nitrogen

6 Replicates

6 Replicates

6 Replicates

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

Field Studies - 

Indeed, in 2021 we did experience 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 7 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 8 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 9 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. 

Nitrogen derived from the atmosphere (NDFA - %) and total nitrogen fixation by native rhizobia at the West River Research Farm in Sturgis, SD
Figure 7. Nitrogen derived from the atmosphere (NDFA - %) and total nitrogen fixation by native rhizobia at the West River Research Farm in Sturgis, SD
Figure 8. Nitrogen derived from the atmosphere (NDFA - %) and total nitrogen fixation by native rhizobia at the Dakota Lakes Farm near Pierre, SD
Figure 8. Nitrogen derived from the atmosphere (NDFA - %) and total nitrogen fixation by native rhizobia at the Dakota Lakes Farm near Pierre, SD
Figure 9. Combined yield (bu/ac) from both study sites in 2021.
Figure 9. Combined yield (bu/ac) from both study sites in 2021.

Research conclusions:

NA

Participation Summary
2 Farmers participating in research

Project Activities

Field Days
Native Rhizobia Screening
Rhizobia Field Day

Educational & Outreach Activities

3 On-farm demonstrations
3 Workshop field days

Participation Summary:

40 Farmers
30 Ag professionals participated
Education/outreach description:

Field days and demonstrations were described in the previous section

Learning Outcomes

Key areas taught:
  • NA

Project Outcomes

Key practices changed:
  • NA

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.