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Identifying rhizobia partners for cold-hardy vetch

Cover crops provide multiple benefits on organic farms, but there are few cover crop options that successfully overwinter in the Upper Midwest. Hairy vetch is the most reliable winter-hardy cover crop option, and recent breeding efforts have improved its winter survival, but cold temperatures still put pressure on its ability to fix nitrogen. My research focuses on ways to improve nitrogen fixation in hairy vetch. In this article, I will share new results on nitrogen fixation in hairy vetch at low temperatures, along with takeaways on the applicability of controlled environment experiments to field outcomes.


Nitrogen is an essential nutrient for plant growth, but it is often limiting on organic farms. Farmers face a challenge in providing nitrogen without contributing to nutrient runoff and eutrophication. As a result, cover crop mixtures that include legumes have become an increasingly popular management tool for nitrogen provision.

Most plants take up nitrogen from the soil, but the legume family of plants can derive nitrogen directly from the air (and remember: air is almost 80% nitrogen gas!). Legumes can’t do this alone, however. They must associate with soil bacteria called rhizobia to engage in the process of biological nitrogen fixation. In this process, rhizobia form specialized organs on the legumes’ roots called nodules, which are ideal environments for the conversion of atmospheric nitrogen into a nitrogen form the plant can use. This makes legumes valuable sources of protein (think of soybeans, lentils, chickpeas, and more) as well as valuable sources of soil nitrogen fertility.

One useful method to incorporate leguminous cover crops into a rotation is by using winter annual plants. Winter annual cover crops are planted in late summer to take advantage of the early fall growing season before becoming dormant through winter. They then grow again in the spring until termination and incorporation into the soil. This system prevents nutrient competition between the cover crop and summer cash crops and suppresses weed growth in the early growing season. In most regions, the cover crop’s senescence also coincides with the cash crop’s nutrient requirements. However, cover crop cultivation in the upper Midwest is a significant challenge due to the region’s short growing season and extremely cold temperatures.

Of the winter annual cover crop legumes available to organic producers, hairy vetch has shown higher cold tolerance than others. Like other cool season cover crops, hairy vetch in the upper Midwest is sown in the fall after cash crop harvest, provides cover throughout winter, and is terminated then incorporated in spring. Breeding efforts over the last ten years have led to varieties that are cold-tolerant enough to survive winter as far north as Roseau, Minnesota. However, low temperatures negatively affect biological nitrogen fixation, so achieving consistent nitrogen accumulation in these cold-hardy vetch remains a challenge.

In my research, I am trying to better understand what limits nitrogen fixation in vetch at low temperatures, while also seeking solutions to improve nitrogen fixation. Successful nitrogen accumulation depends on the relationship between the legume and the rhizobia, but cold temperatures hamper the development of nodules. The soil contains a huge diversity of rhizobia—some which fix nitrogen better than others, some which can establish nodules on a plant faster—and this diversity forms the basis for my research questions. If I can determine which traits in rhizobia are most important for nitrogen fixation at low temperatures, that information can inform inoculant development and hairy vetch breeding efforts that might improve nitrogen accumulation.

One trait that interests me is how quickly rhizobia establish nodules on plants. I predicted that rhizobia strains that establish nodules on a plant faster would ultimately accumulate more nitrogen. For fall planted crops, temperatures gradually decrease until it is too cold for any nitrogen fixation; I hypothesized that plants which developed nodules faster would have more time to accumulate nitrogen and thus lead to higher total nitrogen in the plant. If time to nodulation proved to be a good indicator of how well rhizobia fix nitrogen at low temperatures, it would be very useful for developing an inoculant; a researcher could just check legumes’ roots after a short period of growth and find good rhizobia candidates for cold-climate inoculant development.


I decided to test my ideas about nodulation speed in a growth chamber experiment, rather than in the field, so that I could control for temperature without worrying about any other environmental factors. Hairy vetch plants were inoculated with one of six unique rhizobia strains, which I previously confirmed to have different nodulation speeds—in other words, the number of days to the onset of the first nodule differed across the different rhizobia strains. Plants were grown in modified magenta units (Figure 1) to make sure they would only be nodulated by our applied rhizobium inoculum strains. The bottom well of the magenta unit was filled with a nutrient solution containing all the macro- and micronutrients plants need except for nitrogen, to ensure that plants only receive nitrogen through their symbiosis with rhizobia.

The positive nitrogen control plants (represented as N+ in figures) are given nutrient solution containing nitrogen, but they aren’t inoculated with rhizobia; this allows us to compare each rhizobia treatment’s nitrogen accumulation to a plant receiving as much nitrogen as it needs.
Because the roots are not visible through the soil substrate in the magenta units, I needed to harvest plants at an early time point and late time point to check how many nodules had developed. As a result, I harvested plants after six weeks and ten weeks, to see how nodule number at an early stage influenced biomass in more mature plants. To measure biomass and nodule size, plant shoots and nodules were dried in an oven and weighed.


Contrary to my prediction, rhizobia strains that developed nodules on the plants faster did not lead to greater vetch biomass at ten weeks. The following figure (Figure 2) shows the number of nodules on each hairy vetch plant at six weeks, versus the plant’s size at ten weeks. Because these experimental plants receive all their nitrogen from biological nitrogen fixation with rhizobia, we know that the size of the plant depends on how much nitrogen is fixed by rhizobia. My results show that some rhizobia strains do lead to higher biomass accumulation than other strains, but I can deduce that nodulation speed does not account for this difference in performance. Determining what enables certain rhizobia strains to fix nitrogen better at low temperatures than others remains an important question for inoculant development. In future experiments, I will investigate rhizobia traits other than nodulation speed, including how efficient strains are at fixing nitrogen, to determine what enables better nitrogen accumulation at low temperatures.

Although my experiment did not set out to test the reliability of using growth chambers, my results indicate some problems with this method that haven’t been well-explored. Growth chamber experiments are often used as a first step in testing an idea at a small scale before running a full field experiment to save costs and time. This technique, of course, differs greatly from field experiments, and results should not be extrapolated from a growth chamber experiment directly to explain something in the field. However, growth chamber experiments are useful because realities in the growth chamber can translate into realities in the field. Growth chamber experiments are frequently run for short periods of time (3 – 6 weeks), especially when researchers test nitrogen fixation by legumes. In my experiment, I harvested hairy vetch plants at both six weeks and ten weeks, measuring their biomass at both time points (Figure 3). Harvesting plants at two different time points is somewhat unusual in a growth chamber experiment, but I needed to check plant roots earlier than my harvest date.

Interestingly, I found that rhizobia strains that led plants to grow well at six weeks (i.e. plants with high biomass) did not necessarily lead plants to grow well, or have high biomass, at ten weeks. I was really surprised to discover this, because many growth chamber experiments only run for six weeks. If I had only run my experiment for six weeks, I would have a completely different understanding of which rhizobia strains led to the best plant growth. My results suggest that growth chamber experiments that depend on plant age, rather than plant growth stage, might create misleading results. For example, many farmers terminate cover crops when 50% of plants are flowering, so it would be more appropriate to terminate plants at that growth stage than at a given number of weeks of growth.


There are a few big takeaways from this experiment. First, even though it would be efficient and useful to use rhizobia strains that develop nodules the fastest as inoculant strains, my results suggest that would not be the best method. My data suggest that rhizobia strains that develop many nodules early on are not necessarily the strains that lead to the most plant growth over time. The other important takeaway from my research is that growers and researchers performing field-based experiments need a healthy dose of skepticism when translating results from the growth chamber into the field. While more experiments are needed to test growth chamber effects on plants other than hairy vetch, my results suggest that terminating an experiment based on plant age, rather than plant vegetative growth stage, might give misleading results about the effect of a treatment.

Rebecca Fudge is a Ph. D. student at the University of Minnesota in the plant and microbial biology program.

Issue: May 2022
By: Rebecca Fudge, first place winner, 2022 Organic Research Forum