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Measuring fitness benefits to rhizobia from symbiosis with legumes

This week's paper is "Measuring the fitness of symbiotic rhizobia", published in the journal Symbiosis by Will Ratcliff, who earned a PhD with me, Kyra Underbakke, who did a prize-winning science fair project in our lab when she was in high school and has done undergraduate research with us since then, and me.
Alfalfa nodules containing nitrogen-fixing rhizobia. Photo by Alex May.

Rhizobia are soil bacteria, best known for infecting the roots of legume plants, reproducing inside swellings called nodules, and converting nitrogen gas from the soil atmosphere into forms their plant hosts can use. Since about 2000, I've been asking why they do these things.

Every "why" question in biology has the same general answer, although details differ. Living things do what they do largely because they inherited a tendency to do so, from ancestors whose survival and reproduction depended on doing something similar. So rhizobia infect legume roots and "fix" (take up) nitrogen inside nodules because ancestors who did that had greater fitness (proportional representation in the next generation), relative to otherwise similar bacteria that didn't do these things. (Many of the ancestors of a given rhizobial cell may have spent their lives in soil, never infecting a legume root. But those that did had so many more descendants that the trait has persisted.)

Infecting a root and reproducing inside a nodule seems like a no-brainer, which is convenient, since rhizobia don't have brains. But why use resources to fix nitrogen that the rhizobia could have used for more reproduction, instead. We hypothesized (Denison 2000, West et al. 2002), and then confirmed experimentally (Kiers et al. 2003, Oono et al. 2011), that legumes (in particular, soybeans, alfalfa, and pea plants) treat nodules that fail to fix nitrogen differently, in ways that presumably keep the legumes from wasting resources, and incidentally reduce the reproduction of rhizobia inside. We have called these plant responses "sanctions", without any implication that plants are self-aware or that sanctions will change the behavior of rhizobia, except via evolutionary decreases in the frequency of rhizobial "cheaters" over generations.

Moderate cheating (fixing less nitrogen than the best strains, but still some) may or may not trigger sanctions (Kiers et al. 2006, Simms et al. 2006, Heath and Tiffin 2009). But how can we tell? Some researchers have found a correlation between rhizobia/nodule and nodule weight, then looked to see whether strains that are more beneficial make larger nodules, presumably containing more rhizobia. We've based our conclusions on actual counts of rhizobia, worrying that correlations might be misleading.

For example, Gubry-Rangin and colleagues (Gubry-Rangin et al. 2010) found that nodules containing a strain that couldn't fix nitrogen were smaller (consistent with the host imposing sanctions), yet they contained similar numbers of rhizobia as nodules containing a good nitrogen fixer (so those sanctions might not always affect rhizobial evolution the way we've hypothesized). They noted that:

"These results may therefore contrast with the positive correlation between the size and the viable rhizobia found in M. truncatula (Heath & Tiffin 2007). As discussed by Oono et al. (2009), this relationship may vary among different rhizobia..."

This week's paper provides additional evidence of this. There was a good correlation between nodule weight and rhizobia/nodule for each of the two strains in the figure, individually, but this relationship differed between strains.
Even actual measurements of numbers of rhizobia/nodule may not be a complete measure of the fitness benefits rhizobia gain from symbiosis. We found that a rhizobial strain that was less beneficial to its plant host accumulated more resources (specifically, polyhydroxybutyrate or PHB) per rhizobial cell. How much more? Enough to reproduce without external resources. Correcting for PHB showed that the less-beneficial strain gained twice as much fitness from symbiosis as the better strain, whereas ignoring PHB would have led to the incorrect conclusion that there was no difference in fitness between the strains.

There may be more to this story. Having enough PHB to reproduce without external resources only matters if external resources are limiting. Under starvation conditions, rhizobia can definitely use PHB to survive and even to reproduce (Ratcliff et al. 2008). But do nodules containing higher-PHB rhizobia release more rhizobia to the soil (because they use the PHB to reproduce inside dying nodules) or do rhizobia released still have extra PHB? If the latter, how much does this extra PHB affect survival and reproduction in soil? If the National Science Foundation funds our next grant proposal, we will find out. If they don't, I still appreciate their support of my past research.


Denison R. F. 2000. Legume sanctions and the evolution of symbiotic cooperation by rhizobia. American Naturalist 156:567-576.

Gubry-Rangin C., M. Garcia, and G. Bena. 2010. Partner choice in Medicago truncatula-Sinorhizobium symbiosis. Proceedings of the Royal Society B 277:1947-1951.

Heath K. D., P. Tiffin. 2009. Stabilizing mechanisms in a legume-rhizobium mutualism. Evolution 63:652-662.

Kiers E. T., R. A. Rousseau, and R. F. Denison. 2006. Measured sanctions: legume hosts detect quantitative variation in rhizobium cooperation and punish accordingly. Evolutionary Ecology Research 8:1077-1086.

Kiers E. T., R. A. Rousseau, S. A. West, and R. F. Denison. 2003. Host sanctions and the legume-rhizobium mutualism. Nature 425:78-81.

Oono, R., R.F. Denison, E.T. Kiers. 2009. Tansley review: Controlling the reproductive fate of rhizobia: How universal are legume sanctions? New Phytologist 183:967-979.

Oono, R., C. G. Anderson, and R. F. Denison. 2011. Failure to fix nitrogen by non-reproductive symbiotic rhizobia triggers host sanctions that reduce fitness of their reproductive clonemates. Proceedings of the Royal Society B 278:2698-2703.

Ratcliff,W.C., K. Underbakke, R.F. Denison. 2012. Measuring the fitness of symbiotic rhizobia. Symbiosis 55: 85-90.

Ratcliff W. C., S. V. Kadam, and R. F. Denison. 2008. Polyhydroxybutyrate supports survival and reproduction in starving rhizobia. FEMS Microbiology Ecology 65:391-399.

Simms E. L., D. L. Taylor, J. Povich, R. P. Shefferson, J. L. Sachs, M. Urbina, and Y. Tausczik. 2006. An empirical test of partner choice mechanisms in a wild legume-rhizobium interaction. Proceedings of the Royal Society B 273:77-81.

West S. A., E. T. Kiers, E. L. Simms, and R. F. Denison. 2002. Sanctions and mutualism stability: why do rhizobia fix nitrogen? Proceedings of the Royal Society B 269:685-694.


This isn't really a comment on this post, per se, but I'm wondering what you think of the possibility/desirability of engineering N2 fixation in cereals, either directly or by engineering a bacterial symbiont.

I think it could be beneficial to both food security and the environment IF the cereals limited their nitrogen fixation rate to the amount of nitrogen they actually need, as legumes do. But I don't expect to see cereals that can meet > 50% of their nitrogen needs from fixation by 2025. It's just too hard. I've written about why:

Just so interesting to read about your research! I am just hooked up on legumes for the moment - have just put up a page on my site on alternative fuel sources. http://www.best-alternative-fuel-sources.com/legume-trees.html I believe the discussion on legumes is a bit forgotten in the biofuel debate.
I wish you good luck with your research and hope you get more happening!
Cheers Anna

Thanks for the link to your interesting site. I question whether biofuels are a better solution to energy problems than conservation, wind, etc. But we also depend on oil as the main raw ingredient (feedstock) to make lubricants, synthetic fibers and other plastics, etc. As oil becomes more expensive, we will have to switch to plants, as your site mentions. And legumes have a lot to offer there.

Your site says that "If a product (whatever that is, chocolate or fuel) pollutes a river or replaces people on its way, we can't call it sustainable. Or ethically acceptable." I would say that moderate levels of pollution may be "sustainable" (possible to continue indefinitely) but not necessarily ethically acceptable.

Great post to read. But its a little bit confusing about the possibility of N2 fixation. My confusion is about that whether it will be good or bad for nature.
It is obvious that in near future we will be in crisis of oil, petrol, oaten or whatever other sources.
I also think that needs of nitrogen can not be fulfill within 2025 or 2050.
By the way, Great research work.

Generally, N2-fixing crops reduce their fixation rate when soil N is high, so probably less pollution than with fertilizers (including organic ones, like manure), unless the fertilizer is very carefully managed. Based on errors in what you wrote, you might want to consider a career other than writing.

Quite interesting to read. Since schooling we learnt about legumes and symbiosis. But why they do so is a curious question. By the way, since bacteria have no brains, are there chances that they can be selfish and take more benefits from plant roots?:-) Thanks for posting this article..

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