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A collaborative research team from The Chinese University of Hong Kong (CUHK) and the French National Research Institute for Sustainable Development (IRD) has made a remarkable biological discovery: certain soil bacteria known as Bradyrhizobium acquired the capacity to fix nitrogen on their own long before they evolved symbiotic relationships with legumes. Published in the leading international journal Proceedings of the National Academy of Sciences (PNAS), the findings overturn the long-held belief that the nitrogen-fixing capacity of a prevalent bacterial ally of legume plants such as soybean and peas primarily evolved within legume root nodules. This discovery could pave the way for the development of natural biofertilisers for major staple crops such as rice, wheat and corn.

Today, nearly half of the global food production relies on chemical nitrogen fertilisers. However, their manufacturing is energy-intensive, leading to greenhouse gas emissions as well as water pollution. While legumes can form symbiotic relationships with bacteria in their root nodules to fix atmospheric nitrogen, staple crops including rice, wheat and corn lack this symbiotic capability and remain heavily dependent on chemical fertilisers.

Uncovering genetic evolution of nitrogen-fixing bacteria

Led by Professor Luo Haiwei, Associate Professor in CUHK’s School of Life Sciences, the team included Dr Ling Lu, a former PhD student of Professor Luo who carried out this work as her doctoral research. They collected root and soil samples from non-legume plants in rice paddies, forests and wetlands across Hong Kong and the Chinese Mainland, isolating 88 strains of Bradyrhizobium. The team discovered that these bacteria not only survive freely in the soil but also settle on the roots of non-legume plants while continuing to fix nitrogen without requiring legume nodules. In other words, they can independently produce natural nitrogen fertiliser.

Using advanced genomic analysis, the team reconstructed the evolutionary history of the bacteria’s nitrogen-fixing genes. The results showed that the ancestors of these bacteria had already been capable of fixing nitrogen in the soil long before they partnered with legumes, revealing that the symbiotic relationship with legumes evolved independently multiple times later.

The ‘protective shield’ gene enabling bacteria’s independent survival

The researchers further identified a key gene, glbO, that is commonly found in free-living strains. This gene produces a protein that protects the oxygen-sensitive nitrogen-fixing enzyme from oxygen damage, acting like a “protective shield”. By contrast, bacteria living inside legume nodules are protected by the low-oxygen environment provided by the host plant, leading many symbiotic strains to lose this gene through evolution.

To verify this mechanism, the CUHK team collaborated with Dr Eric Giraud and his colleagues at IRD to study the unique Bradyrhizobium strain ORS285, which can fix nitrogen both inside legume nodules and in the free-living state. When the researchers disabled the glbO gene in this strain, the bacteria’s ability to fix nitrogen in the soil declined significantly, while its performance inside legume nodules remained unaffected. Conversely, restoring the gene to a strictly symbiotic strain that had previously lost it significantly improved its capacity to fix nitrogen independently in soil.

New opportunities for sustainable natural biofertilisers and global food security

Professor Luo said: “Our study shows that Bradyrhizobium bacteria were already able to fix nitrogen on their own long before they evolved symbiotic relationships with legumes. The symbiotic, legume-dwelling lifestyle evolved independently multiple times from these free-living ancestors. While mainstream research has focused on nitrogen fixation within legume root nodules, our findings suggest that the ancestral, free-living bacteria represent the original evolutionary starting point of this process. By understanding how these bacteria protect their nitrogen-fixing machinery in soil, we may be able to develop or screen strains that help non-legume crops such as rice and wheat produce their own fertiliser.”

Dr Ling, the study’s first author, added: “Chemical fertilisers are costly and environmentally damaging. Yet nature has already evolved bacteria capable of supplying plants with nitrogen at no cost. The challenge now is learning how to harness these beneficial microbes effectively.”

By demonstrating that free-living nitrogen-fixing Bradyrhizobium are both widespread and diverse in nature, this study provides a robust scientific foundation for future agricultural biotechnology. In the future, researchers may be able to use genetic engineering and microbial screening techniques to introduce these naturally protected bacteria into rice and wheat fields, thereby improving global food security while reducing agriculture’s environmental footprint.

The full research article is available here: https://www.pnas.org/doi/10.1073/pnas.2604918123