Nitrogen is the nutrient that most often limits crop productivity, and modern agriculture supplies it overwhelmingly through synthetic fertilizers produced by the energy-intensive Haber-Bosch process. Legumes such as soybeans and peas solve part of the problem naturally, hosting rhizobia bacteria in root nodules that convert atmospheric dinitrogen gas into biologically usable ammonia. Cereals, however, including rice, wheat and maize, lack this symbiotic machinery, so they depend almost entirely on applied nitrogen. Global nitrogen budgets for cereal systems show that a large share of applied fertilizer is never captured by the crop, escaping into waterways or the atmosphere as potent pollutants. For decades, scientists have therefore pursued associative nitrogen fixation, the idea that free-living soil bacteria could be encouraged to colonize the roots of cereals and share some of the nitrogen they fix. The new study adds a striking piece to that puzzle by showing that a fungal endophyte can reshape the soil environment to favor exactly those bacteria.

The research team, led by Jun-Feng Ouyang and Chuan-Chao Dai, worked with two genetically distinct rice subspecies: a japonica variety called WYJ23 and an indica variety called HLY996. Each variety was grown under low-nitrogen conditions both with and without inoculation by the endophytic fungus B3, a strain of Phomopsis liquidambaris that the group had previously shown to improve nitrogen cycling in the rice rhizosphere. The central question was deceptively simple: if the fungus changes which nitrogen-transforming microbes gather around rice roots, does it do so by changing the chemistry of the rhizosphere, the narrow zone of soil directly influenced by root secretions? To answer it, the researchers collected the metabolites exuded into the rhizosphere soil and subjected them to untargeted metabolome analysis, a technique that profiles the full spectrum of small molecules present in a sample without preselecting which compounds to look for.

The metabolomic comparison revealed that infection with B3 substantially reshaped the rhizosphere metabolite profiles of both rice subspecies. Among the many compounds whose abundance shifted, one stood out for its consistency: p-hydroxyphenylacetic acid, a phenolic acid, was upregulated in the rhizosphere of both japonica WYJ23 and indica HLY996 plants carrying the endophyte. That cross-subspecies consistency matters, because it suggests the fungus is not merely nudging the idiosyncratic chemistry of one rice genotype but is instead steering a shared metabolic pathway that operates across rice diversity. Phenolic acids are well known players in root-soil signaling, and previous studies have implicated related compounds, such as benzoic acid in maize and flavones in rice and poplar, in the recruitment of diazotrophic, or nitrogen-fixing, bacteria. The new findings place p-hydroxyphenylacetic acid squarely within this growing catalog of root-derived molecules that function as microbial attractants.

Establishing that the fungus changes root chemistry was only the first step. The team then asked whether those changed chemicals were actually responsible for recruiting the nitrogen-fixing community. In a soil conditioning experiment, they treated soil with the rhizosphere metabolites harvested from B3-inoculated rice and from uninoculated controls, then measured the resulting microbial communities. Soils receiving metabolites from endophyte-colonized plants exhibited markedly higher abundances of nitrogen-fixing bacteria. High-throughput sequencing of the conditioned soils sharpened the picture further: metabolites from B3-inoculated plants of both rice subspecies significantly enriched the bacterial taxon Enterobacter, a genus that includes well-documented plant-associated diazotrophs. In other words, the chemical signature left by the fungus-colonized roots was sufficient, on its own, to summon the very bacteria capable of fixing atmospheric nitrogen.

To test the mechanism at the level of individual molecules and microbes, the researchers turned to co-culture assays, growing nitrogen-fixing bacterial strains in the presence of p-hydroxyphenylacetic acid. The compound proved to be a highly bioavailable substrate for the diazotrophs that B3 inoculation had enriched, meaning the bacteria could readily take it up and use it to fuel their growth. Among the tested taxa, strains of Enterobacter showed the most pronounced responses, with measurable increases in both growth and biofilm formation. Biofilm formation is particularly significant in this context: dense bacterial films on root surfaces are thought to create the oxygen-limited microenvironments that nitrogenase, the oxygen-sensitive enzyme responsible for nitrogen fixation, requires to function. A molecule that simultaneously feeds diazotrophs and encourages them to coat the root in a biofilm is therefore doing double duty as both a nutrient and an architectural signal.

The final and arguably most consequential phase of the study asked whether this fungal-chemical-bacterial cascade actually benefits the plant. In greenhouse assays, the team applied Enterobacter strains and a microbiome modified by p-hydroxyphenylacetic acid to rice plants and measured growth and nitrogen accumulation. Both treatments significantly promoted rice biomass and nitrogen content, confirming that the recruited bacteria deliver more than mere company. The standout performer was Enterobacter strain T-9, which produced the largest gains of any isolate tested: increases of 30.05 percent in dry biomass and 24.6 percent in nitrogen accumulation relative to controls. Those are substantial effect sizes for a single bacterial inoculant, and they demonstrate that the enrichment strategy does not merely shift community composition but translates into measurable plant performance under nitrogen-limited conditions.

The study’s design deserves attention because it carefully disentangles correlation from causation at each step. Metabolomics identified candidate compounds; soil conditioning showed that those compounds reshape communities; sequencing pinpointed the enriched taxa; co-culture verified substrate preference and growth responses; and plant assays confirmed functional benefits. This chain of evidence addresses a persistent weakness in rhizosphere research, where studies often stop at showing that a treatment and a community shift co-occur. By isolating p-hydroxyphenylacetic acid and demonstrating its direct effects on Enterobacter physiology, the researchers provide a mechanistic link rather than a statistical association. The work also builds on a broader literature showing that root exudates act as signals and nutrients in plant-microbe interactions, and that specific exudate compounds can be manipulated, whether by endophytes, by breeding, or by genetic engineering of flavone biosynthesis, to enhance associative nitrogen fixation in cereals.

The agricultural implications are considerable. If a single, chemically defined compound can be applied to soil to recruit nitrogen-fixing bacteria around cereal roots, growers could potentially reduce fertilizer inputs without sacrificing yield, cutting both costs and the environmental externalities of nitrogen pollution. The compound itself, p-hydroxyphenylacetic acid, is a small organic acid that could in principle be manufactured and applied as a soil amendment, or its production could be encouraged by inoculating crops with endophytes such as B3. The finding that the mechanism worked across both japonica and indica subspecies suggests a degree of generality that would be essential for any practical deployment across the enormous diversity of cultivated rice. At the same time, the authors emphasize the tight association between rhizosphere metabolites and nitrogen-fixing bacteria as a general principle, hinting that similar metabolite-mediated recruitment may operate in other crops and with other beneficial taxa, a possibility already supported by studies of peach, soybean, maize and poplar systems.

There remain, of course, the usual distances between greenhouse results and open-field reality. Soil conditioning experiments and co-culture assays are controlled settings, and the behavior of p-hydroxyphenylacetic acid in complex paddy soils, with their distinctive flooded chemistry and competing microbial populations, will need direct testing. The stability, dosage and timing of any metabolite-based amendment would also require optimization, and the ecological consequences of systematically enriching Enterobacter populations in paddies would need assessment. Still, the study represents a compelling proof of concept: an invisible fungal partner inside the root can reprogram the chemical conversation between plant and soil, and that reprogrammed conversation recruits bacteria that pull nitrogen from the atmosphere and hand it to the crop. As the search for nitrogen-efficient cereals accelerates, the most transformative tools may prove to be not genes or fertilizers, but the small molecules that mediate who gets invited to the root’s dinner table.

Subject of Research: How an endophytic fungus alters rice rhizosphere metabolites to recruit nitrogen-fixing bacteria

Article Title: Endophytic Phomopsis liquidambaris recruits nitrogen-fixing taxa by altering the rhizosphere metabolites of Oryza sativa L.

Article References: Ouyang, J.-F., Zhang, Y., Fei, Y.-J., Jiang, L., & Dai, C.-C. (2026). Endophytic Phomopsis liquidambaris recruits nitrogen-fixing taxa by altering the rhizosphere metabolites of Oryza sativa L.. Plant and Soil. https://doi.org/10.1007/s11104-026-09087-1

Image Credits: AI Generated

DOI: 10.1007/s11104-026-09087-1

Keywords: rice, endophytic fungus, Phomopsis liquidambaris, nitrogen fixation, rhizosphere metabolites, p-hydroxyphenylacetic acid, Enterobacter, soil microbiome, root exudates, metabolomics, sustainable agriculture, Plant and Soil