Arbuscular mycorrhizal fungi are celebrated allies of legumes, helping roots gather phosphorus that fuels the energy-hungry nodules where rhizobial bacteria fix nitrogen. A new greenhouse study of alfalfa (Medicago sativa) shows, however, that this celebrated partnership has an Achilles heel: when warming and drought strike together, the fungi stop boosting nodulation and redirect their contribution toward maintaining the phosphorus status of the host’s shoots. The findings, published in Ecological Processes, come from a factorial experiment conducted at Western Sydney University’s Hawkesbury Campus in Richmond, New South Wales, Australia, and they carry a caution for anyone counting on soil microbes to buffer legume crops against a changing climate.
The research team, led by Xiaoning Dong, Xue Han and colleagues including Haiyang Zhang, grew alfalfa cultivar SARDI 7 Series 2 in 64 pots distributed across four greenhouse chambers. Three factors were crossed: two watering regimes (well-watered versus drought), two temperature regimes (ambient at 26/18 °C day/night versus warming at 30/22 °C day/night, representing a predicted 4 °C regional increase by the end of the century), and inoculation with or without the arbuscular mycorrhizal fungus Rhizophagus irregularis. All pots received a compatible rhizobial inoculant, so the experiment isolated the mycorrhizal contribution to a tripartite symbiosis that also included the nitrogen-fixing bacteria. The experiment ran from mid-April to early August 2018 and lasted four months.
The substrate was deliberately engineered to make the plants dependent on their microbial partners. Sandy-loam soil was mixed with quartz sand at a 7:3 ratio, gamma-irradiated at 50 kGy to eliminate the indigenous microbiome including native arbuscular mycorrhizal fungi, and then reseeded with a standardized microbial wash. The wash, prepared by wet-sieving field-fresh soil through a 20 µm nylon mesh, retained ambient bacteria and microflora while excluding arbuscular mycorrhizal spores roughly 40 to 140 µm in diameter. Soil pH was 5.6, with plant-available nitrogen of 55 mg per kg and plant-available phosphorus of 39 mg per kg, concentrations the authors describe as strategically low to promote symbiotic dependence. A non-legume companion species grown in the same substrate showed leaf nitrogen and phosphorus levels indicative of severe nutrient limitation in temperate grasses.
Watering was initially maintained at 100 percent water holding capacity, corresponding to a gravimetric water content of 17.3 percent, with irrigation every two days. Drought treatments began three weeks before the final harvest: water was withheld until water holding capacity dropped to 40 percent over three to four days, pots were held at that level for a week, then rewatered to 100 percent for a week, and subjected to a second week-long drought phase before harvest. Well-watered pots stayed at 100 percent throughout. Plants grew under a 15-hour light and 9-hour dark cycle with natural daylight supplemented by fluorescent lamps to roughly 400 µmol per square metre per second of photosynthetic photon flux density, at 60 percent relative humidity. Aboveground material was trimmed once about eight weeks after transplanting to mimic pasture clipping, then allowed to regrow for another eight weeks before the final harvest.
At harvest, the researchers separated nodules from a representative subsample of fresh roots, counted and weighed them, and extrapolated the nodule-to-root ratio to total root biomass to estimate total fresh nodule biomass and nodule number per pot. Root biomass allocation to nodules was defined as the proportion of total belowground biomass dedicated to nodules. Fresh nodules were incubated with acetylene for one hour, and ethylene production was measured to estimate nitrogenase activity via the acetylene reduction assay. Shoots and roots were dried, ground, and analyzed for total nitrogen, nitrogen isotope composition, and total phosphorus, with phosphorus quantified by X-ray fluorescence spectrometry. The percentage of nitrogen derived from biological fixation was calculated using the δ15N natural abundance method, with Festuca arundinacea grown in the same substrate serving as the non-fixing reference plant and a published value of −1.14‰ used for the nitrogen isotope ratio of alfalfa relying entirely on fixation.
The results were striking. Under control conditions, mycorrhizal inoculation increased root biomass allocation to nodules by 59.9 percent and fresh nodule biomass by 72 percent compared with non-mycorrhizal plants. Under warming alone, allocation rose 63.9 percent and nodule biomass 53.5 percent; under drought alone, the corresponding figures were 45 percent and 43.2 percent. Under combined drought and warming, however, neither metric showed a significant mycorrhizal effect.
Phosphorus emerged as a central currency in this trade. Across all four climate treatments, fresh nodule biomass correlated negatively with both shoot and root phosphorus concentration, in mycorrhizal and non-mycorrhizal plants alike. In the mycorrhizal treatment, root colonization was negatively correlated with root phosphorus concentration and marginally positively correlated with fresh nodule biomass. The authors interpret the negative nodule-phosphorus relationship as evidence that nodulation imposes a substantial phosphorus cost, while acknowledging it may partly reflect a size-related dilution effect, since plants with greater nodule biomass also tended to have higher shoot biomass and consequently lower phosphorus concentrations.
The most telling shift appeared in shoot phosphorus. Drought alone decreased shoot phosphorus concentration and content, and drought also reduced shoot nitrogen concentration and content, the percentage of nitrogen derived from fixation, and the total amount of fixed nitrogen. But under combined warming and drought, mycorrhizal inoculation increased shoot phosphorus concentration and content, an effect absent under control conditions and under the single stresses. The authors conclude that compound climate stress redirects the mycorrhizal contribution away from supporting energy-intensive nodulation and toward maintaining the host’s shoot phosphorus status. When hosts are not stressed, the fungi preferentially direct acquired phosphorus toward nodules; under combined stress, that preferential routing apparently ceases, and the phosphorus the fungi deliver instead shows up in the shoots.
Why would combined warming and drought, but not either stress alone, erase the nodulation benefit? The authors propose a carbon-allocation trade-off: both symbionts draw on the same photosynthate pool, and climate stress that reduces carbon supply intensifies competition between them, shifting the mycorrhizal contribution away from nodulation. They note that warming alone did not alter mycorrhizal colonization or nodule biomass in their experiment, suggesting belowground carbon allocation was not substantially changed by that single factor. Drought, by contrast, is known to suppress rhizobial infection, nodule development, and nitrogenase activity through metabolic carbon limitation, and to restrict extraradical fungal hyphae and soil phosphorus mobility.
The study has limitations the authors acknowledge candidly. Mycorrhizal colonization was assessed on only four randomly selected replicates per treatment after some samples were lost during storage, a smaller sample than the six to eight used for biomass measurements, and colonization levels varied substantially among samples, with means ranging from 21.2 percent under drought to 44.0 percent under warming. The researchers also could not fully decouple the specific contributions of arbuscular mycorrhizal fungi to plant nitrogen status from those of the legume-rhizobia symbiosis, and they call for future work incorporating non-nodulated controls, wider ranges of inoculation densities, direct measurements of photosynthesis and carbon flux, and quantification of extraradical mycelium and specific intraradical structures.
Subject of Research: How combined warming and drought alter the effects of arbuscular mycorrhizal fungi on nodulation in the legume Medicago sativa.
Article Title: Warming combined with drought removes the beneficial effects of arbuscular mycorrhizal fungi on legume (Medicago sativa) nodulation
Article References: Dong, X., Han, X., Bo, Y., Plett, J. M., Wang, G., Yang, J., Miao, K., Li, Z., Shao, W., Zhang, X., Jiang, L., Han, X., & Zhang, H. (2026). Warming combined with drought removes the beneficial effects of arbuscular mycorrhizal fungi on legume (Medicago sativa) nodulation. Ecological Processes, 15(1), Article 82. https://doi.org/10.1186/s13717-026-00743-z
Image Credits: AI Generated
DOI: 10.1186/s13717-026-00743-z
Keywords: arbuscular mycorrhizal fungi, rhizobia, legumes, alfalfa, Medicago sativa, nodulation, nitrogen fixation, phosphorus, drought, warming, climate stress, greenhouse experiment
News Source: Roger Howard. (October 30, 2026). Warming plus drought strips mycorrhizal fungi of their nodulation benefits in alfalfa. Scienmag.
