Endophytic N2 fixation in sweet potato: responses to N, P, and K inputs and visualization of 15N2 utilizing bacterial cells via Raman spectroscopy

Abstract

Fertilizer-N strongly limits non-leguminous crop yields; however, sweet potato (Ipomoea batatas) is an exception, likely due to its ability to acquire atmospheric N2 via endophytic diazotrophs. Using Raman spectroscopy, we found that in 15N2-fed sweet potato, some endophytic bacteria contained 15N, providing direct evidence of N2 fixation in planta. To assess N2-fixing capability, pot experiments were conducted by varying N, P, and K fertilizer inputs. Sweet potato showed higher N content than the non-N2-fixing Ipomoea aquatica; additionally, it showed increased N content which was 1.4-fold higher than the fertilizer-N input. Its δ15N values were closer to those of N2-fixing soybean, with an estimated 11–56% of plant N derived from N2. The estimated amount of fixed-N in sweet potato was negligible without fertilizer-N; however, a gradual accumulation leading to an immediate saturation was observed with increasing fertilizer-N. During this state, increasing P supply linearly enhanced the capability, reaching 13 g N m−2, comparable to that of legumes. However, K inputs affected neither N2 fixation nor growth owing to strong K acquisition from the soil even without fertilizer-K. Our results indicate extensive N2 fixation in sweet potato, depending on its nutritional status, particularly P; resonance Raman spectroscopy facilitates the visualization of active N2-fixing bacteria on a single-cell scale.