This study examined how a riboflavin-requiring mutant of Rhizobium trifolii forms nodules on red clover and how the supply of riboflavin affects nodule development and nitrogen fixation. The mutant strain (T1/D-hisT-15) produced ineffective or partially effective nodules lacking sufficient nitrogen-fixing bacteroids when grown without added riboflavin, but developed normally when riboflavin was supplied. Riboflavin was most critical during days 7-9 after inoculation, coinciding with bacteroid formation from vegetative bacteria. Using light and electron microscopy, researchers documented that without riboflavin, infected cells contained primarily vegetative rods instead of functional bacteroids, and large accumulations of polysaccharide material impeded bacterial release from infection threads. When riboflavin was provided early (at inoculation or 3 days post-inoculation), nodules developed normally. Remarkably, riboflavin could be withdrawn after 6-8 days post-inoculation without sacrificing nodule effectiveness, suggesting that once critical early development is supported, the plant can subsequently sustain the bacteria. Acetylene reduction assays confirmed these structural observations, showing nitrogen fixation rates recovered when riboflavin was supplied at appropriate developmental windows.

Key findings

• Riboflavin requirement is most critical 7-9 days after inoculation, coinciding with the conversion of vegetative bacteria to nitrogen-fixing bacteroids in nodule cells.
• Without external riboflavin supply, the mutant strain fails to properly form bacteroids and produces nodules with only ~15% of parent strain nitrogen fixation activity.
• Riboflavin supplied early in development (by day 3-4 of inoculation) can be subsequently withdrawn after day 6-8 without impairing nodule effectiveness, indicating critical early-stage vitamin dependence.
• Absence of riboflavin causes abnormal accumulation of polysaccharide material around bacteria in infection threads, impairing their release into host cells.
• Delayed riboflavin addition causes proportional delays in nodule formation and nitrogen fixation, with recovery possible if vitamin is provided by day 8 post-inoculation.