Effects of Different Sources of Nitrogen on Endophytic Colonization of Rice Plants by Azospirillum sp. B510

Azospirillum sp. B510, a free-living nitrogen-fixing bacterium isolated from the stems of rice (Oryza sativa cv. Nipponbare), was investigated to establish effective conditions for the colonization of rice plants. We analyzed the effects of the nitrogen sources KNO₃, NH₄Cl, urea (CO[NH₂]₂), and NH₄NO₃ at different concentrations (0.01-10 mM) on this colonization. Nitrogen promoted plant growth in a concentration-dependent manner, with minor differences being observed among the different nitrogen sources. Bacterial colonization was markedly suppressed on media containing NH₄⁺ concentrations higher than 1 mM. Since concentrations of up to and including 10 mM NH₄⁺ did not exhibit any antibacterial activity, we analyzed several factors affecting the NH₄⁺-dependent inhibition of endophytic colonization, including the accumulation of the reactive oxygen species H₂O₂ and the secretion of the chemotactic substrate malic acid. The accumulation of H₂O₂ was increased in rice roots grown on 1 mM NH₄Cl. The amounts of malic acid secreted from NH₄-grown rice plants were lower than those secreted from plants grown without nitrogen or with KNO₃. Although the bacterium exhibited chemotactic activity, moving towards root exudates from plants grown without nitrogen and KNO₃-grown plants, this activity was not observed with root exudates from NH₄⁺-grown plants. NH₄⁺, but not NO₃^-, caused the acidification of growth media, which inhibited plant bacterial colonization. These NH₄⁺-dependent phenomena were markedly suppressed by the stabilization of medium pH using a buffer. These results demonstrate that the type and concentration of nitrogen fertilizer affects the colonization of rice plants by Azospirillum sp. B510.