Abstract:[Background] Associative nitrogen-fixing bacteria are non-host specific, and widely distributed in soil and phyllosphere environments, playing an important role in nitrogen supply for ecosystem. They also can promote plant growth indirectly via producing hormone for host plants or protecting plant from disease attacking, therein have great potential for agriculture application. However, the application of microbial inoculant into soil is limited owing to the unstable efficiency in field, due to the competition from indigenous microorganisms and inhibition effect of soils. Compared to soil habitat, phyllosphere is relatively simple and constitute large areas. Foliar application of associative nitrogen fixing bacterial inoculant provides an alternative way instead of soil application in agriculture. [Objective] To optimize the liquid inoculant of an associative nitrogen-fixing bacteria strain W12 by adding different additives, and to track its colonization on the phyllosphere of maize after foliar application. [Methods] Firstly, the 16S rRNA and nifH gene of strain W12 were sequenced to identify its taxonomy. The effects of carboxymethyl cellulose (CMC) and glycerol (Gly) used as liquid inoculant additives on the growth and nitrogenase activity of strain W12 were evaluated in liquid culture and the liquid inoculant was prepared based on the optimized condition, and then the colonization of strain W12 on the phyllosphere of maize after spraying of liquid inoculant were estimated by plate counting on a low-nitrogen agar. After harvest, the grain yield and nitrogen content of grain, stem and leaf of maize in field were measured. Meanwhile, we also inspected the shelf life of inoculant under different storage conditions. [Results] The phylogeny analysis based on 16S rRNA and nifH gene both suggested that strain W12 is closely related to the species Klebsiella variicola. CMC and glycerol addition in liquid media showed no significant influence on the growth of strain W12, but remarkably improved the nitrogenase activity by 3.7 and 24.2 times, respectively. After the foliar application of liquid inoculant via leaf spraying, the recovered strain W12-similar colonies on low-nitrogen media were up to 1.7×105 and 4.3×105 CFU/g leaf from maize phyllosphere in pot and field experiments, significantly higher than the control without inoculation, while the nitrogen content of grain, stem and leaf of maize in inoculation treatment were higher than those treatments without inoculation. The number of viable strain W12 decreased over the storage time while storage at 4 °C maintained much higher viable bacteria than at room temperature, with the viable bacteria number more than 1.0×108 CFU/mL after storage at 4 °C for 90 days. [Conclusion] CMC and glycerol used as additives of liquid inoculant are favorable for bacteria adhesion on leaves and could remarkably improve nitrogenase activity of strain W12. Storage at 4 °C can sustain long shelf life of strain W12 liquid inoculation, compared with room temperature. After foliar application in field, strain W12 could successfully colonize in the phyllosphere and inoculation significantly improved the nitrogen content of maize grain and plant. Our results exhibit new insights on the preparation and application of foliar inoculant, and set a good example for field application of nitrogen fixing bacteria to reduce the nitrogen fertilizer application and stabilize crop yield.
