Acetobacter diazotrophicus, a nitrogen-fixing bacterial endophyte of sugarcane: Analysis of nifHDK genes, plant colonization, and growth promotion

Abstract

Acetobacter diazotrophicus, a nitrogen-fixing bacterial endophyte, is believed to be responsible for biological nitrogen fixation (BNF) in sugarcane. However, no monocot has yet been unequivocally shown to receive fixed N through BNF. The main question addressed in this dissertation is whether A. diazotrophicus promotes plant growth, and if so, whether growth promotion is via BNF. Another question is whether the growth benefits can be extended to other grasses. To answer these questions, the nifHDK genes encoding the protein subunits of the nitrogenase enzyme were first isolated and sequenced. Secondly, Nif⁻ mutant strains were constructed by inserting a gene cassette in nifD. The growth of sugarcane plants inoculated with A. diazotrophicus wild type and Nif⁻ mutant strains were compared in growth chamber, greenhouse, and field experiments. A. diazotrophicus was also tagged with marker genes to investigate the colonization process in sugarcane and other grasses. The effect of A. diazotrophicus on the growth of other grasses was also determined. Analysis of the A. diazotrophicus NifHDK sequence revealed features typical of proteobacterial nifHDK genes and gene products. Phylogenetic analysis established the close relationship of A. diazotrophicus with the α-proteobacteria and the β-proteobacterium, Herbaspirillum seropedicae, another sugarcane endophyte. Nif⁻ mutant strains established endophytically in sugarcane plants equally well as wild type strains. ¹⁵N₂ incorporation experiments demonstrated that wild type strains but not the Nif⁻ mutants fixed N inside sugarcane plants with decreased fixation when plants were grown in medium with fixed N. In N-deficient conditions, sugarcane inoculated with wild type strains grew better and had higher total N content than either uninoculated or plants inoculated with Nif⁻ mutants. When N was not limiting, growth enhancement was observed in plants inoculated with either wild type or the Nif⁻ mutants. These results suggest that depending on the nitrogen condition, A. diazotrophicus promotes sugarcane growth via nitrogen fixation and other growth promoting factor. The results also indicated a possible strain-cultivar specificity in growth promotion. A. diazotrophicus colonized other grasses through different entry sites but was limited in the root. Under N-deficient conditions, wild type strain but not the Nif-- mutant promoted rice seedling growth indicating the beneficial effects of A. diazotrophicus to other grasses.