Genotypic Variation of Endophytic Nitrogen-Fixing Activity and Bacterial Flora in Rice Stem Based on Sugar Content

Enhancement of the nitrogen-fixing ability of endophytic bacteria in rice is expected to result in improved nitrogen use under low-nitrogen conditions. Endophytic nitrogen-fixing bacteria require a large amount of energy to fix atmospheric nitrogen. However, it is unknown which carbon source and bacteria would affect nitrogen-fixing activity in rice. Therefore, this study examined genotypic variations in the nitrogen-fixing ability of rice plant stem as affected by non-structural carbohydrates and endophytic bacterial flora in field-grown rice. In the field experiments, six varieties and 10 genotypes of rice were grown in 2017 and 2018 to compare the acetylene reduction activity (nitrogen-fixing activity) and non-structural carbohydrates (glucose, sucrose, and starch) concentration in their stems at the heading stage. For the bacterial flora analysis, two genes were amplified using a primer set of 16S rRNA and nitrogenase (NifH) gene-specific primers. Next, acetylene reduction activity was correlated with sugar concentration among genotypes in both years, suggesting that the levels of soluble sugars influenced stem nitrogen-fixing activity. Bacterial flora analysis also suggested the presence of common and genotype-specific bacterial flora in both 16S rRNA and nifH genes. Similarly, bacteria classified as rhizobia, such as Bradyrhizobium sp. (Alphaproteobacteria) and Paraburkholderia sp. (Betaproteobacteria), were highly abundant in all rice genotypes, suggesting that these bacteria make major contributions to the nitrogen fixation process in rice stems. Gammaproteobacteria were more abundant in CG14 as well, which showed the highest acetylene reduction activity and sugar concentration among genotypes and is also proposed to contribute to the higher amount of nitrogen-fixing activity.

Influence of environmental factors and exogenous nitrogen on nitrogen fixation by intact thallus and excised cephalodia of Peltigera aphthosa

The present study investigates the effects of various abiotic environmental factors: air humidity, moisture content, oxygen and exogenously supplied nitrogen on acetylene reduction by intact thallus and excised cephalodia of Peltgera aphthosa. Intact thallus and excised cephalodia of Peltigera aphthosa were incubated at various conditions of air humidity, moisture contents, oxygen tensions, and addition of exogenous nitrogen, and comparative nitrogen fixation by the intact thallus and excised cephalodia was used as a method for assessment. Acetylene reduction (nitrogen fixation) was enhanced at conditions of lowered oxygen tension and at initial addition of nitrogen to the medium. However, prolonged incubation of Peltigera aphthosa in the medium with combined nitrogen addition resulted in fluctuation of nitrogenase synthesis. Acetylene reduction rates were stimulated in an atmosphere of 100% relative humidity (RH) and moisture content range of 570-620% of dry weight of Peltigera aphthosa. The decrease of nitrogenase activity measurable by acetylene reduction of thallus after prolonged incubation in the medium with combined nitrogen addition shows susceptibility of the symbiosis (lichen) since the thallus showed signs of disintegration at this time. Furthermore, air humidity and moisture content of the thallus influenced nitrogenase synthesis of Peltigera aphthosa considerably on separation of cephalodia such approach decreased nitrogenase activity and also elicited differences in their response to the various treatments. Applicability of this technology would enhance plant sustainability and yield in agricultural farms.