Diazotrophic abundance and community structure associated with three meadow plants on the Qinghai-Tibet Plateau

Abstract

Symbiotic diazotrophs form associations with legumes and substantially fix nitrogen into soils. However, grasslands on the Qinghai-Tibet Plateau are dominated by non-legume plants, such as Kobresia tibetica. Herein, we investigated the diazotrophic abundance, composition, and community structure in the soils and roots of three plants, non-legume K. tibetica and Kobresia humilis and the legume Oxytropis ochrocephala, using molecular methods targeting nifH gene. Diazotrophs were abundantly observed in both bulk and rhizosphere soils, as well as in roots of all three plants, but their abundance varied with plant type and soil. In both bulk and rhizosphere soils, K. tibetica showed the highest diazotroph abundance, whereas K. humilis had the lowest. In roots, O. ochrocephala and K. humilis showed the highest and the lowest diazotroph abundance, respectively. The bulk and rhizosphere soils exhibited similar diazotrophic community structure in both O. ochrocephala and K. tibetica, but were substantially distinct from the roots in both plants. Interestingly, the root diazotrophic community structures in legume O. ochrocephala and non-legume K. tibetica were similar. Diazotrophs in bulk and rhizosphere soils were more diverse than those in the roots of three plants. Rhizosphere soils of K. humilis were dominated by Actinobacteria, while rhizosphere soils and roots of K. tibetica were dominated by Verrumicrobia and Proteobacteria. The O. ochrocephala root diazotrophs were dominated by Alphaproteobacteria. These findings indicate that free-living diazotrophs abundantly and diversely occur in grassland soils dominated by non-legume plants, suggesting that these diazotrophs may play important roles in fixing nitrogen into soils on the plateau.

Keywords: Qinghai-Tibet Plateau; biological nitrogen fixation; diazotrophs; grassland ecosystems; nifH gene; non-leguminous plants.

Figure 1

Diazotrophic abundance indicated by the nifH gene in the bulk soils (A), rhizosphere soils (B), and the roots of the three plant species (C). Error bars denote the standard error of the mean. Different lowercase letters indicate a significant difference at p < 0.05 among the bulk and rhizosphere soils, and roots of three plants. Kh, Kt, and Oo stand for Kobresia humilis, Kobresia tibetica, and Oxytropis ochrocephala, respectively.

Figure 2

Diazotrophic community structure assessed by principal component analysis (PCA) based on terminal restriction fragment length polymorphism profiles. The shapes of the symbols indicate sample type: bulk soils (triangle), rhizosphere soils (rectangle), and the roots (cross). The percentage of the variation explained by the plotted principal coordinates is indicated on the axes. Kh, Oo, and Kt represent Kobresia humilis, Oxytropis ochrocephala, and Kobresia tibetica, respectively.

Figure 3

Neighbor-joining phylogenetic tree of nifH gene sequences. Clones are shown in bold with sample name, followed by the corresponding length of terminal restriction fragments and accession number. Bootstrap values (>50) determined from 1,000 iterations are indicated at branch points.

Figure 4

Relative abundance and distribution of the most dominant diazotrophic terminal restriction fragments obtained by terminal restriction fragment length polymorphism analysis. Kh, Oo and Kt represent Kobresia humilis, Oxytropis ochrocephala, and Kobresia tibetica, respectively.