Desert is one of the harshest terrestrial ecosystems on earth, characterized by high solar radiation level, low rainfall and extremely high temperature. In addition, the character of desert soil is low water retention, low nutrition level and high salinity(1). Desert ecosystems cover most of the earth’s land surface and are characterized by extremely low productivity and limited availability of water and nitrogen (N) as always(2). Due to human activities and climate change, a larger proportion of these arid lands are facing the threat of continuous desertification(3). Desert ecosystems are generally considered to be inanimate habitats under extreme environmental conditions, despite the fact that they are colonized by a few microorganisms(4). Desert ecosystems may change due to global climate change and nitrogen (N) deposition. The effects of precipitation and increased nitrogen deposition on plant growth and nitrogen cycle largely depend on nitrogen distribution and nitrogen recovery efficiency in plant-soil ecosystems, but the researches on desert ecosystems are limited(5).
In desert ecosystems, the distribution of higher plants and animals is limited by extreme environmental conditions in deserts or arid areas. Microorganisms are considered to be the main driving force of ecosystem services and can regulate key ecosystem processes(6, 7). Gramineae plants growing in sandy/rocky desert soil have developed a root system called “rhizosheath” as a trait to adapt to drought and absorb nutrients(8). The rhizosheath is defined as a part of the soil that physically adheres to the root system and can encase the entire root system of certain plants(9). Rhizosheath-related microorganisms of desert plants can promote plant growth and enhance stress tolerance, especially nitrogen-fixing related microorganisms(10, 11). Stipagrostis pennata (Gramineae) has advantages in effectively resisting long-distance diffusion and occupying the surrounding optimized environment(12). Our previous experimental results show that the strategy of stipagrostis pennata against harsh environment is related to the nitrogen fixation ability of root sheath(13). Nitrogenase is a protein complex gene encoded by nifH, nifD and nifK(14). NifH is the most widely used part to study the diversity and composition of nitrogen-fixing microbial communities(15). Some factors may also affect the composition of bacterial communities that actually express nifH and/or the number of nifH transcripts(16).
The sequence of nifH is highly conserved, so nifH could be used to study the diversity of nitrogen-fixing microorganisms in soil. In this paper, we identified the sequence of nifH in rhizosheath at different growth periods of Stipagrostis pennata by nifH sequencing. Based on the species and quantity of nitrogen-fixing microorganisms obtained from rhizosheaths, the possible factors for Stipagrostis pennata to become the pioneer population in desert were evaluated.
Collection of Stipagrostis Pennata Rhizosheaths
To collect samples of Stipagrostis pennata rhizosheaths from same hinterland of Junggar Basin in Xinjiang and ensure the representativeness of samples in different growth periods. The randomly collected samples were as follows: Returning Green Period (A) Sample Numbers: TYZ-10, TYZ-48, TYZ-51; Flowering Period (B) Sample Numbers: TYZ-8, TYZ-24, TYZ-27; Filling Period (S) Sample Numbers: TYZ-3, TYZ-43, TYZ-45; Fruit Period (M) Sample Numbers: TYZ-14, TYZ-21, TYZ-23; Withering Period (W) Sample Numbers: TYZ-6, TYZ-36, TYZ-37, i.e. 3 duplicate samples were set for each growth period.
The Extraction of DNA and Amplification of NifH
Power SoiLTM DNA Isolation kit (MOBIO, US) was used for total DNA extraction according to the manufacturer’s instructions. The concentration and quality of DNA were detected by NanoDrop spectrophotometer. The nifH was amplified by using polF/polR primers through PCR.PCR products(Liu, Liberton et al. 2018) were purified by using Ambion DNA-free kit (Life Technologies, USA).
The PCR product was purified and cloned into pGEMs-T Easy Vector System kit (Promega, France), and the positive clone was sequenced (Biofidal, Vaulx-en-Velin, France). According to the requirements of Illumina library preparation scheme, sequencing samples were prepared by TruSeq DNA kit, then applied to Illumina Miseq system, sequenced by Reagent Kit v2 2×250 bp.
Sequencing data were processed using qiime pipeline-version 1.7.0 (http://qiime.org/). The sequence with high quality was used for subsequent analysis. Uchime algorithm was used to check the aligned nifH gene sequence, and the sequences were removed from the data set before the OTU table was constructed. High quality sequences were clustered into Operational Taxonomic Units with 97% identity or threshold. Diversity analysis of α and β diversity was calculated, and metastats group significant difference analysis was used to access whether OTU has differences by Kruskal-Wallis rank sum test. Species with significant differences were analysed by LDA Effect Size in abundance between groups.