Introduction
Soils, plants and animals, as the primary objects of the grassland study, have been investigated in the traditional researches separately. Nevertheless, these issues are all the interconnected components in the entirety of grassland ecosystem. Thus, new insight of internal connections among biotopes of soil, phyllosphere and faeces in the ecosystem are urgently needed to investigate the grassland. To evaluate the components of the grassland ecosystem, the term “biotope” has been applied for a long history. The concept of biotope has evolved with quite different definitions along the development of ecology theory (Dennis, Dapporto, & Dover, 2014; Olenin & Ducrotoy, 2006). With the evolution of relative accompanying ideas, a new definition of the concept biotope has been accepted extensively with the contemporary meaning of the combination of substantial environment and assembled organism species (Kobori, 2009; Rodiek & Thomas, 2007). Abiotic components are attributed to the “habitat”, which means the physical and chemical environment with the distinctive physiographic features and geographical locations. The biotic components are attributed to the “community”, which means groups of organism species in a particular environment with the interaction of each other or the interconnection between the organisms and environment (Dennis et al., 2014; Olenin & Ducrotoy, 2006). Notably, compared to the entire ecosystem, Olenin and Ducrotoy pointed out that the summation of all the biotopes does not involve the energy and other linkages between the ecosystem components (Olenin & Ducrotoy, 2006). In another words, apart from the biotopes, the connections, especially the microbial linkages among the biotopes also serve for the ecosystem running.
In a temperate grazed steppe, the ecosystem could be divided into three biotopes of great importance: soil, phyllosphere and faeces. All of the biotopes include the distinctive environmental features of abiotic conditions. These abiotic conditions could also regulate and control the distribution and migration of microbial community among the three biotopes. Thus, every biotope with unique abiotic and biotic characteristics is shaped into a sub-system of the pastureland ecosystem. Concurrently, all these sub-systems interact with each other by the web of food and predation. In such an environment, microbes play a role of the vector and transporter for the energy flow (Andrés et al., 2016; Bardgett & van der Putten, 2014; Wardle et al., 2004). This function made microbiota as an important inhibitor for the environmental disturbance to regulate and maintain the ecosystem (de Vries et al., 2012).
As the main assembled organisms, soil, phyllosphere and fecal microbiota play an essential role to regulate and control the ecosystem of the grazed steppe. Initially, soil microbiota is deeply engaged participants in the grassland ecosystem. Soil microbiota is the engine of the biogenic elements in the soil-plant system to immobilize and regulate the migration and transportation of nutrient elements owing to its role of decomposer. Concurrently, soil microbiota plays a key role in the soil assimilation process to the contaminant because the microbial biotransformation is considerably correlated to the occurrence and fate of the soil contaminants (Adrian, Szewzyk, Wecke, & Gorisch, 2000; Bunge et al., 2003; Giller, Witter, & McGrath, 2009; Zhu, Yoshinaga, Zhao, & Rosen, 2014). The last function of soil microbiota is regulation of the greenhouse gas emission by affecting the biogeochemical process of the elements (Brauer, Cadillo-Quiroz, Yashiro, Yavitt, & Zinder, 2006; Erkel, Kube, Reinhardt, & Liesack, 2006; Lu & Conrad, 2005; Mahecha et al., 2010; Yvon-Durocher et al., 2012). Another critical microbial community controlling the grazed steppe ecosystem is phyllosphere microbiota. Phyllosphere microbiota is termed as the microbes who colonize in the aerial habitat on the surface of leaves (Steven E. Lindow & Leveau, 2002; Vacher et al., 2016). The primary phyllosphere microbial modification is the alteration to the properties of plant surface (S. E. Lindow & Brandl, 2003). The substantial heterogeneous aggregates of microbes also modify the traits of their microhabitat to develop the nutrient availability expression of phenotypes by the phylloplane (Horton et al., 2014; Monier & Lindow, 2003; Rastogi, Coaker, & Leveau, 2013; Whipps, Hand, Pink, & Bending, 2008). The phyllosphere epiphytic microbiota modify the plant by controlling on plant disease and frost injury as well (Gourion, Rossignol, & Vorholt, 2006; Knief, Ramette, Frances, Alonso-Blanco, & Vorholt, 2010; Steven E. Lindow & Leveau, 2002; Melotto, Underwood, Koczan, Nomura, & He, 2006). In addition, the microbial adaptation regulates the resistance to the stress of the phyllosphere (Delmotte et al., 2009; Vacher et al., 2016; Vorholt, 2012; Zimmerman & Vitousek, 2012). The last crucial compound to regulate the pasture is fecal microbiota. The fresh fecal microbiota originates from the intestinal tracts of large livestock. In a grazed grassland, 60-99% of the nutrients ingested by the herbivorous livestock are returned into soil as the excreta (Yanjiang Cai, Chang, & Cheng, 2017). After entering to the soil through the faeces coverd patches, the fecal microbiota could regulate the decomposition process of the faeces and control the release of the nutrient element (Lin et al., 2009; Liu et al., 2018; Saggar, Bolan, Bhandral, Hedley, & Luo, 2010). Furthermore, the physiochemical properties, biological activities and plant growth are also influenced by fecal microbiota (Y. Cai, Zheng, Bodelier, Conrad, & Jia, 2016; Jost, Joergensen, & Sundrum, 2012). In the pasture, the fecal microbial composition and the behavior of microbial community is a vital attribute to impact the strategy and the method of livestock utilization and management (Jost et al., 2012; Liu et al., 2018; Saggar et al., 2010). Unfortunately, there is surprisingly little knowledge about the migration and cycle of the microbial community among three substantial biotopes: soil, phyllosphere and faeces in a grazed grassland. Although the studies on soil, phyllosphere and fecal microbiota have been carried out separately, the lack of the demonstration for microbial transportation among these three biotopes limits the insight to the link among components and energy flow path in the ecosystem. Neither any study for how these microbial linkages served for the stability of the ecosystem has been made.
In this study, we investigated the distribution of soil, phyllosphere and fecal microbiota to figure out the microbial migration and cycle among these biotopes using molecular method such as high-throughput sequencing and real-time Polymerase Chain Reaction (qPCR) combining with genomic analysis and machine learning classification. The hypotheses are advanced as following: (1) Are there significant different distributions among soil, phyllosphere and fecal microbiota? (2) What are the interactions of the microbiota among three biotopes? and (3) What are the dominant microorganisms playing key role in this migration and cycle?