1.Introduction
Legumes are important components of intercropping systems and fix atmospheric nitrogen in root nodules, which can be used by associated crops (Lipman, 1912). Alfalfa (Medicago sativa L.) is a widely distributed forage legume and rich in protein (Baslam, Antolín, Gogorcena, Muñoz, & Goicoechea, 2014), it has become one of main forage sources for animal husbandry. Intercropped with alfalfa could increase microbial activity and diversity in the rhizosphere of Siberian wild rye (Elymus sibiricus L.) (Y. M. Sun et al.) and Mulberry (Morus alba L.) (Zhang, Wang, Hu, & Sun). Mulberry, a forage deciduous tree, has been recognized as a suitable alternative for supplementing live-stock diets (Sanchez, 1999; Wang, Yang, Bo, Ding, & Cao, 2012) in sustainable animal production systems (e.g. agro-forestry or agro-silvopastoral systems), with a higher biomass amount than that of most traditional forages (Sanchez, 1999). In terms of digestible nutrients, mulberry leaves and young stems are highly palatable and digestible (70-90%) for herbivorous animals and can also be fed to monogastrics. Alfalfa intercropped with mulberry is a new agroforestry system in China and has become an important and economic feasible planting pattern (Zhang et al., 2018). The two plants are both perennial forages with high protein contents; they are mowed two to three times a year and remove large amounts of soil nitrogen. Nitrogen supply is a determining factor in agricultural systems and affects biomass quantity and quality of alfalfa and mulberry. It has been reported that the percentage of N obtained through alfalfa N2 fixation in a pure stand is only 33 to 80% (Heichel & Vance, 1979). Moreover, alfalfa mainly relies on the uptake of nitrogen from the soil to meet its N demands during the seedling stage (not forming nodules) or after mowing (low photosynthetic capacity) (Teuber, Levin, Sweeney, & Phillips, 1984). The promoting effect of nitrogen application on alfalfa yield is obvious in the first planting year. Microbial communities are considered the drivers of soil functions and are involved in organic matter formation and decomposition (Condron, Stark, O’Callaghan, Clinton, & Huang, 2010), respiration (Liu et al., 2018), nutrient mineralization and cycling as well as water infiltration (Chu & Grogan, 2010). Any decrease in microbial diversity or abundance may adversely affect nutrient absorption from the soil (Giller, Witter, & Mcgrath, 1998), while in turn, the structure and diversity of the soil microbial communities are affected by vegetation composition and diversity (Ladygina & Hedlund, 2010), and in continuous cultivation systems, microbial communities show a reduced diversity (Jiao, 2006). Therefore, intercropping is an important approach to increase microbial diversity and quantity (Tang et al., 2014), resulting in increased productivity (Mao et al., 2012).
At present, the effects of nitrogen application and intercropping on the soil microbial diversity of the mulberry and alfalfa rhizosphere in the first planting year are not clear. The Biolog EcoPlate contains three replicate wells of 31 of the most useful carbon sources for soil community-level physiological profiling (CLPP) of heterotrophic bacterial assemblages capable of being metabolically active and growing in plates (Stefanowicz, 2006). This method is based on the fact that different microorganisms use different carbon sources, which enables the determination of the functional diversity of a microbial community (Amador & Görres, 2007). In comparative studies using CLPP and high-throughput sequencing (16S rRNA and ITS rRNA) (Huang et al., 2017), similar results were found in terms of the effect of land-use change on the soil microbial community structure (Dong, Yao, De-Yong, & Huang, 2008); however, the advantage of the Biolog approach is that it can be performed simply and conveniently and does not require substantial costs and resources (Classen, Boyle, Haskins, Overby, & Hart, 2003; Jay L. Garland, 1997).
In this study, we used the Biolog EcoplateTMmicroplate technique to assess the effects of nitrogen and intercropping on the microbial community structure and function of mulberry and alfalfa rhizosphere soil. In addition, we analyzed the relationship between the diversity of mulberry and alfalfa rhizosphere microbes (in terms of carbon source use) and the soil physiochemical properties. We anticipate that our findings will provide a reference for the adequate cultivation and management of agroforestry systems of mulberry intercropped with alfalfa.