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.