4.Discussion
Mulberry leaves and young stems have high protein contents and are rich in numerous active ingredients (Sanchez, 1999). Mulberry can be used as a high-quality feed source for livestock and poultry (Kitahara, Shibata, & Nishida, 2002) and is characterized by a rapid growth, high yield, and strong resistance, enabling it to adapt to various regions (MACHII, Koyama, & Yamanouchi, 2001). Previous research has demonstrated that mulberry intercropped with alfalfa increased bacterial richness and diversity (Zhang et al., 2018). For example, Li et al. (Li, Wu, & Chen, 2007) found that intercropping and intercropping with nitrogen promoted available nutrient enrichment in mulberry rhizosphere soil. In this experiment, intercropping with nitrogen increased available N (AN) contents both in mulberry and alfalfa treatments, while AN contents were significantly higher in alfalfa than in mulberry treatments. Alfalfa has been described as a strong competitor for available N (Tomm, Walley, Kessel, & Slinkard, 1995). However, AN contents were significantly decreased in intercropping systems without nitrogen application, mainly because of the relatively small photosynthetically active area of alfalfa (Savoie, Pouliot, & Sokhansanj, 1995), limiting biological nitrogen fixation after mowing in mid-July. However, mowed alfalfa also absorbs AN from soils and therefore does not act as an N source or sink (Tomm et al., 1995). In this experiment, intercropping increased the OM contents in mulberry rhizosphere soil, but decreased OM in alfalfa rhizosphere soil. Nitrogen application decreased the OM contents in mulberry and alfalfa rhizosphere soils. Fog et al. (Fog, 1988) have reported that nitrogen application decreased the OM contents in soil. Motavalli et al. (Motavalli, Palm, Parton, Elliott, & Frey, 1995) found that soil organic matter turnover is affected by soil acidity, and previous studies have found that the cultivation of alfalfa can increase soil pH (Su & Evans, 1996). In our experiment, nitrogen application increased the pH value both in rhizosphere soil of mulberry and alfalfa, while intercropping resulted in a decreased pH, especially when in combination with nitrogen. This indicates that intercropping could stimulate acid metabolic substances both in alfalfa and mulberry cultivation, which is in agreement with Latati (Latati et al., 2014), who stated that intercropping improved organic acid amounts in the rhizosphere, along with soil phosphorus availability. These acid substances were the main substances for osmotic pressure adjustment, resulting in higher soil water contents (SWC) in intercropping than in monoculture systems. Enzymes catalyze all biochemical reactions and are an integral part of nutrient cycling in the soil (Bandick & Dick, 1999). Urease catalyzes urea conversion in the soil and therefore has a significant effect on urea use (Delgado‐Baquerizo, Grinyer, Reich, & Singh, 2016). Therefore, the activity of urease directly affects the use ratio of urea. Intercropping and nitrogen application (urea) increased urease activity both in mulberry and alfalfa. However, the activity of polyphenoloxidase decreased in these treatments. The high activity of polyphenoloxidase hinder the further synthesis of humus by the intermediate products of phenol, produced via the mineralization of organic matter, causing the accumulation of phenolic compounds and, consequently, poisoning. Nitrogen application increased the activity of POD in mulberry treatments, but decreased it in alfalfa treatments with nitrogen application and intercropping. The activities of the main enzymes were lower in alfalfa treatments with nitrogen and intercropping, especially in intercropping systems with nitrogen application.
Among the 31 carbon sources on the Biolog-ECO plates, 9 were root exudates. The capability of microorganisms to use different carbon sources was measured by average well color development (AWCD )(J. L. Garland & Mills, 1991). Samples with larger AWCD values have a higher carbon source use capability and tend to have a higher microbial abundance (Jay L. Garland, 1997). Previous research has indicated that intercropping enhances the diversity of the mulberry rhizosphere soil microbial community and changed the main carbon source types of alfalfa (Zhang et al., 2015). In our study, nitrogen application increased the AWCD values of mulberry intercropped with alfalfa systems. While, intercropped with mulberry and nitrogen application decreased the AWCD values of alfalfa treatments. The changed trends of the diversity indices between mulberry and alfalfa were in accordance with the AWCD values. The Shannon diversity index (H) is greatly influenced by species richness (Y. H. Sun, Yang, Zhao, & Li, 2012), and higher values indicate a higher metabolic functional diversity (Strong, 2016). The Simpson index (D) is greatly reflected by most common species because it gives more weight to common or dominant species (Simpson, 1949). Interestingly, there were no significant differences of mean AWCD and H and D between monoculture mulberry treatments with nitrogen application (MNE) and intercropped with alfalfa treatments without nitrogen (M0), indicating they use similar carbon sources. The results of the PCA showed that the treatments MNE and M0 were similar in terms of the soil microbial community diversity, showing clusters in using the main carbon source closely related the two principal components in the scattered plots. The relative use rates of six types of carbon sources were significantly different between treatments of mulberry and alfalfa, indicating that these species strategically and complementarily use these carbon sources. According to previous studies, this is mainly related to the advantages of intercropping systems in terms of nitrogen use (Chalk, 1998; Willey, 1979). Compared to monoculture systems, intercropping systems show an improved use of available resources, resulting in higher yields (Hauggaardnielsen & Jensen, 2001). Cultivars suitable for intercropping should enhance the complementary effects between species (Davis & Woolley, 1993). In this study, we confirmed that mulberry and alfalfa are suitable for intercropping systems, complementarily using various carbon sources. However, the relative use rates of 2-hydroxybenzoic acid, α-D-lactose, γ-hydroxybutyric acid, L-threonine, and α-ketobutyric acid were less than 2% in mulberry, while the relative use rates of 2-hydroxybenzoic acid and α-D-lactose were less than 2% in alfalfa treatments. In previous studies, these carbon sources were hardly degraded in plant rhizosphere soil (Deveryshetty, Suvekbala, Varadamshetty, & Phale, 2010; Wu & Wang, 2007). Nitrogen application and intercropping increased the numbers of carbon sources with use rates of more than 4% in mulberry treatments, while intercropping decreased these numbers in alfalfa treatments. This indicates that intercropping and nitrogen application created a more favorable environment for certain microbial groups in mulberry rhizosphere soil (Garau, Silvetti, Deiana, Deiana, & Castaldi, 2011). Redundancy analysis showed that certain soil environmental factors greatly influence the microbial function of intercropped mulberry/alfalfa treatments. Soil physico-chemical parameters were positively related with mulberry treatments, in particular in intercropped mulberry treatments with nitrogen application, while in alfalfa treatments with nitrogen application, we observed a negative correlation. This indicates that nitrogen application and intercropping result in lower carbon metabolic activities of alfalfa soil microbial communities, which is in accordance with the results of Zhao et al. (Zhao, Zeng, He, Chen, & Wang, 2015). Available nitrogen contents and soil water contents were negatively related with treatments of alfalfa with nitrogen application. However, further studies are needed to evaluate the long-term effects of nitrogen application and intercropping on the functionality of soil microbial communities in mulberry and alfalfa intercropping systems.