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.