Xiuli Zhang·Zhiyuan Teng·Huihui Zhang·Dunjiang Cai·Jingyun Zhang·Fanjuan Meng·Guangyu Sun

Abstract Intercropping of mulberry (Morus alba L.)and alfalfa (Medicago sativa L.) is a new forestry-grass compound model in China,which can provide high forage yields with high protein.Nitrogen application is one of the important factors determining the production and quality of this system.To elucidate the advantages of intercropping and nitrogen application,we analyzed the changes of physicochemical properties,enzyme activities,and microbial communities in the rhizosphere soil.We used principal components analysis (PCA) and redundancy discriminators analysis to clarify the relationships among treatments and between treatments and environmental factors,respectively.The results showed that nitrogen application significantly increased pH value,available nitrogen content,soil water content (SWC),and urea (URE) activity in rhizosphere soil of monoculture mulberry.In contrast,intercropping and intercropping+N significantly decreased pH and SWC in mulberry treatments.Nitrogen,intercropping and intercropping+N sharply reduced soil organic matter content and SWC in alfalfa treatments.Nitrogen,intercropping,and intercropping+N increased the values of McIntosh diversity(U),Simpson diversity (D),and Shannon—Weaver diversity(H′) in mulberry treatments.However,PCA scatter plots showed clustering of monoculture mulberry with nitrogen(MNE) and intercropping mulberry without nitrogen (M0).Intercropping reduced both H′ and D but nitrogen application showed no effect on diversity of microbial communities in alfalfa.There were obvious differences in using the six types of carbon sources between mulberry and alfalfa treatments.Nitrogen and intercropping increased the numbers of sole carbon substrate in mulberry treatments where the relative use rate exceeded 4%.While the numbers declined in alfalfa with nitrogen and intercropping.RDA indicated that URE was positive when intercropping mulberry was treated with nitrogen,but was negative in monoculture alfalfa treated with nitrogen.Soil pH and SWC were positive with mulberry treatments but were negative with alfalfa treatments.Intercropping with alfalfa benefited mulberry in the absence of nitrogen application.Intercropping with alfalfa and nitrogen application could improve the microbial community function and diversity in rhizosphere soil of mulberry.The microbial community in rhizosphere soil of mulberry and alfalfa is strategically complementary in terms of using carbon sources.

Keywords Mulberry intercropped with alfalfa·Nitrogen application·Principal components analysis·Redundancy discriminators analysis·Rhizosphere soil

Introduction

Mulberry (Morus albaL),a deciduous tree,has been used as a supplementary livestock feed (Sánchez 2002;Wang et al.2012) in agroforestry systems.It enables sustained animal production at higher rates of biomass accumulation than do most traditional forages (Sánchez 2000).Mulberry leaves and young stems are highly digestible (≥ 70%) and palatable for ruminant and monogastric herbivores.Legumes are important components of intercropping systems that fix atmospheric nitrogen in root nodules,enabling its uptake by associated crops (Ashworth et al.2015).Alfalfa (Medicago sativaL.) is a widely distributed forage legume rich in protein (Baslam et al.2014).It is one of the main forage sources for animal husbandry.Microbial activity and diversity in the rhizosphere of mulberry are increased by intercropping with alfalfa (Zhang et al.2018).Alfalfa intercropped with mulberry is a new agro-silvo-pastoral system in China and has become an important and economically feasible planting pattern (Zhang et al.2018).Mulberry and alfalfa are perennial species with high protein content that can be mowed two to three times per year,resulting in removal of large amounts of soil nitrogen.However,nitrogen supply is a determining factor in agricultural systems and affects biomass quantity and quality of alfalfa and mulberry.Heichel and Vance(1979) reported that only 33%—80% of biological nitrogen fixation is absorbed by growing alfalfa itself.Alfalfa mainly takes nitrogen from the soil to meet its N demands during the seedling stage when it does not form nodules and after mowing when plants have reduced photosynthetic capacity(Teuber et al.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 the formation and decomposition of organic matter (Condron et al.2010),respiration (Liu et al.2018),nutrient mineralization,and cycling,as well as water inf iltration (Chu and Grogan 2010).Changes in microbial diversity or abundance can affect nutrient uptake from the soil (Giller et al.1998).In turn,the vegetation composition and diversity affect the structure and diversity of the soil microbial communities (Ladygina and Hedlund 2010).Continuous cultivation reduces the diversity of microbial communities (Narita 1983;Yao et al.2006).Intercropping is an important approach to increase microbial diversity and abundance (Tang et al.2014),and thereby improve productivity (Mao et al.2012).But the effects of nitrogen application and intercropping on soil microbial diversity in the mulberry and alfalfa rhizosphere during the planting year have not been fully described.

The Biolog Ecoplate™ is a tool for analyzing microbial response to the presence of 31 of the most useful natural carbon sources (Garland and Mills 1991).By inoculating ecoplates in three replicates with field soil samples containing populations of microbes,researchers are able,after 2—5 days to quantify characteristics of the sampled microbial communities as seen in their responses to individual carbon sources.This approach is soil community-level physiological prof iling (CLPP) (Stefanowicz 2006),which has proven effective in describing spatial and temporal changes in microbial communities.The principle of this method is that different microorganisms use different carbon sources,which enables the determination of the functional diversity of a microbial community (Amador and Gorres 2007).Huang et al.(2017)found that the results of CLPP-based PCA and high-throughput sequencing (16S rRNA and ITS rRNA) have a similar changing tendency in soil microbial community diversity.This approach has been proven to be simple and convenient,and it requires fewer costs and resources (Garland 1997;Classen et al.2003).

In order to clarify the response of mulberry and alfalfa to nitrogen application and plant pattern,we used the Biolog Ecoplate™ microplate technique to assess the effects of nitrogen and intercropping on the microbial community structure and function of mulberry and alfalfa rhizosphere soil.We also analyzed the relationship between the diversity of mulberry and alfalfa rhizosphere microbes (based on carbon source use) and soil physicochemical properties.Our study objective was to provide a reference for the adequate cultivation and management of agroforestry systems of mulberry intercropped with alfalfa.

Materials and methods

Material,field and design

Mulberry (Morus albaL.cv.Qiuyu) used in this study was a high-yield,cold-and drought-resistant variety provided by the Heilongjiang Institute of Silkworm Breeding.In order to maintain consistent initial growth,seedlings of one-year-old mulberry plants were pruned to retain the main stem and root at 5 cm each.These seedlings were transplanted to the field on 11 April 2016.Alfalfa (Medicago sativaL.cv.Zhaodong) seeds with strong overwintering ability and high yield were sown by drilling also on 11 April 2016.The seeds of alfalfa were obtained from the Institute of Crop Development of the Heilongjiang Academy of Land Reclamation Science.Study plots were located at the institute (latitude,46°17.02′N;longitude,46°53.16′E;a mean elevation of 81.2 m a.b.s;northeast China).Study plot soil was classified as meadow soil:pH 6.5,available nitrogen 89.2 mg kg−1,available phosphorus 128.2 mg kg−1,available potassium 106 mg kg−1.

The experimental design consisted of a split-plot experiment with two factors (monoculture and intercropping).The treatments included nitrogen application and no nitrogen application.Total study plot area was 1400 m2.Each intercropping treatment covered 60 m2(5 m × 12 m).The conf igured lines of intercropped mulberry and alfalfa were two and four,respectively (Fig.1).The planting distance of mulberry was 45 cm,with a row spacing of 60 cm.For alfalfa,row spacing was 30 cm.To avoid nitrogen leaching and seedling burn,nitrogen was applied at the bud stage of alfalfa through an opening ditch on 8 May 2016).Phosphorus,potassium,and other essential elements were applied during land preparation on 10 April 2016.We applied 34.5 kg ha−1of superphosphate and 10 kg ha−1of potassium chloride.To avoid interference by other elements,urea (46% N) as nitrogen was applied.In both systems (monoculture and intercropping),the same amounts of nitrogen were applied.For alfalfa,we applied 8.78 kg ha−1of urea and,for mulberry,33 kg ha−1were applied.

Fig.1 The conf igured lines of intercropped mulberry and alfalfa with nitrogen application.In the monoculture and intercropping,the line space of mulberry and alfalfa is 60 cm and 30 cm,respectively.The distance of nitrogen location between mulberry and alfalfa is 20 cm and 10 cm,respectively.While,there are four lines of alfalfa and two lines of mulberry in the intercropping system,the line space between mulberry and alfalfa is 45 cm.Note monoculture mulberry without nitrogen (MN0),monoculture mulberry with nitrogen(MNE),intercropping mulberry without nitrogen (M0),intercropping mulberry with nitrogen (ME),monoculture alfalfa without nitrogen (AN0),monoculture alfalfa with nitrogen (ANE),intercropping alfalfa without nitrogen (A0),intercropping alfalfa with nitrogen (AE)

Soil sampling and determination

On 4 July 2016,at the mid-bloom stage of alfalfa in the planting year,rhizosphere soils of mulberry and alfalfa were sampled.Samples were divided into two parts,one for analysis of soil properties,and the other for determination of carbon sources used in Biolog Ecoplates™.For extraction,10 g of soil was dissolved in 100 mL of Tris buffer (0.1 M,pH 7.5) and shaken for 10 min,and then centrifuged at 2500 g for 15 min.The supernatant was diluted 1000 times,and 150 mL was inoculated onto Biolog Ecoplates™ (Biolog,Hayward,CA,USA).The plates were kept in a dark place and incubated at 28 °C.The metabolic substrates used by microbial communities were monitored by an automated plate reader (ELX808,Lab systems,Helsinki,Finland),which measured absorbance at 590 nm every 24 h for 192 h.The indices of metabolic diversity and their calculation methods followed Garland (1997).Average wellcolor development (AWCD) was calculated by averaging the final absorbance values for all substrate wells :AWCD=∑(Ci−R)/n,where Ciis the absorbance value of each reaction at different incubation times at 590 nm,excluding the control well;R is the absorbance value of the control well,andnis the total number of carbon sources (n=31).The Shannon index (H) was calculated as follows:H=−∑(Pi×lnPi),Pi=(Ci−R)∕∑(Ci−R),whereP iis the proportion of the corrected absorbance value of each well to the sum of absorbance values of all wells.McIntosh diversity (U) was calculated as:The Simpson diversity index(D)was determined via the following equations:D=1 −∑Pi2.

The soil sample used for physicochemical properties analyses was passed through a 2 mm soil sieve and then airdried at room temperature.The soil water content (SWC)was determined by the method of drying and weighing.The pH values of soil were determined with combined electrodes in a suspension of soil and water (1:2.5) using a pH meter(delta320,Mettler Toledo,Switzerland).The contents of soil organic matter (OM) were determined by 1/6 potassium dichromate titration (Janzen 1987).Available nitrogen (AN)was measured by the alkali hydrolysis diff usion (Jenkinson 1968).The activities of urease were determined by sodium phenol colorimetry and polyphenol oxidase was determined as described by Saiya-Cork et al.(2002).Catalase activity was measured by the colorimetric method,following(Trasar-Cepeda et al.1999).Five ml of 0.3% H2O2solution was added into the mixture of soil (0.5 g) and water (40 mL).Next,5 mL 3-M H2SO4was added to the suspension and then filtered.Aliquots (100 μL) of the extracts were neutralized by addition of phosphate buffer (900 μL,110 mM,pH 7.4).The resulting solution was treated with 9 mL of the indicator reagent (peroxidase-catalyzed decomposition),which yielded O2that was then used to drive an oxidative coupling between 4-aminoantipyrine and phenol,yielding a colored product that absorbed at 505 nm.

Statistical analysis

Means were compared using two-way analysis of variance(ANOVA),according to the generalized linear model procedure of SPSS version 12.0.1 for windows.Duncan′s multiple range test was used for the analysis of variance (P<0.05).The Canoco 4.5 (Microcomputer Power,Ithaca,NY,USA)was used for principal components analysis (PCA) and detrended correspondence analysis (DCA),the maximum value of gradient lengths in four axes was 0.436,which is less than 3,so the redundancy discriminatory analysis(RDA) was selected to sort the data.In PCA,the differences in the utilization of carbon sources by samples from treatments with nitrogen,treatments with intercropping,and treatments with intercropping and nitrogen were analyzed by use of the ADONIS (PERMANOVA) test.Correlations between treatments and environmental variables were analyzed using RDA (Monte Carlo test with 499 random permutations).The absorbance data after 96 h were analyzed to quantify the relative rates of six kinds of carbon source utilization.The ratio of the data per sample and the maximum data in all samples were regarded as the related-use ratio of carbon sources,and via a comparison matrix,we determined the use regularity of the sole carbon sources.Results of these analyses were used to draw a heatmap using Microsoft Excel 365.

Results

Soil physicochemical properties

Rhizosphere soils from the eight treatments differed significantly in terms of physicochemical characteristics (Table 1).Mulberry rhizosphere soils had higher pH value than alfalfa soils.However,compared to the monocultures,pH waslower in the intercropped system under the same planting pattern and fertilizer.Application of nitrogen significantly reduced soil organic matter (OM) in mulberry and alfalfa soils in monoculture.In intercropped systems,the effect of nitrogen on OM was alleviated.Nitrogen increased available nitrogen content (AN) in soils of mulberry and alfalfa in monoculture,while intercropping reduced AN.SWC was higher in mulberry than in alfalfa soils under the same planting pattern and fertilizer.Nitrogen application and intercropping significantly reduced SWC in alfalfa soil.The results of the two-way ANOVA test showed that N and plant pattern × N had significant effects on pH values of mulberry and alfalfa soils.N and plant pattern had significant effects on OM in mulberry treatments,while pattern and pattern × N had significant effects on OM in alfalfa treatments.In contrast,N,plant pattern,and plant pattern × N had no effect on AN and SWC in mulberry treatments.

Table 1 The effects of different treatments on the physicochemical properties of mulberry and alfalfa rhizosphere soil

Soil enzymatic activities

Nitrogen,intercropping,and intercropping+N significantly increased urea activity (URE) in rhizosphere soil of mulberry (Table 2).While intercropping+N significantly increased URE in alfalfa.Nitrogen application significantly increased peroxidase (POD) activity in mulberry soils but reduced POD activity in alfalfa soil.Nitrogen,intercropping,intercropping+N significantly decreased catalase(CAT) activity in mulberry treatments,but had no effect on alfalfa treatments except for intercropping+N.Polyphenol oxidase (PPO) activity declined significantly with nitrogen,intercropping,and intercropping+N in mulberry and alfalfa soils.Two-way ANOVA test showed that N,plant pattern,and plant pattern × N affected activities of URE and PPO,while,N and plant pattern × N affected POD activity in mulberry soils.N and plant pattern × N affected activities ofURE,POD,CAT,and PPO,and plant pattern affected the activities of CAT and PPO in rhizosphere soil of alfalfa.

Table 2 The effects of different treatments on enzymatic activities of mulberry and alfalfa rhizosphere soils

Average well color development

During incubation,the average rate of increase in average well color development (AWCD) slowed after 5 days(Fig.2).AWCD peaked on day 8 and was stable afterward.Among the eight treatments,Alfalfa in monoculture with nitrogen (ANE) showed the highest metabolic rate.The AWCD of ANE increased from 0 to 1.32 after 8 days.Mulberry in monoculture without nitrogen (MN0) showed the lowest metabolic rate,and AWCD increased from 0 to 0.86 after 8 days,indicating less efficient carbon substrate use.During the stable period,there were significant differences in AWCD among the microbial communities in alfalfa treatments,following the decreasing order:monoculture alfalfa+N (ANE) >intercropping alfalfa+N (AE) >monoculture alfalfa (AN0) >intercropping alfalfa (A0),suggesting that nitrogen application enhanced the metabolic activity of the microbial community in treatments of alfalfa.AWCD differed among the microbial communities of the mulberry treatments,following the order:intercropping mulberry+N(ME) >intercropping mulberry (M0) >monoculture mulberry+N (NME) >monoculture mulberry (NM0),which indicated that intercropping increased the metabolic activity of the mulberry microbial community.

Metabolic functional diversity indices

Nitrogen and intercropping significantly increased Mean-AWCD,Shannon diversity index (H’),Simpson diversity index (D),and McIntosh diversity index (U) in rhizosphere soil of mulberry treatment (Table 3).The order is ME >M0 ≥ MNE >MN0.There were no significant differences between M0 and MNE for the above parameters,indicating that microbial abundance,species richness,and the most common species were similar in M0 and MNE.In alfalfa,application of nitrogen significantly increased mean AWCD,H’,U and D,but intercropping reduced these indices.The rank order was AN0 >ANE >A0 >AE.There were no significant differences between AN0 and ANE,AE,and A0 in H’ and D.Two way-ANOVA showed that N,plant pattern,plant pattern × N affected mean AWCD,H’,U,and D in rhizosphere soil of mulberry treatments.In rhizosphere soil of alfalfa treatments,N and plant pattern affected these indices (except for D),while plant pattern × N had no interaction effects on these indices.

Principal components analysis of carbon source metabolization

The eight treatments (ADONIS;R2=0.948;P=0.001) distributed separately at 55.64% and 13.24% on the principal component analysis (PCA) vector 1 and 2 axes (Fig.3).M0 and ME (ADONIS;R2=0.75,P=0.007) were separated along both PC1 and PC2 axes.MN0 and MNE (ADONIS;R2=0.019,P=0.759) were separated along the PC1 axis.Especially,the samples in M0 and MNE (ADONIS;R2=0.883,P=0.536) were closer to each other on the two axes.Alfalfa samples were compositionally different from mulberry samples (ADONIS;R2=0.614;P=0.016),and alfalfa samples in monoculture separately differed from alfalfa samples in intercropping (ADONIS;R2=0.972;P=0.047) along PC1 and PC2 axes.The treatments of AN0 and ANE (ADONIS;R2=0.394,P=0.116) were located positive axis of PC1,while,A0 and AE (ADONIS;R2=0.972,P=0.058) were located negative axis of PC1.The sample distributions along the principal components axes were related to carbon utilization by the microbial community (Table 4).The carbon sources utilized by microbes in mulberry treatments were directly related to PC1,including amino acids,carbohydrates,and a carboxylic acid.The microbial community in alfalfa mainly used carbon sources was related to PC2.Based on the PCA results,17 carbon sources constituted PC1,including six amino acids,three carbohydrates,three carboxylic acids,two polymers,two miscellaneous substances,and one amide.Only four carbonsources accounted for PC2,including two carboxyl acids,one polymer,and one carbohydrate.

Table 4 Loading factors of principal components analysis

Fig.3 Principal component analysis for carbon source use of soil microbial communities in mulberry and alfalfa treatments.Note Data from three replication per treatment.Note monoculture mulberry without nitrogen (MN0),monoculture mulberry with nitrogen(MNE),intercropping mulberry without nitrogen (M0),intercropping mulberry with nitrogen (ME),monoculture alfalfa without nitrogen (AN0),monoculture alfalfa with nitrogen (ANE),intercropping alfalfa without nitrogen (A0),intercropping alfalfa with nitrogen (AE)

Table 3 Comparison of metabolic functional diversity indices of mulberry and alfalfa rhizosphere soil

Utilization of six types of carbon sources

There were notable differences in utilization of the six types of carbon sources between mulberry and alfalfa treatments (Fig.4).The proportional use of carbohydrates(31%—38%),polymers (21%—24%),and miscellaneous substrates (15%—19%) in mulberry treatments was higher than in alfalfa treatments,namely carbohydrates (28%—31%),polymers (14%—15%),and miscellaneous substrates (6%—7%).Proportional use of carboxylic acids (19%—20%),amino acids (23%—26%),and amines/amides (4—5%) in alfalfa treatments was higher than in mulberry treatments,namely acids (11%—19%),amino acids (7%—8%),and amines/amides(3%—6%).These results indicate that mulberry and alfalfa complementarily utilize the six types of carbon sources.

Fig.4 Utilization of ratio of six types of carbon sources intercropping and nitrogen on carbon source used in treatments of mulberry and alfalfa.Note monoculture mulberry without nitrogen (MN0),monoculture mulberry with nitrogen (MNE),intercropping mulberry without nitrogen (M0),intercropping mulberry with nitrogen (ME),monoculture alfalfa without nitrogen (AN0),monoculture alfalfa with nitrogen (ANE),intercropping alfalfa without nitrogen (A0),intercropping alfalfa with nitrogen (AE).Note Data represent mean ± SE,values followed by different lowercase letters represent significant differences (P <0.05)

Relative use rate of sole carbon substrate

The relative use rate of carbon substrate differed significantly among all treatments (Fig.5).Specifically,nitrogen,intercropping,and intercropping+N increased the number of carbon sources with high relative use rates (>4%)in mulberry treatments,while reducing the number in alfalfa treatments.Nitrogen,intercropping and intercropping+nitrogen increased the relative use rate of L-arginine and α-cyclodextrin in mulberry treatments (>4%),while reducing the relative use rate of pyruvic acid methyl ester and D-xylose in alfalfa treatments (>4%).Nitrogen increased the relative use rate of putrescine and D-galactonic-acid-lacton in mulberry treatments (>4%),and of α-cyclodextrin,and itaconic acid in alfalfa treatments(>4%).Intercropping increased the relative use rate of D-glucosaminic acid,glucose-l-phosphate,and Tween 80 in mulberry treatments (>4%),while reducing the relative use rates of 4-hydroxy benzoic acid,putrescine,D-galactonic acid lactone,glucose-1-phosphate,Tween 80,and glycogen in alfalfa treatments (>4%).The relative rate was less than 2% including γ-hydroxybutyric acid,L-threonine,and α-ketobutyric acid,2-hydroxy benzoic acid and D,L-α-lactose in mulberry treatments,and including 2-hydroxy benzoic acid and D,L-α-lactose in alfalfa treatments.The relative use rates of L-asparagine,D-glucosamine acid,L-serine,D-mannito,D-cellobiose,N-acetyl-D-glucose,exceeded 4% in all treatments.The relative use rates of D-malic acid,phenylethylamine,and glycyl-L-glutamic acid were <4% in all treatments.

Fig.5 The utilization rate of the carbon source by microbial communities in different treatments.Note Data represent mean ± SE,values followed by different lowercase letters indicate significant differences at the 0.05 level.Note monoculture mulberry without nitrogen(MN0),monoculture mulberry with nitrogen (MNE),intercropping mulberry without nitrogen (M0),intercropping mulberry with nitrogen (ME),monoculture alfalfa without nitrogen (AN0),monoculture alfalfa with nitrogen (ANE),intercropping alfalfa without nitrogen(A0),intercropping alfalfa with nitrogen (AE)

Redundancy analysis

Redundancy discriminators analysis (RDA) was used to analyze relationships between treatments and environmental factors in terms of soil microbial functional structure(Fig.6).The first ordination RDA axis (axis 1,horizontal),which was strongly related to URE and AN,explained 46.9%of the variability in use of carbon sources.The second ordination RDA axis (axis 2,vertical) was mainly related to pH and SWC and explained 34.9% of the variability.The greatest differences were in the correlation of environmental factors (pH,OM,SWC,and AN) with treatments of mulberry and alfalfa.pH and OM were positively related to M0,while NME and were negatively related to AN0 and A0.SWC and AN were positively correlated with MN0,but negatively related to ANE and AE.Moreover,responses of nitrogen application and plant plant pattern in mulberry and alfalfa were remarkable different.The greatest differences were related to application of nitrogen to mulberry and alfalfa treatments.Mulberry and alfalfa without nitrogen and with nitrogen were located on positive and negative sides of axis 1,both in monoculture and intercropping treatments.Alfalfa treatments without nitrogen (AN0 and A0) and with nitrogen(ANE and AE) showed the greatest positive vs.negative separation along axis 1.URE,POD,CAT,and PPO were positively related to AN0 and A0,and were negatively related to M0 and MNE.Especially,URE was positively related to ME,while it was negatively related to M0.

Fig.6 Ordination plots of redundancy discriminatory analysis(RDA) showing the relationship between and treatments and the environmental variables.The soil water content (SWC),available N(AN),organic matter (OM),and pH of alfalfa mulberry rhizosphere soil were used as environmental variables.The direction of an arrow indicates the steepest increase in the variable,and the length indicates the strength relative to other variables The direction of an arrow indicates the steepest increase in the variable,and the length indicates the strength relative to other variables Note Black circle with closed dots:mulberry monoculture with nitrogen (MNE);gray circle with closed dots:mulberry monoculture without nitrogen (MN0);black circle:mulberry intercropping with nitrogen (ME);gray circle:mulberry intercropping without nitrogen (M0);black triangle with closed dots:alfalfa monoculture with nitrogen (ANE);gray triangle with closed dots:alfalfa monoculture without nitrogen (AN0);black triangle:alfalfa intercropping with nitrogen (AE);gray triangle:alfalfa intercropping without nitrogen (A0)

Discussion

Mulberry leaves and young stems have high protein content and are rich in numerous active ingredients (Sánchez 2020).As a high-quality feed source for livestock and poultry,mulberry is characterized by rapid growth,high yield,and strong resistance (Kitahara et al.2002).These characteristics enable it to adapt to various regions (Machii et al.2001).Mulberry intercropped with alfalfa can increase bacterial richness and diversity (Zhang et al.2018).Li et al.(2007) found that intercropping and intercropping with nitrogen could promote nutrient enrichment in mulberry rhizosphere soil.In our trials,intercropping with nitrogen increased AN both in mulberry and alfalfa treatments,while AN was significantly higher in alfalfa than in mulberry treatments.Alfalfa has been described as a strong competitor for available N(Tomm et al.1995).However,AN declined significantly in intercropping systems without nitrogen application,possibly due to the relatively small photosynthetically active area of alfalfa,which limited biological nitrogen fixation after mowing in mid-July.However,mowed alfalfa also took up AN from soils,which therefore do not act as an N sink (Tomm et al.1995).In this experiment,intercropping increased OM content in mulberry soils,but reduced OM in alfalfa soils.Nitrogen application reduced OM contents in mulberry and alfalfa soils.Fog (1988) reported that nitrogen application decreased OM content in soil.Motavalli et al.(1995) found that soil organic matter turnover was affected by soil acidity,while Su and Evans (1996) reported that the cultivation of alfalfa can increase soil pH.In our experiment,nitrogen application increased the pH value of mulberry and alfalfa soils,while intercropping resulted in lower pH,especially in combination with nitrogen.This indicates that intercropping could stimulate acid metabolic substances both in alfalfa and mulberry cultivation.This is consistent with Latati et al.(2014) who reported more favorable pH levels following intercropping.These acid substances were the main substances for osmotic pressure adjustment,resulting in higher SWC in intercropping than in monoculture systems.

Enzymes catalyze all biochemical reactions,which are an integral part of nutrient cycling in the soil (Bandick and Dick 1999).Urease catalyzes urea conversion in the soil and therefore has a significant effect on utilization rate (Delgadoâ Baquerizo et al.2016).Therefore,the urease activity directly affects the use ratio of urea.Intercropping and nitrogen (urea) increased urease activity in both mulberry and alfalfa.However,the polyphenol oxidase activity declined in these treatments.High activity of polyphenol oxidase hinders the further synthesis of humus by the intermediate products of phenol,produced via organic matter mineralization.Nitrogen application increased POD activity in mulberry soils but reduced it in alfalfa soils with intercropping.The activities of the main enzymes were lower in alfalfa treatments with nitrogen and intercropping,especially in intercropping systems with nitrogen applications.

Samples with higher AWCD values have greater carbon source use capability,indicating higher microbial abundance(Garland 1997 a).Intercropping enhances the diversity of the mulberry rhizosphere soil microbial community and changes the main carbon source types of alfalfa (Zhang et al.2018).In this study,nitrogen and intercropping had significantly positive effects on AWCD values of mulberry,while intercropping and nitrogen had negative effects on AWCD values of alfalfa treatments.The change trends of the diversity indices between mulberry and alfalfa were also in agreement with the AWCD values.The diversity index H is greatly influenced by species richness (Sun et al.2012),with higher values indicating higher metabolic functional diversity (Strong 2016).The index D gives more weight to common or dominant species (Simpson 1949).Interestingly,there were no significant differences in mean AWCD,H,and D between MNE and M0,which indicated that microbes had similar capacities to utilize carbon substrates.PCA results showed that the treatments MNE and M0 were similar in terms of soil microbial community diversity.This indicated that nitrogen could be transferred from alfalfa to mulberry in the intercropping system.Our findings were in accord with the principles of agricultural practice.Intercropping of legumes and nonlegumes is popular in many countries(Hector 1999;Van Der Heijden et al.2006).There were significant differences between mulberry and alfalfa in the relative use of six types of carbon sources,indicating that the two intercrops had complementary effects on the utilization of carbon sources.According to previous studies,this is mainly related to the advantages of intercropping systems in terms of nutrient use (Willey 1979;Chalk 1998).Compared to monoculture systems,intercropping systems show improved use of available resources,and higher crop yield(Hauggaard-Nielsen and Jensen 2001).Cultivars suitable for intercropping should enhance the complementary effects between species (Davis and Woolley 1993).In this study,we conf irmed that mulberry and alfalfa are suitable for intercropping systems,complementarily using carbon and other sources.However,the relative use rates of 2-hydroxybenzoic acid,α-D-lactose,γ-hydroxybutyric acid,L-threonine,and α-ketobutyric acid were below 2% in mulberry,while the relative use rates of 2-hydroxybenzoic acid and α-Dlactose were below 2% in alfalfa treatments.In previous studies,these carbon sources were hardly degraded in plant rhizosphere soil (Deveryshetty et al.2007;Wu and 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 et al.2011).Nitrogen application and intercropping result in lower carbon metabolic activities of alfalfa soil microbial communities,as reported by Zhang et al.(2015).Redundancy analysis showed that certain soil environmental factors greatly influence microbial functional diversity in treatments of mulberry and alfalfa.It is recognized that pH (Xu et al.2014) and SWC (Wei et al.2019) are important factors affecting soil microbial communities.In our study,pH and SWC were negatively related to treatments of alfalfa,were strongly positively correlated with mulberry except for ME.This may result in microbial communities in alfalfa soils that differ from those in mulberry soils.The difference of microbial community diversity in rhizosphere soil between mulberry and alfalfa,resulted in complementary use of carbon sources in the intercropping system.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.

Conclusions

The microbial communities in rhizosphere soil of mulberry and alfalfa showed complementarity in their use of carbon sources.Intercropping and nitrogen application changed pH values and SWC both in mulberry and alfalfa rhizosphere soil.The rhizosphere soil microbial communities and available nitrogen content were largely similar in mulberry intercropped in alfalfa without nitrogen and monoculture mulberry with nitrogen.Intercropping with alfalfa benefited uptake of nitrogen by mulberry.