Effects Of Intercropped With Mulberry And Nitrogen Application On Soybean Growth And Nodulation And Nitrogen Fixation

In China,soybean(Glycine max L.)is the main grain crop,oil crop,and feed crop.However,the low yield and poor quality of soybeans severely limit the development of China’s soybean industry.Developing and promoting high-yield cultivation measures for soybeans is one of the effective measures to increase soybean yield.Intercropping could promote nitrogen fixation efficiency and increases soybean yield through enhancing efficient utilization of environmental resources.The high protein forage mulberry,which can be cut 2-3 times per growing season,which mean output a large amount of nitrogen from the soil,leading to a prominent problem of excessive nitrogen fertilizer input in production.Intercropping forage mulberry could create rhizosphere soil "nitrogen deficiency" environment for nitrogen fixation of neighboring soybeans,could promote nitrogen fixation efficiency of soybeans,and have the potential to achieve "nitrogen fertilizer reduction";Meanwhile,during the regeneration period after cutting,resulting in sufficient time and space are exposed for soybean growth to capture light energy and avoid long-term shading of soybean lodging.Forage mulberry requires a large amount of nitrogen.Although soybean has the ability to combine nitrogen fixation,its root nodules are not yet mature before flowering,and nitrogen needs to be absorbed from the soil;While,the nitrogen fixed by soybean nodulation can be transferred to adjacent feed mulberry,therefore,the intercropping of feed mulberry and soybean has a special demand for nitrogen fertilizer,and the demand pattern for nitrogen fertilizer is still unclear.Therefore,this study explores the effects of intercropping mulberry feed and nitrogen application on soybean nodulation and nitrogen fixation.This experiment was carried out the experimental field of the Liming Experimental Teaching Base of Northeast Forestry University in 2022.Six treatments were set up,including soybean monoculture without nitrogen(SN0),soybean monoculture nitrogen reduction(SN2/3),soybean monoculture with total nitrogen(SN1),soybean intercropping forage mulberry nitrogen free(IN0),soybean intercropping forage mulberry nitrogen reduction(IN2/3),and soybean intercropping forage mulberry total nitrogen(IN1).In order to study the effects of intercropping forage mulberry and nitrogen application on soybean growth indicators,yield,and nitrogen metabolism,nodule characteristics and gene expression.Analyzing the comprehensive effects of intercropping and nitrogen application on soybean growth and production,which could enrich and develop the theory of leguminous nitrogen fixation in intercropping systems,and provide reference data for constructing efficient biological nitrogen fixation cultivation measures for soybeans and reducing nitrogen input.The main research findings are as follows:1.Intercropping forage mulberry and nitrogen application significant promote the aboveground growth and development of soybean.At the same nitrogen application level,intercropping significantly increased soybean plant height,fresh weight,dry weight,and yield compared to soybean monoculture;Meanwhile,intercropping total nitrogen great promote soybean stem diameter,while its promoting effect on soybean leaf area is more obvious in its later growth stage;At the soybean monoculture mode,compared with the no nitrogen treatment,nitrogen applicationsignificant promote soybean growth indicators.The total nitrogen treatment significantly increases soybean plant height,while the nitrogen reduction treatment promotes significantly on the parameters of soybean leaf area,fresh weight,dry weight and so on;At different planting modes,nitrogen application increased the stem diameter and yield of soybean,showing SN2/3>SN1>SN0,IN1>IN2/3>IN0.2.Intercropping forage mulberry and nitrogen application affect the growth and development of soybean underground nodules.At the same nitrogen application level,the planting mode had no obvious difference on the number of soybean nodules,dry weight of nodules,and nitrogenase activity in nodules.At the same planting mode conditions,nitrogen application significantly increased the number of soybean nodules,fresh nodule weight,dry nodule weight,and nodule nitrogenase activity;The interaction between planting mode and nitrogen application level have significant difference on the number of soybean root nodules,fresh weight of root nodules,dry weight of root nodules,and nitrogenase activity of root nodules.3.The effects of intercropping forage mulberry and nitrogen application on the activity of nitrogen metabolism enzymes and the content of nitrogen compounds in soybean leaves are different.Comparing different planting modes,nitrogen application significantly increased the nitrate reductase activity(NR)and ammonium nitrogen content in soybean leaves during the initial flowering,full flowering,and full pod stages;The inhibitory effect on glutamate synthase activity(GOGAT),glutamine synthase activity(GS),and nitrate nitrogen content is significant.The NR enzyme activity of soybean leaves decreases with the advancement of growth period;The activities of GS and GOGAT in soybean leaves increase as the growth period progresses.In monoculture mode,the response of soybean leaf NR and GS to nitrogen application was SN1>SN2/3>SN1,while nitrogen application significantly inhibited soybean leaf GOGAT activity and nitrate nitrogen content.In intercropping mode,nitrogen application can significantly increase the activity of NR and GS in soybean leaves,as well as the content of ammonium nitrogen;However,nitrogen application significantly inhibited the GOGAT activity and nitrate nitrogen content in soybean leaves.4.RNA-seq technology was used to detect the gene expression in the root system of soybean during the initial flowering period.There were 2220 differentially expressed genes(DEGs)(1339 up-regulated and 881 down-regulated)and 2142 differentially expressed genes(1113 up-regulated and 1029 down-regulated)in compared group of the SN0 and IN0,IN0 and IN1,respectively.In the SN0/IN0 group,the expression level of the isoflavone 2’-hydroxylase gene(Gm Cyp81D1)in isoflavone biosynthesis was slightly down-regulated,but the difference was not significant.The expression levels of glutamate decarboxylase gene(Gm GAD),chalcone synthase gene(Gm TT4),and chalcone--isomerase gene(Gm NOT4C),which interact with plant pathogens,are up-regulated.In the IN1/IN0 group,the expression level of phosphoenolpyruvate carboxyl kinase gene(Gm PCK1)for pyruvate metabolism was down-regulated,while the expression levels of plant pathogen signaling calmodulin gene(Gm CML11),iron transporter gene(Gm NRAMP3),nitrate transporter gene(Gm NRT2.4),and chalcone synthase gene(Gm TT4)were all up-regulated.Integrating and analyzing the growth indicators,physiological indicators and transcriptome sequencing in this experiment,intercropping with mulberry and nitrogen application are beneficial to the growth and development,high yield and nitrogen reduction of soybean.Which could promote the expression of soybean nodulation and nitrogen fixation genes,increase the number of nodules,enhance the activity of nitrogen metabolic enzymes,improve the nitrogen fixation efficiency of soybean.