Studies On The Effects Of Stover And Biochar Application On Diazotroph Communities In The Field And The Contribution Ratio Of Biological Nitrogen Fixation For Peanut

Biological nitrogen fixation（BNF）is an important method to reduce the use of chemical nitrogen（N）fertilizers by converting atmospheric dinitrogen（N₂）to replenish soil N pool.N₂-fixing microorganisms play a dominant role,and symbiotic nitrogen fixation by legumes is one of the most important ways of BNF.Agronomic practice for legume cultivation often includes organic material applications,and maize（Zea mays）stover and biochar are frequent additions to field amendments.The practice often induces the growth of N₂-fixing bacteria（diazotrophs）and stimulate BNF,with concomitant effects on symbiotic nitrogen fixation in legumes.However,there is uncertainty regarding the effect of stover and biochar application on the community and activity of diazotrophs in soil,including deep soils,and on the contribution of BNF（%Ndfa）and the amount of nitrogen fixed(N_fix)in legumes.Peanut（Arachis hypogaea）is an important Oilseed legume with international competitiveness in China,and we established a long-term field trial for Peanut in 2013,using maize stover and biochar for fertilization and soil amendment,and established treatments including no fertilizer（CK）,chemical fertilizer（NPK）,biochar（BC）,BC+chemical fertilizer（BNPK）,corn stover（MS）and MS+chemical fertilizer（SNPK）.Here,soil samples were collected from bulk soil at different depths（range:0-40 cm）in 2018 and 2020 the peanut growing seasons and maturity period,16S r RNA and nif H gene abundance were investigated using Real-time PCR,diazotroph community structure was explored by high-throughput sequencing（Illumina Mi Seq）,and R_Nfix was assessed with the help of acetylene reduction method（ARA）;samples of whole peanut and reference plants were collected at peanut maturity in 2018 and 2020 to calculate%Ndfa and N_fix of peanut using the¹⁵N natural abundance method and measured peanut biological yield（PBY）.In the studied peanut field,the vertical stratification of diazotroph communities and their activity were revealed,the abundance of diazotrophs and bacteria as well as the structure and RNfix of the diazotroph community were highly correlated.Stover strongly increased nif H gene abundance in the 0-10 cm layer and RNfix in most soil layers and affected diazotroph community structure succession in the 0-20 cm soils.It was consistent with the effects of stover on soil properties such as DOC,DON,SOM,and C/N,whereby the SOM played the dominated role in governing the abundance,structure and activity of diazotroph communities and the BNF process in the soil.Maize stover incorporation caused significant positive changes in the vertical stratification of soil diazotroph communities,and these effects decreased with soil depth and were minor when stover was combined with chemical fertilizers.Biochar application was associated with increased SOM and pH,which resulted in an increased abundance of bacteria and diazotrophs in the 0-20 cm and enhanced BNF activity.Soil carbon（C）sources（e.g.,SOM,C/N ratio,and DOC）and pH were the main environmental drivers of increased abundance of diazotrophs and BNF activity in 0-20 cm in the biochar treatment,while soil nitrogen（N）sources（e.g.,TN and NO₃^--N）were the main factors affecting the abundance of bacteria and diazotrophs in the 20-40 cm soil.Chemical fertilizers negatively affected BNF and led to a significant decrease in R_Nfix in the 20-40 cm soil by a mechanism that may be related to changes in the community structure of diazotrophs rather than abundance.The addition of biochar induced an increase in core diazotroph communities（e.g.,Rhizobiales）and had a significant positive effect on BNF,despite hindering the growth of bacteria in the 20-40 cm soil,in addition to the combined application of biochar and fertilizer partially offset their negative effects when applied alone.The B value of whole peanut（variety:Fuhua No.12）was determined to be-0.26‰by a N-free sand culture experiments in a greenhouse.SNPK reduced peanut N%and C%.The variation observed in the ¹⁵N/¹⁴N ratio of peanut in different tissues was not related to the nutrient distribution of peanut N concentration among the tissues but more to the treatment.Water use efficiency（WUE）of peanut for the growing season as judged byδ¹³C,where SNPK significantly increased peanut WUE.The range of peanut%Ndfa when stover and biochar were applied alone was at 74.8-84.4%and 70.8-83.9%,respectively,which were not significantly different from the blank treatment,and the application of chemical fertilizer significantly reduced peanut%Ndfa by 35.3-56.2%,but both chemical fertilizer with stover or biochar application significantly increased peanut%Ndfa by 47.7-68.4%and 43.4-63.5%,respectively.Stover application was effective in increasing peanut PBY,and biochar with chemical fertilizer application was effective in increasing nitrogen accumulation(N_BY)in peanut.In conclusion,we found that the application of maize stover and biochar in a peanut field for consecutive years could significantly increase SOM and TN content.Maize stover incorporation caused significant positive changes in the vertical stratification of soil diazotroph communities;although these effects decreased with soil depth and were minor when stover was combined with chemical fertilizers,they still highlighted the crucial role of stover return in enhancing BNF in soil under the legume.Biochar induces the succession of dominant diazotroph community,rather than abundance,by increasing soil carbon content and pH,which in turn enhances the BNF activity,which enriches our knowledge of biochar application affecting diazotroph communities and their activity.The application of chemical fertilizers remains a vital practice to ensure PBY and N_BY accumulation,but the application of stover and biochar significantly reduced the inhibitory effect of mineral N on peanut BNF and significantly increased the amount of N_fixin peanut,which is essential for sustainable yield increase in legumes.This provides a theoretical basis for the measures that can increase the benefits of legumes in the field by applying organic fertilizers and increasing soil organic matter,coupled with reducing the application of N fertilizers.