Signalling Effect And Intrinsic Mechanism Of Nitrogen Application On Rhizosphere Colonization Of Beneficial Microbe

Nitrogen（N）is an important limiting factor for primary productivity in agricultural production.N application plays a crucial role in guaranteeing the world’s grain yield;especially in China,the N application has increased more than tripled since1980,resulting in a nearly 70%increase in crop yield.The biochemical cycle of N in soil involves two processes as nitrification and denitrification.During the nitrification process,the nutritional effects of ammonium and nitrate on soil,plants,and microorganisms have received much attention;besides that,nitric oxide（NO）,a precursor of greenhouse gas that produced during denitrification,is an important signaling molecule that can regulate diverse physiological processes.Unfortunately,little attention has been paid to the signaling effects of soil N,especially its transformation intermediates after N application,on biological interactions in rhizosphere.Since rhizosphere beneficial microbes are important for the growth and health of plants,this study was designed to explore the signaling effects and intrinsic mechanism of NO that produced by nitrogen denitrification,in regulating plant-microbe rhizosphere interactions.In the present study,a typical plant-beneficial rhizobacteria Bacillus velezensis SQR9 was selected as the testing strain.The effect of nitrogen application on the rhizosphere colonization of strain SQR9 and its biological mechanism were investigated under natural soil conditions,the denitrification process was further confirmed under hydroponic conditions and the effect of the produced NO on the rhizosphere colonization of strain SQR9,and finally the effect of NO gas on the biofilm formation of strain SQR9 and the underlying mechanism were investigated.The main results are as follows:1.Nitrogen（N）fertilization significantly affected the rhizosphere colonization by strain SQR9 under soil condition,which was mediated by NO that serves as an intermediate of soil N-cycle.The effects of different N application levels on rhizosphere colonization by strain SQR9 was investigated by pot experiments in soil system,and it was found that appropriate N application（0.15 g/kg soil）significantly promoted the colonization of SQR9,which was 5.3 times higher than that of the control group without N fertilization,whereas excessive N application（over 0.3 g/kg soil）inhibited the rhizosphere colonization of SQR9 to the same level as that of the control.There was a significant linear positive correlation between soil NO production and N fertilization at different N application levels.The cumulative amount of NO was 18.3PPB（this unit is the unit of magnitude detected in this experimental system and only serves as the trend representative）of control（without N application）;an appropriate amount of N application（0.15 g/kg）produced a moderate amount of NO（about 53.9PPB）,which was 39.4%less NO than that produced by excessive nitrogen application（0.3 g/kg）.The denitrification potential and the abundance of denitrification functional genes nir S/nir K also increased with increasing N fertilization.Furthermore,addition of c-PTIO（a NO obligate scavenger）decreased soil NO production and attenuated the effects of N application levels on rhizosphere colonization of strain SQR9,indicating that the effects of N inputs on the SQR9 colonization is mediated by NO.2.NO produced by simulated denitrification pathway affects rhizosphere colonization by strain SQR9 under hydroponic condition.Chryseobacterium rhizoplanae XL97 and Acinetobacter baumannii XL380,were selected as the denitrifier strains from the microbial agents stock of our laboratory,based on the presence of gene nir K/nir S that encode nitrite reductase.Both strains can reduce the substrate Na NO₂and produce NO,among which strain XL97 showed a stronger NO₂^-reducing ability.The NO production by strain XL97 gradually increased with the concentration of nitrogen source,and the maximum cumulative amount of NO was observed after incubation in Na NO₂（4 m M）for 24 h（864 PPB）.The effect of NO produced by denitrification strain XL97 at different concentrations of Na NO₂ on rhizosphere colonization by strain SQR9 was investigated under hydroponic condition.Results showed that inoculation with denitrifier XL97 alone or addition of Na NO₂ did not affect the colonization of strain SQR9 in the rhizosphere of Arabidopsis and cucumber.Simultaneous inoculation of XL97 and provision of 1μM Na NO₂ significantly increased the colonization of strain SQR9 in Arabidopsis rhizosphere,which was about1.5 times of that in the Control;simultaneous inoculation with XL97 and supplement of 10μM Na NO₂ significantly increased the colonization of strain SQR9 in cucumber rhizosphere,which was about 2.7 times that in the Control.When strain XL97 was inoculated and the Na NO₂ concentration was increased to 10μM,the colonization of strain SQR9 in Arabidopsis rhizosphere decreased to be similar with Control;in addition,when strain XL97 was inoculated and the Na NO₂ concentration was increased to 1 m M,the colonization of SQR9 in cucumber rhizosphere was significantly lower than that in Control.Moreover,exogenous addition of DETA NONOate,an NO donor,at a final concentration of 1μM,was able to significantly enhance the colonization by SQR9 in the rhizosphere of Arabidopsis and cucumber,which increased by 6.8 and 3.0times as compared with the relevant Control（NO donor free）,respectively.Importantly,addition of c-PTIO abolished the increase of rhizosphere colonization by strain SQR9caused by co-addition of denitrification strain XL97 and Na NO₂,or supplement of DETA NONOate.These results demonstrated that NO produced in the denitrification pathway can affect the rhizosphere colonization of strain SQR9 in a signaling effects,which showing promotion at low concentration but inhibition at high concentration.3.NO affects biofilm formation and rhizosphere colonization of strain SQR9by regulating the synthesis of extracellular matrixγ-polyglutamic acid（γ-PGA）.The effects of NO produced by strain XL97 after cultivation in the Na NO₂ substrate on biofilm formation by strain SQR9,was studied by using the plate consisting of an inner chamber placed in the center of an outer chamber.Inoculation of XL97 alone or supplying Na NO₂ in the outer chamber,had no effects on biofilm formation of strain SQR9 in the inner chamber.Meanwhile,inoculation of XL97 and supplying Na NO₂substrate at 0.5 m M or 1 m M significantly promoted biofilm formation of SQR9,while elevating Na NO₂ concentration to 4 m M inhibited the biofilm formation of SQR9.Addition of c-PTIO abolished the above-mentioned effects of inoculating strain XL97and providing Na NO₂ on strain the biofilm formation by strain SQR9.These results indicated that denitrifier XL97 affected biofilm formation of strain SQR9 by producing NO when grown in Na NO₂ substrate,showing that low concentration of NO generated by denitrification under low concentration of nitrogen source promoted biofilm formation,while high concentration of NO generated by denitrification under high concentration of nitrogen source inhibited biofilm formation.The promoting effect of NO on strain SQR9 biofilm formation has been reported to be attributed to the regulation of the synthesis of extracellular matrixγ-polyglutamic acid（γ-PGA）,therefore we quantified the expression ofγ-PGA synthesis gene cluster yws C-ywt A-ywt A in the cells of rhizosphere colonized SQR9.Compared with the control without N application,an appropriate amount of nitrogen application（0.15 g/kg）significantly upregulated the expression of cluster yws C-ywt A-ywt A,whereas addition of the c-PTIO significantly downregulated the expression of this cluster at all N application levels.Therefore,the effect of different N application on rhizosphere colonization of strain SQR9 is mediated by the regulation ofγ-PGA synthesis through NO.In conclusion,NO produced during N denitrification affected the rhizosphere interaction between plants and beneficial microbes through signaling effects,showing moderate N application promote while excessive inputs inhibit the rhizosphere colonization of PGPRs.This study highlights a pathway model of plant-microbe interaction regulated by NO produced during soil N transformation,and provides a theoretical basis to guide the rational application of chemical N fertilizer and utilization of the growth-promotion effects of plant-beneficial rhizobacteria.