A Study On The Promotion Of Nitrogen Fixation In Continuous Cropping Peanuts By P. Philippine B3 And Alleviation Of Leaf Senescence

Peanut（Arachis hypogaea L.）is an important oil crop in the world.Due to arable land scarcity,peanut is usually continuously monocropped on the same filed.The continuous cropping of peanut will lead to continuous cropping obstacles,such as the outbreak of soil borne diseases,the declines of yield and quality.We previously reported that an endophytic fungus,Phomopsis liquidambaris B3,originally isolated from the inner bark of Bischofia polycarpa,can promote peanut growth,enhance disease resistance and increase pod yield under continuous cropping condition.The leaves are the main place for plant carbon fixation,and the delayed leaf senescence can provide more assimilation for the pods.However,whether Ph.liquidambaris affects peanut leaf senescence and consequently increases peanut yield,and the underlying mechanism is still unknown.Given the importance of carbon and nitrogen in regulating leaf senescence,this study explored the effect of endophytic fungus Ph.liquidambaris on peanut leaf senescence and its mechanism from the perspective of plant carbon and nitrogen metabolism.First of all,we conducted an outdoor pot experiment.Peanut was planted in continuous cropping red soil,which was divided into endophytic fungus Ph.liquidambaris inoculation and non-inoculation treatment.Different physiological indexes related to senescence of peanut leaves were measured at different time points after flowering.The results showed that the inoculation of Ph.liquidambaris significantly promoted the growth and yield of peanuts under continuous cropping conditions.Compared with the control,the Ph.liquidambaris inoculation significantly reduced the degradation of chlorophyll,the level of membrane ion leakage and the expression level of senescence-associated genes,and improved the antioxidant capacity of the leaves at the same time.In addition,Ph.liquidambaris significantly increased the photosynthetic rate of peanut leaves,but had no significant effect on different sugar content.After inoculation with Ph.liquidambaris,peanut leaves maintained higher total nitrogen and soluble protein contents with more nodule numbers and higher nitrogen metabolism related enzyme activities.Further experiments showed that chlorophyll content and ion leakage increased and decreased,respectively after exogenous nitrogen treatment.In conclusion,our results suggest that Ph.liquidambaris may alleviate leaf senescence of continuous cropping peanut by promoting nitrogen uptake.Although inoculation of Ph.liquidambaris increased the photosynthesis of peanut,it did not affect the sugar content of peanut leaves.Rhizobial symbiosis in root zone is a costly process,which takes substantial photosynthetically-fixed C to sustain their growth and N₂-fixing.We assumed that the symbiosis of Ph.liquidambaris with peanut increased sink strengthen of roots,which in turn altered distribution of carbohydrates and enhanced nodulation.To verify our speculation,we carried out subsequent greenhouse experiments.Ph.liquidambaris inoculation increased the photosynthesis and total carbon content of peanut roots.Further analysis the content of carbon,soluble sugar and starch in different tissues and found that the inoculation of Ph.liquidambaris promoted allocation of assimilates to roots,resulting in higher carbohydrates in roots.In addition,Ph.liquidambaris inoculation increased the sucrose concentration in phloem sap and roots.Furthermore,the experiments with the promotor and inhibitor of carbohydrate biosynthesis found that the enhanced nodulation and N₂-fixing by Ph.liquidambaris were due to the increased sugar content in roots.These results suggested that Ph.liquidambaris drives nodulation enhanced,which is related to enhanced sugar allocation to roots.In order to explore the effect of Ph.liquidambaris on carbon partitioning of peanut and its related mechanism,peanut was planted in sterilized and moist vermiculite substrate after germination.Our results showed that inoculation with Ph.liquidambaris increased the contents of soluble and total sugars in the root,as well as sucrose in the phloem sap.In addition,auxin（IAA）and abscisic acid（ABA）are important hormones regulating carbon metabolism.Therefore,we quantified the ABA and IAA in peanut root and found that Ph.liquidambaris inoculation increased the level of indole acetic acid（IAA）,but had no significant effect on the content of ABA.Meanwhile,we found that IAA could affect the accumulation of different sugar components in peanut roots by exogenous application of IAA and IAA response inhibitors（PCIB）.In addition,compared with the control,Ph.liquidambaris increased the activities of acid invertase,sucrose phosphate synthase andβ-amylas,while the application of PCIB decreased the activities of acid invertase and sucrose phosphate synthase in roots.Furthermore,PCIB diminished Ph.liquidambaris-induced increased of sugar transporters.These results indicated that the increase of sugar distribution and accumulation in root induced by Ph.liquidambaris may be related to the accumulation of IAA in root.Taken together,endophytic fungus could alleviate the senescence of peanut leaf under continuous cropping condition.We consider the mechanisms are related to the inoculation of Ph.liquidambaris increases the accumulation of auxin in root,which in turn enhances the expression of carbon metabolism and sugar transporter gene,and then promotes the transport and accumulation of sugar to root.The accumulation of root sugars provided more energy for peanut nodulation and nitrogen fixation,which promotes the absorption of nitrogen and thereby alleviating leaf senescence.