Physiology And Phytohormones Response Behind Phomopsis Liquidambari B3-mediated Peanut Nodulation Enhancement

The establishment of legume-rhizobia symbiosis requires the series biochemical and physiological responses from symbiotic partners.Meanwhile,plant hormones play vital roles in the nodule organogenesis.Previous studies had reported that the plant broad-spectrum fungal endophyte Phomopsis liquidambari strain B3 could increase root nodule number and improve pod yield of peanut plants in a continuous cropping system.However,the mechanisms behind the fungal endophyte-mediated nodule number increase remain largely unknown.Furthermore,it is uncertainty whether that P.liquidambari could enhance peanut-rhizobia nodulation in a pure culture system.Given the importance of plant physiology and hormones in nodulation,we aim to reveal the mechanisms behind the P.liquidambari-mediated nodulation enhancement from these two aspects.The pot study was performed to investigate the effects of P.liquidambari and P.liquidambari fragments on peanut-soil rhizobia nodulation and symbiotic N2-fixation,and reveal the physiological mechanisms behind P.liquidambari-mediated nodulation and symbiotic efficiency alterations when peanuts were grown in continuous cropping soil.The results showed that P.liquidambari could colonize peanut root as an endophyte.Compared to control and P.liquidambari fragments,P.liquidambari enhanced not only nodule initiation and nodule development,but also symbiotic efficiency.Although P.liquidambari fragments enhanced nodule initiation at the early stage of symbiosis,it did not affect final nodule number and nodule nitrogen fixation capability.Both P.liquidambari and its fragments elevated peanut root defence enzymes activities,including the flavonoids synthesis-related enzymes.Fungal endophyte rather than endophyte fragments increased plant leaf photosynthesis and sugar content.Furthermore,the nodules in P.liquidambari treated plants showed higher nodule carbon metabolites content and carbon metabolites related enzymes activities than those in nodules of P.liquidambari fragments and control treatments.These results indicated that the enhancement of nodulation and symbiotic efficiency of peanut by P.liquidambari is likely linked to the enhancement of plant photosynthetic activity and carbon pool.In order to determine the detailed effects of P.liquidambari on peanut-bradyrhizobium nodulation and symbiotic N2-fixation,peanuts were grown in sterilized vermiculite.The experiments included four treatments:uninoculated control;inoculated with bradyrhizobium;inoculated with P.liquidambari and co-inoculation.One week after planting,the peanut roots were inoculated with corresponding microbes.The results showed that P.liquidambari could endophytically colonize in peanut roots and did not produce any adverse effects on peanut growth.The colonization of P.liquidambari promoted the amount of bradyrhizobial root colonization.Compared to bradyrhizobium only inoculated peanuts,co-inoculated peanuts showed enhancement of nodule initiation and subsequent nodule development rather than nodule function.Furthermore,co-inoculated peanuts showed more lateral root formation(peanut rhizobial invasion sites)and root flavonoids(rhizobial nodulation gene inducers)secretion.These results indicated that P.liquidambari co-inoculated with bradyrhizobium could enhance peanut nodulation and this enhancement might be associated with the alterations of plant physiology.To analyse the effects of P.liquidambari on nodulation-related plant hormones,the peanuts were placed on sterized plant nutrient medium.The spot inoculation of fungal endophyte and bradyrhizobium elevated the levels of local salicylic acid(SA),indoleacetic acid(IAA),ethylene(Eth)and abscisic acid(ABA).Meanwhile,the exogenous application of IAA,IAA analogues and IAA transport inhibitors confirmed the involvement of IAA in peanut-bradyrhizobium nodulation.In addition,both fungal endophyte and IAA increased the expression of a vital nodulin gene,CCaMK,which is an important component of common symbiosis signaling pathway.These results indicated that P.liquidambari-peanut interaction could increase the plant hormones,which are in favor of peanut nodulation.In conclusion,the broad-spectrum plant fungal endophyte Phomopsis liquidambari strain B3 could enhance peanut-rhizobia nodulation.We consider that the mechanisms behind fungal endophyte-mediated symbiosis enhancement are realted to the alterations of nodulation associated plant physiology and plant hormones.