| The peanut(Arachis hypogaea L.)is an important oil and cash crop in China,and the China has the highest peanut production in the world though its planting area is second.Because the soybean production had a lower competitiveness compared with other countries,so the maintenance of peanut yield played an important role in the stabilization of edible oil production in China.However,peanuts are usually continuously monocropped on a large scale in the same field for many years without any crop rotation due to the limited arable land and the requirement for intensifying agricultural production.Therefore,the continuous cropping obstacles in monoculture fields are a major production constraint for peanuts.The long-term peanut continuous cropping significantly decreased peanut germination,podding and yield,and this restriction could be aggravated with the extension of the continuous cropping period.Many studies demonstrated that allelochemical accumulation,enzyme activity disruption,and microbial community deterioration are the main factors that cause peanut continuous cropping obstacles.For this problem,many methods have been used to overcome peanut continuous obstacles,including crop rotation,soil disinfection,and application of organic fertilizer and beneficial microorganisms.However,all of these methods are different to be used under the practical field conditions,since they are restricted by region,tradition and technology.In our previous works,we isolated a broad-spectrum endophytic fungus called Phomopsis liquidambari from the inner bark of Bischofia polycarpam’s stem.Pot experiment have demonstrated that inoculation of P.liquidambari can significantly improve soil enzyme activities and microbial community structures,which is contribute to alleviate peanut continuous cropping obstacles.Further analysis found that the increase in nodulation and N2 fixation may play an important role in P.liquidambari enhancement of peanut yield.Peanut growth needs nitrogen,which about 40-50%obtained from nodules.Therefore,the promotion of peanut nodulation and N2 fixation plays an important role in improving peanut yield.Due to the increased nodules,peanut growth strong,and then contribute to resist the occurrence of disease.Therefore,we believed that the increase of nodulation and N2 fixation should be considered as an important reason for overcoming continuous cropping obstacles.However,the mechanisms by which P.liquidambari improve peanut-Bradyrhizobium interaction,nodulation and N2 fixation are still unclear.The high concentration of phenolic acid is one of the main characteristics of long-term peanut continuous cropping soil.Previous studies have shown that high concentrations of phenolic acids can lead to the imbalance of soil microbial community structure,and may also have a strong inhibitory effect on the growth of soil rhizobia.However,a lower phenolic acids concentration in soil may beneficial to peanut-Bradyrhizobium interaction,nodulation and N2 fixation.Many previous studies have found that the number and diversity of soil rhizobium could be reduced in those soils contained a lot of phenolic acids,which further decreased the interaction of peanut-Bradyrhizobium,and finally aggravated the continuous cropping obstacles of peanut.Therefore,the maintenance of low concentration phenolic acids in continuous cropping soil is a prerequisite to ensure the biological activities of soil Bradyrhizobium.They also provide important guarantee for increasing the yield of peanut and alleviating the continuous cropping obstacles.Therefore,screening for an appropriate microorganism for phenolic acids degradation is meaningful for alleviating peanut continuous cropping obstacles.In this study,it was found that the low concentrations of 4-hydroxy benzoic acid,vanillic acid,cumaric acid,cinnamic acid,sinapic acid and ferulic acid could significantly stimulate the growth,biofilm formation and nodC gene expression of Bradyrhizobium,but the high concentrations of phenolic acids significantly inhibited these biological activities,and finally reduced peanut nodulation rate.Therefore,we investigated whether endophytic fungus P.liquidambari was able to exhibit a broad-spectrum degradation potential for a variety of phenolic acids in the soils.Results demonstrated that endophytic fungus P.liquidambari could effectively degrade allechemical cinnamic acid,sinapic acid and ferulic acid as its sole source of carbon and energy for growth,and glucose did not significantly affect its degradation efficiency.The degradation pathway of cinnamic acid,sinapic acid and ferulic acid was proposed based on metabolites identified by GC-MS and HPLC-MS.Further studies found that a variety of degradation enzymes were involved in endophytic fungal degradation of phenolic acids,including phenolic acid decarboxylase,laccase and dioxygenase.qRT-PCR results demonstrated that these degradation enzyme activities and their gene transcription dynamics were consistent with the changes in related intermediate product concentrations.Moreover,the generation of laccase significantly improved the degradation rate of cinnamic acid,sinapic acid and ferulic acid.Further studies found that even in a complex soil environment,this strain could also efficiently degrade cinnamic acid,sinapic acid and ferulic acid.Therefore,these results demonstrated that P.liquidambari could adapt to the in vitro environment and with a broad-spectrum potential for degrading phenolic acids allelochemicals,which is helpful to eliminante the inhibitory effect of phenolic acids accumulation on soil Bradyrhizobium and finally contribute alleviate peanut continuous cropping obstacles.Many studies have demonstrated that the development of legume-rhizobium symbiosis involves the activities of a variety of endogenous signalling molecules in host plants,which are important for controlling or mediating symbiotic responses.Among these signalling molecules,particularly hydrogen peroxide(H2O2)and nitric oxide(NO)have been widely accepted as the signalling response to biotic and abiotic stress processes,and there is currently compelling evidence that H2O2 and NO play important role in signaling processes during the establishment of legume-Rhizobium symbioses.However,whether H2O2 and NO involved in endophytic fungal promotion of nodulation and N2 fixation in peanut is still unclear.This work showed that compared with only Bradyrhizobium inoculation,co-inoculation with P.liquidambari significantly elevated endogenous H2O2 and NO levels in peanut roots.Pre-treatment of seedlings with specific scavengers of H2O2(CAT)and NO(cPTIO)blocked P.liquidambari-induced nodulation and N2 fixation.CAT not only suppressed the P.liquidambari-induced nodulation and N2 fixation,but also suppressed the enhanced H2O2 and NO generation.Nevertheless,the cPTIO did not significantly inhibit the induced H2O2 biosynthesis,implying that H2O2 acted upstream of NO production.These results were confirmed by observations that exogenous H2O2 and SNP(sodium nitroprusside)reversed the inhibition of P.liquidambari-increased nodulation and N2 fixation by the specific scavengers.The transcriptional activities of the symbiosis-related genes SymRK and CCaMK of peanut-Bradyrhizobium interactions also increased significantly in response to P.liquidambari,H2O2 and SNP treatments.The pot experiment further confirmed that the P.liquidambari infection-enhanced H2O2 and NO signalling pathways were significantly related to the increase in peanut nodulation and N2 fixation.This is the first report that endophytic fungus P.liquidambari can increase peanut-Bradyrhizobium interactions via enhanced H2O2/NO-dependent signalling crosstalk,which is conducive to the alleviation of continuous cropping obstacles via an increase in nodulation and N2 fixation.Soil microorganism is an important indicator of soil health and fertility,and the deteriorating soil microbial community structure is one of the main causes of peanut continuous cropping obstacles.Previous studies have shown that there is a certain correlation between soil nutrient status such as NH4+ and NO3-concentrations and legume nodulation efficiency,and the activity of ammonia-oxidizing microbial communities plays an important role in the transformation of soil N.The soil N2 fixation bacterial community structure,mainly including Rhizobium population,its number and diversity play important role in the nodulation frequency of legume-rhizobia interaction.Moreover,the metabolic function diversity of soil microbial community can further provide an advantageous soil environmental condition for promoting legume-rhizobia interaction,nodulation and N2-fixaiton.However,few studies have pointed out that the potential role of soil functional microbial community in promoting peanut nodulation and N2-fixaiton.Plant root exudates contain a wide variety of nutrients and secondary metabolites,which are considered to be important factors affecting soil nutrients status and microbial community structure.This work demonstrated that the addition of P.liquidambari-infected peanut root exudates significantly increased microbial metabolic activity and metabolic function diversity.qRT-PCR and DGGE results found that P.liquidambari-infected root exudates significantly inhibited the abundance and diversity of soil ammonia-oxidizing microbial community,but significantly increased the abundance and diversity soil diazotroph community.Further cloning sequencing analysis showed that endophytic fungus P.liquidambari-infected root exudates inhibited soil ammonia-oxidizing archaea Thaumarchaeota archaeon;however,soil diazotrophs such as Enterobacter sacchari,Azospirillum halopraeferens,Bradyrhizobium lablabi,Rhodopseudomonas palustris,Bradyrhizobium icense,Azohydromonas australica and Bradyrhizobium oligotrophicum were significantly stimulated by P.liquidambari-infected root exudates.Further analysis showed that a variety of bacteria in the stimulated diazotroph community belonged to bradyrhizobium,and their increase was directly related to the increase of peanut nodulation.Moreover,soil nutrient status such asH4+ and NO3-concentrations can also reflect the dynamic changes of these functional microbial communities from the side.In addition,the pot experiment also provided evidences that P.liquidambari-infected root exudates could significantly promote peanut-rhizobium symbiosis and nodulation.The detection of root exudates indicated that the increased soluble sugar,amino acids,total carbon,and total nitrogen may be a main factor for improving soil microbial activity,and the increase in the concentrations of organic acids,phenolics and flavonoids in root exudates may be another reason for inhibiting ammonia-oxidizing microorganisms and stimulating diazotroph community by P.liquidambari inoculation.Therefore,we speculate that endophyttic fungus P.liquidambari infection can improve soil microbial community structure by changing the components of root exudates,especially to increase the diversity of diazotroph in the continuous cropping soils,which contribute to increase peanut-rhizobium interaction and nodulation,and finally beneficial to alleviate peanut continuous cropping obstacles.Many studies have shown that there are many factors related with peanut continuous cropping obstacles,including soil nutrient imbalance,enzyme activity decrease,toxic allelochemical accumulation,microflora deterioration and soil-borne pathogen increase.Previous pot experiment indicated that the inoculation of endophytic fungus P.liquidambari could effectively improve soil enzyme activiey,microbial community,and contribute to increase peanut production.Meanwhile,we also found that P.liquidambari inoculation significantly decreased the accumulation of allelochemicals produced by litter decomposition.Therefore,these results demonstrated that endophytic fungus P.liquidambari should have the potential to alleviate peanut continuous cropping obstacles.However,few studies have reported the use of beneficial microbes to successfully alleviate the problems associated with continuous peanut cropping under field conditions.In this study,the impacts of endophytic fungus Phomopsis liquidambari inoculation on rhizosphere soil micro-ecological properties,peanut nutrient uptake and disease control were first evaluated under field plot conditions.P.liquidambari colonization was detected and significantly improved the rhizosphere soil microbiological and chemical properties,enhanced N,P and K assimilation and suppressed the incidence of peanut disease compared with the non-inoculated treatment.Statistical analysis demonstrated that the yield enhancement was significantly correlated with the improvement in the rhizosphere soil micro-ecological environment and the peanut’s physiological status after the P.liquidambari inoculation.In addition,further study found that in comparison with the control,the inoculation of P.liquidambari also significantly improved the peanut quality.Therefore,our results evidenced that the endophytic fungus P.liquidambari could have strong potential for practical application to alleviate the obstacles associated with continuous peanut cropping under field conditionsIn conclusion,this study demonstrated that endophytic fungus P.liquidambari can effectively increase peanut nodulation and N2 fixation by degrading soil phenolic acids allelochemicals,enhancing peanut’s physiological status,and increasing soil rhizobia number and diversity,which is important to increase peanut yield.Moreover,the field experiment also demonstrated that the inoculation of P.liquidambari significantly improved the rhizosphere soil microbiological and chemical properties,enhanced peanut nutrient assimilation and suppressed the incidence of peanut disease.Therefore,endophytic fungus P.liquidambari could be considered a potential microbial agent for practical applications to alleviate the obstacles associated with continuous peanut cropping. |
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