The Ecological Mechanism Of Rhizosphere Environment Succession Mediated By The Monoculture Of Radix Pseudostellariae And Its Regulation

Replanting disease,or ’Sick Soil Syndrome’,is a problem related to replanting in soils where the same species was previously grown.It has been reported that more than 70%of medicinal plants,especially those with tuberous roots have been attacked by replanting diseases.Radix pseudostellariae L.(Caryophyllaceae)is a common and popular Chinese tuberous roots medicinal plant.High-quality R.pseudostellariae is mainly produced in the Zherong region of Fujian province in southern China,where soil and climate conditions are favorable for its growth.However,the continuously monocultured R.pseudostellariae is prone to severe diseases,which result in reduceing biomass of the plant tuberous roots.Since,a field used for R.pseudostellariae can be replanted once in every three to four years.Therefore,it has become the priority to study and overcome the consecutive monoculture problems,especially exhibited in medicinal plant production.In this study,we discussed the ecological process and mechanism of rhizosphere interaction of R.pseudostellariae from the perspective of rhizosphere microenvironment change,plant-soil-microorganism interaction.In order to provide theoretical basis and technical support for alleviating or overcoming the problems of continuous cropping of R.pseudostellariae and other crops.Moreover,our study also evaluated the effect of biochar amendment on replanting disease of R.pseudostellariae.The main conclusions were listed as follows:HPLC-MS and HPLC were used to determine phenolic acids and organic acids in the tissue culture plantlets medium and in the rhizosphere soil of R.pseudostellariae at different growth stages in different years of monoculture.We identified nine types of phenolic acids in soil.These were gallic acid,coumaric acid,protocatechuic acid,p-hydroxy benzoic acid,vanillic acid,syringic acid,vanillin,ferulic acid,and benzoic acid.Eight types of organic acids were successfully identified.The identified organic acids were oxalic acid,formic acid,malic acid,lactic acid,acetic acid,citric acid,tartaric acid,butanedioic acid.The phenolic acids and organic acids levels tended to increase in the culture medium as the growth of the tissue culture plantlets increased.Phenolic acids levels in the monocultured rhizosphere soil varied with different plant growth stages.They increased initially and then declined.The concentrations of those organic acids in the monocultured rhizosphere soil showed a fluctuating pattern with initial increases and subsequent decreases to a certain extent in different growth stages.These organic acids generally tended to accumulate in the soil of R.pseudostellariae,and this accumulation began during the early tuberous root expansion stage.This implies that microorganisms might be involved in the variability of root exudates levels in monocultured rhizosphere soil.The qRT-PCR analysis showed the contents of total bacteria and B.pumilus tended to decrease in the rhizosphere soil as the number of monoculture years increased.A significant increase in the amount of total fungi,pathogenic T.helicus and K.sacchari in the rhizosphere of R.pseudostellariae as the number of monoculture years increased,especially around the site of infected R.pseudostellariae(SS).Further,we found that B.pumilus promote the growth of R.pseudostellariae in the second cropping year and suppressed the mycelial growth of T.helicus and F.oxysporum when it was co-cultured with the pathogens.These microorganisms have been shown to be involved in the consecutive monoculture problems of R.pseudostellariae.We tested the influence of several rhizospheric organic acids on the physiological characteristics of T.helicus,F.oxysporum,F.moniliforme,K.sacchar,B.megaterium and B.pumilus in vitro.The results showed that the mixture of organic acids significantly promoted the growth of pathogenic microorganisms(T.helicus、F.moniliforme and K.sacchar),inhibited the growth of beneficial bacteria(B.megaterium and B.pumilus).The production of the 3A-DON toxin increased in T.helicus cultures exposed to the organic acid mixture,and increases in production of the 15A-DON toxin were found to be directly related to the applied concentrations of the organic acid mixture.The mixture of the tested organic acids was also found to increase H2O2 secretion from the harmful fungi((F.oxysporum、F.moniliforme and T.helicus))when compared to the control.The mixed organic acids showed the most significant chemotaxis effect and significantly stimulated biofilm formation on the pathogenic bacterium K.sacchari.Conversely,the negative effects were observed for B.megaterium and B.pumlilus.Further analysis of qRT-PCR showed that the mixed organic acids had negative effects on the transcription of many biocontrol-related genes(srfAA,bmyB,yndJ,bioA,srfAB,yngG,ituD,lpa-14,and fenD)in the beneficial bacteria B.megaterium and B.pumilus.The results showed that the PGPR(B.pumilus and B.megaterium)were able to suppress the mycelial growth of the pathogenic fungi,T.helicus,F.oxysporum and F.moniliforme,when they were co-cultured without the addition of any organic acids.However,the antagonistic activities were attenuated in response to increasing concentrations of the applied organic acids.It increased the contents of pathogenic microorganisms and decreased beneficial bacteria,which resulted in the disturbance of the structure of rhizosphere microbial community,including the recruitment of pathogenic microorganisms,reducing the contents of beneficial bacteria,and exacerbating the harm to the growth of R.pseudo st ellariae.HPLC was used to detect the use of nine phenolic acids by the pathogenic microorganisms,which was detected in the rhizosphere soil of the monocultured R.pseudostellariae.The results showed that T.helicus can use eight types of phenolic acids,i.e.,gallic acid,coumaric acid,protocatechuic acid,p-hydroxybenzoic acid,vanillic acid,vanillin,ferulic acid,and benzoic acid.K.sacchari could only use four types,i.e.,gallic acid,coumaric acid,vanillin,and ferulic acid.Syringic acid and mixed phenolic acids significantly promoted the growth of T.helicus and K.sacchari,and significantly promoted the production of the two types of toxins(3A-DON and 15A-DON)in T.helicus.The 3A-DON toxin,at a low concentration promoted the growth of K.sacchari and inhibited the growth of B.pumilus.K.sacchari was able to produce protocatechuic acid from consumption of vanillin.The protocatechuic acid had a negative effect on the growth of the beneficial B.pumilus.These revealed the chemical ecological mechanism of differential evolution of rhizosphere microorganisms and allelopathic interaction in the R.pseudostellariae rhizosphere mediated by continuous monocultureThe transcriptome sequencing was used to reveal the mechanisms underlying the differential response of pathogenic bacterium Kosakonia sacchari and beneficial bacterium Bacillus pumilus on their interactions with phenolic acids.This study found vanillin as one of the main allelochemicals in the root exudates of the medicinal plants,which enhanced the glycolysis/gluconeogenesis,and pentose phosphate,and PTS pathway,leading to high metabolism and a good balance between carbon and nitrogen in the treated pathogenic K.sacchari.Moreover,the genes associated with the fatty acid biosynthesis,flagellar assembly,and bacterial chemotaxis were upregulated in the pathogenic bacteria under treatment.Furthermore,the metabolite(protocatechuic acid)of K.sacchari had a negative effect on the citrate cycle,novobiocin biosynthesis,and phenylalanine,tyrosine,and tryptophan biosynthesis of the beneficial B.pumilus.It also promoted the fatty acid metabolism and inhibited the biofilm formation of B.pumilus under treatment.The results suggested that the metabolite(protocatechuic acid)of K.sacchari in the rhizosphere soil was not conducive to the colonization of B.pumilus and significantly reduced its antagonistic ability against the specific pathogens in the monoculture system,which further elucidated the molecular ecological mechanism of differential evolution of rhizosphere microorganisms mediated by continuous monoculture.High throughput sequencing and qRT-PCR analysis demonstrated that the treatment of phenolic acids significantly decreased the relative abundance of Trichoderma,Penicillium,Pseudomonadales,Xanthomonadales,Streptomycetales,Pseudomonas and Burkholderia spp.The organic acids had a significantly negative effect on the relative abundance of Pseudomonadales and Streptomycetales,and significantly increased abundance of Fusarium,Xanthomonadales,Micrococcales,Gemmatimonadales,F.oxysporum、T.helices,and K.sacchari.The soil sucrase activity and chitinase activity significantly decreased and the dehydrogenase activity,urease activity and acid phosphatase activity significantly increased under the phenolic acids treatment on the different consecutive monoculture years.Furthermore,the organic acids significantly decreased sucrase activity and acid protease activity,the reversal occurred in the case of dehydrogenase activity in the rhizosphere soil of R.pseudostellariae.Analysis based on the non-invasive micro-test technique indicated that the root exudates increased the H+efflux and plasma membrane H+-ATPase activity in the pathogenic fungi and decreased them in the beneficial fungi.The R.pseudostellariae roots exhibited a fluctuating H+influx,whereas F.oxysporum stress caused a stable and constant H+efflux at the apex.Pathogens adapt to different pH conditions and proliferate well under acid environment.But the optimum pH for growth of beneficial bacteria were partial alkalinity.This created an acidic environment for the inhibition of beneficial bacteria and accumulation of specialized plant pathogens.This study explained the mechanisms behind microflora shift and structure disorder,block of nutrient cycle and soil acidification caused by root exudates in a continuously monocultured R.pseudostellariae rhizosphere soil through responses to environmental conditions.The metatranscriptomics sequencing was used to reveal the metabolic changes of soil in R.pseudostellariae rhizosphere soil under different years of monoculture.The results showed that the continuous monoculture of R.pseudostellariae not only led to microbial structure imbalance and soil acidification,but also further inhibited the genes expression of the key metabolic pathway(bacterial chemotaxis pathway,flagellar assembly pathway,TCA cycle pathway,Nitrogen metabolism pathway,Carbon fixation in photosynthetic organisms pathway,Carbon fixation in photosynthetic organisms pathway)in rhizosphere soil.This hindered the growth of microorganisms in the soil,the interaction of microorganisms,energy flow and nutrient cycling.Meanwhile,continuous monoculture also obstructed the expression of most metabolism-related functional proteins in soil,resulting in soil microbes deviating from normal soil seriously.Furthermore,continuous monoculture had some positive effects on the growth of pathogenic microorganisms(Fusarium,Talaromyces,Enterobacter and Xanthomonas),and negative effects on beneficial flora(Burkholderia,Pseudomonas,Trichoderma and Bacillus).This was the first study that\expounded the rhizosphere biology and molecular ecology mechanisms of replant disease in continuous monoculture systems by transcriptomics levels.Here we aimed at elucidating the biochar effects in the continuous monoculture by laboratory assays and greenhouse experiment.The test concentrations of biochar did not show significantly promotional effects towards the culture plantlets of R.pseudostellariae.The slight but insignificant increase in biomass was observed following biochar addition.The biochar addition increased the NO3-and NH4+uptake in treated plants which could help improve the nutrient use efficiency.Moreover,the biochar application increased the abundances of total bacteria and fungi.Conversely,a clear negative effect on pathogenic F.oxysporum,T.helicus and K.sacchari.Meanwhile,the beneficial B.ambifaria,P.chlororaphis and B.pumilus were also inhibited by the biochar.The biochar significantly inhibited the growth of F.oxysporum,F.moniliforme,T.helicus,K.sacchari.Further,the GC-MS-based metabolomics have been used to understand metabolic mechanisms in F.oxysporum and fungus response to biochar addition,respectively.Our data indicate that the biochar affect the metabolism process of F.oxysporum,and then inhibit the mycelium growth and abate the virulence.The results gave a promising strategy for the control of replanting disease by biochar application.The microbial growths were mediated by root exudates of R.pseudostellariae,which have different ecological effects of selective inhibition or promotion.Specific pathogens can reprocess and transform root exudates to inhibit the antimicrobial components of other beneficial bacteria by the interactions of microorganisms.It indicated that the reasons of Consecutive monoculture problems were the interaction between plant,soil and specific microorganisms mediated by root exudates in the rhizosphere soil of R.pseudostellariae.Meanwhile,the study also found that the biochar amendment can be used as a potential regulation to effectively suppress the growth of soil-borne pathogen and alleviate the serious problem of consecutive monoculture problems.The above conclusions have important theoretical and practical significance for deepening and expanding the ecological research of traditional Chinese medicine resources,promoting the sustainable utilization of traditional Chinese medicine resources and the development of related industrial system.