The Modulation Of Pseudostellaria Heterophylla Root Exudates On Rhizosphere Microbial Community Under Consecutive Monoculture

Pseudostellaria heterophylla is a perennial herbaceous plant in the Caryophyllaceae family.The tuberous roots are widely used in traditional Chinese medicine with a long history.However,there are consecutive monoculture problems in the agricultural production of P.heterophylla,such as poor performance,decreased yield and quality,etc.It is urgent to reveal the underlying mechanism of P.heterophylla consecutive monoculture problems and to develop effective bio-control measures.Based on this,cultivar "Zhe Shen No.2" was used as the experimental material in this study,and targeted metabolomics and modern molecular biology techniques were applied to explore plant-microbe interactions in rhizosphere in order to clarify the changes in rhizosphere microbial communities under consecutive monoculture and their action mode.The main findings are as follows:(1)The results from targeted metabolomics analysis showed that the composition and relative abundance of metabolites in tuberous roots and root exudates were distinctly different under consecutive monoculture.In detail,495 kinds of metabolites were detected in tuberous roots and 450 kinds of metabolites were detected in root exudates.Among them,130 kinds of metabolites were co-existed in both tuberous roots and root exudates.Further analysis on coumarins showed that 10 and 8 coumarins were respectively detected in tuberous roots and root exudates.Among them,two coumarins including coumarin and esculin were co-existed in tuberous roots and root exudates.The relative abundance of coumarin in root exudates showed a declining trend under consecutive monoculture,but esculin firstly increased and then decreased.Besides,it was found that coumarin was one of dominant metabolites in root exudates but not in tuberous roots,implying that P.heterophylla might release large amounts of this metabolite into rhizosphere.(2)The effects of specific coumarins on the growth of QS bacteria previously isolated from P.heterophylla rhizosphere soil were further studied.The results showed that different concentrations of coumarin could inhibit the growth of beneficial QS bacteria including Bacillus megaterium(N1),Bacillus aryabhattai(N12),Burkholderia cepacian(N78)and Bacillus thuringiensis(N89).Moreover,coumarin had significant inhibitory effects on the AHL synthesis of QS bacteria N1,N12,N78 and N89.In contrast,low concentrations of coumarin could significantly promoted the growth of pathogenic QS bacteria including Enterobacter hormaechei(T30),Enterobacter cloacae(S131),Enterobacter cancerogenus(S159)and increased the AHL synthesis of T30,S131,S159 and S171.However,high concentrations of coumarin could inhibited the AHL synthesis.(3)Pot experiment by exogenous application of coumarin was carried out to analyze the modulation of coumarins identified in root exudates on the rhizosphere microbial community structure.The results showed that exogenous application of coumarin caused damage to P.heterophylla,leading to poor growth and low yield of tuberous roots.High-throughput sequencing found that the richness of soil bacterial community was higher in newly planted soil(NP)than in control soil(CK)and coumarin-treated soils,such as FMA(0 μmol/g·soil),FMB(0.05 μmol/g·soil),FMC(0.1μmol/g·soil)and FMD(0.2 μmol/g·soil).The richness of rhizosphere bacterial community decreased with the increasing concentrations of coumarin.Further analysis revealed that the relative abundance of Acidobacteriaceae was higher in coumarin-treated soil than in NP soil and CK soil,and Acidobacteriaceae increased with the increasing concentrations of coumarin.The relative abundances of Proteobacteria,Actinobacteria and Gemmatimonadetes were higher in NP soil than in coumarin-treated soils,and they decreased with the increasing concentrations of coumarin.In addition,function prediction showed that the relative abundance of functional potential related to cellular processes was higher in CK soil than in P.heterophylla-planted soils,and they decreased with the increasing concentrations of coumarin.As for fungal community,it was found that the diversity was higher in coumarin-treated soils than in NP soil,and the diversity increased with the increasing concentrations of coumarin.Further analysis found that the relative abundance of beneficial fungi(i.e.,the Penicillium genus)in P.heterophylla rhizosphere decreased under the treatment of coumarin,while the relative abundance of pathogenic fungi(i.e.,the Fusarium genus)increased under coumarin treatment.In addition,function prediction showed that the relative abundances of saprotroph,especially pathotrophsaprotroph were higher in coumarin-treated soils than in CK soil and NP soil.However,the relative abundance of symbiotroph was higher in CK soil and NP soil than in coumarin-treated soils;it decreased with the increasing concentrations of coumarin.In summary,consecutive monoculture of P.heterophylla markedly altered the composition and relative abundance of metabolites in tuberous roots and root exudates.Several specific metabolites(i.e.,coumarins)mediated the differential changes of beneficial bacteria and pathogenic bacteria in P.heterophylla rhizosphere and the imbalance of rhizosphere microbial community structure,which are closely associated with the consecutive monoculture problems existing in P.heterophylla.This study focused on the "rhizosphere environment" and provided theoretical basis for clarifying the complex interactions between plants and microbes,and exploring the underlying mechanism of consecutive monoculture problems and effective alleviation measures.