Screening Of Autotoxin-degrading Bacteria Of Panax Notoginseng And Its Biodegradation Pathway

Panax notoginseng（Burk.）F.H.Chen is one of China’s most important herbal medicines and is often hampered by replanting failure.Plant-soil-micro-organism interaction mediated by root exudates is critical to forming continuous cropping obstacles in P.notoginseng.The autotoxins such as benzoic acid or sap-onin secreted by roots accumulate in soil and inhibit crop growth.The bioreme-diation of allelochemicals by microorganisms is an efficient decontamination pro-cess.In this research,bacteria strain capable of degrading benzoic acid as the sole carbon source were isolated.The degradation characteristics of selected bacteria were also studied.The process of autotoxins degradation by the optimal strain was tracked.The degradation products were also separated and identified,and the degradation pathway was analyzed at the metabolic level.Also,essential degra-dation genes of benzoic acid-degrading bacteria were screened based on the whole genome sequencing.The gene expression was performed to analyze the degrada-tion pathway at the molecular level.Simultaneously,the potential application of degrading bacteria was explored through a series of tests,such as toxicity test of degradation products,simulated soil remediation,and bacteriostatic test.This study’s results have important theoretical and practical significance for developing biological agents,alleviating the obstacles of continuous cropping of P.noto-ginseng,and improving herbal medicines quality.The main research results ob-tained in this paper are as follows:1.Twenty-seven strains capable of degrading benzoic acid or ginsenoside were isolated from rhizosphere soil and root tissue of P.notoginseng by enrich-ment and domestication strategies.The 16s r RNA genes of all strains were ana-lyzed.The results showed that the dominant genus of soil-derived bacteria was Pseudomonas.The dominant genus of bacteria derived from root tissue was En-terobacter and Klebsiella.The dominant genus of total saponin degrading bacteria was Enterobacter.The dominant genera of total saponins and benzoic acid de-grading bacteria were Enterobacter and Pseudomonas,respectively.After re-screening,the six highly efficient degrading bacteria were identified from the as-pects of morphology,molecular biology,physiology,and biochemistry.Finally,Pseudomonas sp.SCB32,Burkholderia sp.PEDB41,Klebsiella sp.ECZ1,En-terobacter sp.EDZ1,Pseudomonas sp.SC2 and Stenotrophomonas sp.SC3 was selected as the materials for subsequent experiments.2.The degradation ability of 4 strains of ginsenoside degrading bacteria with different total saponins concentrations was tested.The degradation characteristics of the optimal degrading bacteria strain SC3 were explored by single-factor ex-periment and optimized degradation conditions.The degradation rate of 200 mg/L ginsenosides by strain SC3 was 88.4%in 120 h.The degradation rate of 400 mg/L ginsenoside Rb₁ by strain SC3 was 90%in 96 h.HPLC separated the three main metabolites produced by the degradation of ginsenoside Rb₁ by strain SC3.The metabolites were identified by ¹³C-NMR,and the degradation pathway of Rb₁ in this study was proposed as ginsenoside Rb₁→gypenoside XVII→ginsenoside F₂→ginsenoside C-K.Besides,this study showed thatβ-glucosidase activity is pos-itively correlated with total saponins’degradation rate.3.The degradation ability of 2 strains of benzoic acid degrading bacteria with different benzoic acid concentrations was tested.The degradation characteristics of the optimal degrading bacteria strain SCB32 were explored by single-factor experiment.The degradation rate of 800 mg/L benzoic acid by strain SCB32 was greater than 97.0%in 24 h.The degradation process was tracked,and identified the metabolites of each degradation period by GC-MS.A total of 3 vital metabolic intermediates were identified.The biodegradation pathway of benzoic acid,the ortho pathway,is proposed for strain SCB32 based on mass spectrometry.4.The genome of the SCB32 strain was sequenced.The genome was 6.3Mbp,with a content GC of 64.6%,and 5,960 coding genes.A total of 55 benzoic acid degradation genes and their homologous genes were discovered from the ge-nome.The results of q RT-PCR showed that genes in the ortho-degradation path-way of benzoic acid were significantly up-regulated to varying degrees at differ-ent time points,which verified that strain SCB32 degraded benzoic acid through the ortho-pathway.Besides,19 proteins encoded by ortho-pathway genes for ben-zoic acid degradation were predicted by bioinformatics.5.This study proves that degrading bacteria have absolute application poten-tial.The degradation products from strains SCB32 and SC3 were less toxic to lettuce seedlings,while seeds in the control group showed significant inhibition of germination.Moreover,the vigor test of P.notoginseng seeds was also showed the same results.In simulated soil degradation,the actual degradation of benzoic acid was 10.5 times of natural degradation,and the actual degradation of Rb₁ was3.2 times of natural degradation.In the pot experiment,the survival rate of P.notoginseng in the continuous cropping soil group was less than 30%,and the survival rate of P.notoginseng increased to 66.7%after inoculation with degrad-ing bacteria.This result indicates a possible application of strain SCB32 and SC3in the bioremediation of autotoxins contamination in agricultural environments.