Panax Notoginseng Allelopathy And Its Microbiological Reduction Mechanism

Sanqi ginseng[Panax notoginseng?Burk.?F.H.Chen]is a precious Chinese herbal medicine.However,continuous cropping obstacle has become a major barrier restricting the sustainable development of Sanqi ginseng industry.Ginsenosides,the main secondary metabolites of Sanqi ginseng,cannot only cause autotoxicity,but also promote the proliferation of soil-borne pathogens,and is the leading factor that causes continuous cropping obstacle of Sanqi ginseng.Therefore,researches on the allelopathy,degradation dynamics,and microbial degradation mechanism of ginsenosides are of great significances for comprehensive understanding the formation mechanism of continuous cropping obstacle of Sanqi ginseng and to effectively alleviate or even overcome it.In this paper,we employ the high performance liquid chromatography analysis?HPLC?,real-time quantitative PCR,BIOLOG microplate assay,and Miseq sequencing,etc.to conduct the following studies.1)Under hydroponic conditions,the allelopathic effects of ginsenosides?R₁,Rg₁,Rb₁,Rh₁,Rd,and their mixture?on Sanqi ginseng seedlings were examined and the underlying mechanisms of ginsenosides-induced oxidative stress on the photosynthesis of seedling leaves were discussed;2)The allelopathic effects of ginsenosides?Rg₁,Rb₁,Rh₁ and their mixture?on soil microbiomes were evaluated through soil incubation experiment and the mechanism of soil microecological imbalance mediated by ginsenosides was revealed;3)Under field conditions,the effects of reductive soil disinfestation?RSD?on ginsenosides and the growth of replant seedlings in Sanqi ginseng mono-cropped soils were studied and the potential mechanism of RSD treatment on the prevention and control of continuous cropping obstacle of Sanqi ginseng was clarified;4)The degradation dynamics of ginsenosides?R₁,Rg₁,Rb₁,Rh₁,and Rd?during RSD process were investigated through laboratory-controlled incubation experiment and the microbiological mechanism of RSD treatment on the ginsenoside degradation was deciphered.The main findings were as follows:Hydroponic experiments showed that ginsenosides exhibited significantly allelopathic inhibition on the growth and development of Sanqi ginseng seedlings.The malonaldehyde?MDA?content,electrolyte leakage,and average temperature of seedling leaves were significantly increased while the water use efficiency was considerably decreased after treated with ginsenosides.In particular,ginsenosides is capable of inhibiting the Sanqi ginseng photosynthesis by inducing an oxidative stress on the photosystem II reaction center?PSII?of its seedling leaves.Correlation analyses showed that the growth and wilting rate of Sanqi ginseng seedlings were significantly and negatively correlated with average temperature of its leaves and the photosynthetic performance,respectively.Regression analyses indicated that MDA content was significantly and positively correlated with the electrolyte leakage of seedling leaves,while the electrolyte leakage of seedling leaves was significantly and positively correlated with its average temperature and had significant and negative correlations with the assimilation rate.These results indicated that the allelopathic inhibition of ginsenosides on Sanqi ginseng photosynthesis may be one of the important reasons for the obstruction on the growth and development of replanted Sanqi ginseng.The addition of exogenous ginsenosides not only significantly altered diversities of bacterial communities,but also significantly improved metabolic activities of soil microbial communities as well as functional diversities.Spearman rank correlation analyses showed that bacterial community diversities?Shannon diversity,richness,PCo1?was positively related with substrate utilization diversity.Particularly,relative abundance of differential bacterial genus,Segetibacter,was significantly and positively correlated with the substrate utilization diversities,with the exception of evenness.Results in this study indicated that ginsenosides-induced variation in soil bacterial community diversities may be involved in the improvement of soil microbial metabolic activities as well as functional diversities.Additionally,the addition of ginsenosides significantly altered structural diversities of soil fungal community,but did not alter alpha diversities.Responses of soil fungal genera to the additions of ginsenosides were species-dependent.Specially,shared and unique fungal microbiomes were significantly altered in different ginsenosides treated soils.In particular,ginsenosides significantly enriched potentially pathogenic taxa,such as Alternaria,Cylindrocarpon,Gibberella,Phoma,and Fusarium,while significantly decreased the accumulative relative abundances of potentially beneficial taxa,such as Acremonium,Mucor,and Ochroconis.Among the pathogenic fungi,the Fusarium genus was most responsive to ginsenoside addition with the abundance.Allelopathic stimulation of ginsenosides on the mycelial growth and conidial germination of F.oxysporum were confirmed through validation tests.In addition,the ginsenoside mixtures exhibited synergistic effects on pathogen proliferation.Regression analyses showed that relative abundances of beneficial taxa were significantly and negatively correlated with those of pathogenic genera.Collectively,this research provides evidence that ginsenosides can disrupt the equilibrium of soil fungal microbiota via the stimulation of potential pathogenic groups and inhibition of beneficial taxa.HPLC showed that short-term maize rotation could reduce the content of ginsenosides Rb₁ and Rh₁,with the degradation rate of 54.3%and 73.5%,respectively.While,the RSD treatments degraded the ginsenosides more efficiently,with the degradation rate up to 82.0-99.4%for Rb₁ and 88.1-99.6%for Rh₁.In addition,RSD treatments significantly reduced the population of F.oxysporum,with the disinfestation efficiency reaching 99.0%.High-throughput sequencing suggested that RSD treatment is capable of restructuring the imbalanced soil microbiome in which induced by Sanqi ginseng cultivation.Particularly,RSD treatment increased soil bacterial diversities as well as the relative abundances of potentially beneficial microbes,such as Bacillus,Streptomyces,Paenibacillus,and Bacteroides,while decreased soil fungal diversities as well as the relative abundances of pathogenic microbes,such as Fusarium.Regression analyses showed that the enriched ginsenosides-degrading microbes and improved microbial activities during RSD process may thereby promote the ginsenosides degradation,improved the survival rate of replant Sanqi ginseng seedlings and alleviated its continuous cropping obstacle.Laboratory-controlled soil experiment showed that RSD treatment significantly degraded the soil ginsenosides and altered the composition and structure of soil microbiomes.After 15 days of RSD treatment,the R₁ and Rb₁were completed degraded,and the Rh₁ content declined significantly with the prolongation of RSD treatment?30 d vs.45 d?,whereas no significant differences in the contents of Rg₁and Rd were observed.After 45 days of RSD treatment,the degradation rates of Rg₁,Rb₁ and Rh₁ were 97%,95%,and 71%,respectively.Correlation analysis showed that relative abundances of the significantly enriched anaerobic microbial genera during RSD treatments,such as Desulfosporosinus,Uc?Ruminococcaceae,Clostridium,Sporomusa,Streptomyces,Oxobacter,Uc?Veillonellaceae,Uc?Enterobacteriaceae,and Talaromyces,activities of FDA hydrolase,urease,and cellulase showed significant and negative correlations with the contents of ginsenosides.Results in this study indicated that lowered soil p H combined with anaerobic conditions improved the enrichment of ginsenoside-degrading anaerobic microbes and enzyme activities,which jointly participated in the microbiological degradation of ginsenosides during the RSD treatment.