| Continuous cropping obstacles limit the sustainability and re-planting of Sanqi(Panax notoginseng)in Yunnan Province,China.This is a difficult problem that merits urgent attention.Pathogens are important factors in the formation of Sanqi continuous cropping obstacles,but the key pathogens and the synergistic mechanism of the pathogens currently remain unclear.Although crop rotation is an effective agricultural measure to reduce continuous cropping obstacles,there is no standard for crop selection and the evaluation of crop rotation effects on Sanqi.In addition,the mechanism of alleviating the continuous cropping obstacles of Sanqi remains unclear.Thus,two aspects of research were primarily examined.The first included an analysis of the pathogens associated with the continuous cropping obstacles of Sanqi.This was achieved by microbial isolation and purification,re-inoculation and identification,and an analysis of the microbiome.The synergism between the pathogens was experimentally investigated through single/mixed inoculations.The second aspect evaluated the role of crop rotation from three aspects: alleviating of indicator microbes,promotion of the probiotic community,and improvement of soil rhizosphere microbial community function in the continuous cropping of Sanqi.Screening of suitable rotation crops and analyzing the mechanism of rotation crop in elleviating pathogens of Sanqi continuous cropping obstacle.The main results and conclusions are as follows:(1)The isolated cultures and re-inoculation experiments confirmed that Pseudomonas sp.SM2-3(OM679226),Stenotrophomonas sp.SM2-13(MW555228)and Fusarium solani SM1-10(OM666621)are the pathogens associated with the continuous cropping obstacles of Sanqi.The mixed inoculation of Pseudomonas sp.SM2-3 and F.solani SM1-10 substantially increased the disease index for root rot in Sanqi.A meta-transcriptome sequencing analysis revealed that mixed inoculation increased both the oxidative stress responses and endoplasmic reticulum stress of Sanqi;promoted programmed cell death,autophagy and associated protein catabolism;and reduced the level of expression of defense response genes in Sanqi,including the calmodulin pathway and salicylic acid pathway targeted to F.solani,while it promoted the expression level of virulence factors in F.solani.These results suggest that there was a synergistic effect between Pseudomonas and F.solani.Highthroughput sequencing technology was used to analyze the endophytic flora in the roots of continuous cropping Sanqi.The results showed that the level of abundance of the bacteria Pseudomonas,Stenotrophomonas,Achromobacter,Sphingobacterium,and Rahnella,and the fungus Clonostachys in the Sanqi continuous cropping group were significantly higher than those of the healthy controls.These microbes also positively correlated,suggesting that they are indicator microbes of the continuous cropping obstacles of Sanqi.(2)Maize(Zea mays),potato(Solanum tuberosum),and Paris polyphylla var.yunnanensis were selected for rotations with Sanqi to test their ability to inhibit the indicator microbes of Sanqi continuous cropping obstacles.Rotation with Pa.polyphylla var.yunnanensis was shown to significantly reduce the abundance of Stenotrophomonas and Clonostachys,as well as that of Pseudomonas.Simultaneously,the abundance of photoheterotrophic bacteria,nitrification and denitrification,aerobic ammonia oxidation and other related bacteria and symbiotic fungi increased following rotation with Pa.polyphylla var.yunnanensis.The inhibitory effects were studied by testing if the root exudates,water extracts and ethanol extracts of Pa.polyphylla var.yunnanensis roots significantly inhibited the Sanqi pathogens Pseudomonas SM2-3and F.solani SM1-10.The ethanol extracts of Pa.polyphylla var.yunnanensis roots significantly inhibited Pseudomonas SM2-3 and F.solani SM1-10.The primary compounds that were enriched in the ethanol extract of Pa.polyphylla var.yunnanensis root included glycerophospholipids,sphingolipids,steroidal saponins,sterols and terpene glycosides.The rhizosphere soil microorganisms of Pa.polyphylla var.yunnanensis were enriched and cultured by adding the root exudates of Pa.polyphylla var.yunnanensis into the culture medium.Strains that were antagonistic to Pseudomonas sp.SM2-3 and F.solani SM1-10 were screened.Two Trichoderma strains that could antagonize F.solani SM1-10 were identified.In addition,two strains of Serratia.sp were identified that could antagonize Pseudomonas sp.SM2-3.One of the strains was found to chemotactically respond to the root exudates of Pa.polyphylla var.yunnanensis,indicating that the root exudates of Pa.polyphylla var.yunnanensis could inhibit the pathogens of Sanqi through chemotaxis.(3)Metagenomic technology was used to analyze and compare the rhizosphere soil microbial functions between the Pa.polyphylla var.yunnanensis rotation group,Sanqi continuous cropping group and Sanqi continuous cropping blank control group,and three conclusions were drawn.1)There were differences in the expression of antibiotic resistance genes between the three groups.The antibiotic resistance genes,including Mex B,Mex K,oqx B,sme E,ceo B,Tri C,and Opr M,that originated from the Sanqi pathogens Pseudomonas,Stenotrophomonas,Escherichia,and Burkholderiacepacia mediated resistance to tetracycline,fluoroquinolones and phenolic antibiotics in the Sanqi continuous cropping group.These resistance genes were down regulated during the Pa.polyphylla var.yunnanensis rotation and were replaced by resistance genes that originated from Nocardia farcinica,Bifidobacterium,Paenibacillus and Streptomyces that mediated resistance to glycopeptide and nitrofuran antibiotics,glycyrrhizic acid and aminocoumarin antibiotics in the Pa.polyphylla var.yunnanensis rotation group.The results indicated that rotation with Pa.polyphylla var.yunnanensis alleviates the presence of antibiotic resistance genes in Sanqi continuous cropping soil.2)There were differences in the carbohydrate metabolic pathways between the three groups.Lysozyme and plant cell wall degrading enzymes,including β-xylosidase,β-1,4-xylanase and polygalacturonase among others,were enriched in the Sanqi continuous cropping,while the enzymes in Pa.polyphylla var.yunnanensis rotation group that were enriched were related to mannan metabolism.The results showed that the carbon source metabolism of microorganisms in the Sanqi continuous cropping primarily involved plant cell wall degradative enzymes,while those of the Pa.polyphylla var.yunnanensis rotation group included the mannan degradation pathway.3)There were differences in the Kyoto Encyclopedia of Genes and Genomes(KEGG)metabolic pathways in the three groups.The Sanqi continuous cropping group significantly increased the bacterial chemotaxis,quorum sensing,biofilm formation of Pseudomonas,two-component system,carbohydrate metabolism,lipid metabolism,energy metabolism and other pathways related to pathogens.Compared with the Sanqi continuous cropping soil blank control group,the levels of the metabolic pathways described above were further significantly reduced in the Pa.polyphylla var.yunnanensis rotation group.In conclusion,the root rot in Sanqi continuous cropping obstacles was caused by a variety of pathogens,including Pseudomonas,Stenotrophomonas and F.solani.This experiment confirmed the synergistic pathogenic effects between Pseudomonas sp.SM2-3 and F.solani SM1-10.The synergistic mechanism functions by reducing the host immune response and enhancing the superposition effect of pathogen virulence factors.In this study,the effects of different crop rotations were evaluated from three aspects,and the results showed that Pa.polyphylla var.yunnanensis is a suitable crop for rotation with Sanqi.In addition,the mechanism of reducing pathogens in Sanqi continuous cropping was discussed from the aspects of improving the antibiotics resistance gene of Sanqi continuous cropping soil,the carbon source metabolism of Sanqi rotation soil,and the active antibacterial substances in root of Pa.polyphylla var.yunnanensis and directly inhibiting the Sanqi pathogens with antagonistic bacteria.These results provide new theoretical and technical support for solving the problem of Sanqi continuous cropping obstacles. |
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