| Panax ginseng C.A.Meyer,known as the "king of Chinese herbs," plays an irreplaceable role in developing traditional Chinese medicine and China’s significant health industry.Ginseng rusty root symptom is one of the main diseases of ginseng.The general incidence is around 20%and can be as severe as 80% or more.The low yield and poor quality of diseased ginseng and the decrease in the content of active ingredients make the effectiveness of ginseng impossible to guarantee,which has become a significant problem for developing the ginseng industry in China.Previous studies on ginseng rusty root symptom mainly focused on phenolic substances metabolism,metal element accumulation,and soil characteristics of ginseng rusty root symptom.The effect of soil environment on ginseng rusty root symptom and ginseng response mechanism was seldom studied.In this study,microbial macro-genome sequencing,whole transcriptome sequencing,and liquid chromatography-mass spectrometry were used to analyze the relationship between ginseng rusty root symptoms and rhizosphere soil,as well as the response pattern of ginseng to this disease at transcriptional and metabolic levels,and to explore the mechanism of “rusty root” symptoms.The main research results were as follows:(1)Comparative analysis of rhizosphere microecology of ginseng with different degree of rusty rootThe rhizosphere soil microecology changes with different levels of disease in the same ginseng farm were studied.The bacterial community’s diversity in the rhizosphere soil of infected ginseng with rusty root area of 25%～50% was significantly higher than that at other stages.The diversity of the rhizosphere fungal community of seriously diseased(rusty root area of 25%～50%)ginseng decreased significantly,but the richness of the rhizosphere fungal community did not change significantly.The composition of soil microbial communities in the inter-rhizosphere of ginseng with different degrees of disease varied and had significantly different taxa.The abundance of potential pathogen Ilyonectria robusta increased with the increase of ginseng disease degree,suggesting that it was closely related to the occurrence of rusty root ginseng in cultivated fields.By establishing a microbial co-occurrence network,it was found that the keystone phyla of ginseng rhizosphere bacteria and fungi remained the same for different disease levels,namely Proteobacteria and Ascomycota.Multiple changes in bacterial community complexity and stability occurred as the disease expanded,and there may be more competition and ecological niche separation in the rhizosphere bacterial communities of diseased ginseng than in healthy ginseng.Interactions in rhizosphere fungal communities were reduced in mildly ginseng(rusty root area of 0～25%),while more complex fungal communities may have formed again in the severe group.Unlike bacterial communities,there may be more interspecies cooperation and niche overlap in fungal networks of severe group ginseng rhizosphere soil.The significant decrease of p H,total phosphorus,available phosphorus,available potassium,catalase,invertase,and phosphatase in the rhizosphere soil of ginseng in the severe group may affect the disease resistance of ginseng.TP,AK,AP,CAT,and INV were the key factors affecting the soil microbial community.(2)Comparative transcriptome analysis revealed the response of ginseng to rusty root symptomComparative transcriptome analysis was performed on diseased and healthy ginseng tissues from diseased ginseng.A total of 9451 differentially expressed genes were screened,among which a large number of genes were involved in the biosynthesis of secondary metabolites and multiple metabolic pathways.The analysis of plant stress-related pathways indicated that up-regulation of critical genes in the phenylpropanoid biosynthesis pathway,regulation of ROS by the peroxisome,up-regulation of resistance genes in the plant-pathogen interaction pathway,and phytohormone signaling such as salicylic acid and jasmonic acid associated with stress response might play essential roles in the response mechanism of ginseng to rusty root.(3)Regulatory role of non-coding RNA in ginseng rusty root symptom tissueThe lnc RNAs,circ RNAs,and mi RNAs in diseased tissues and healthy ginseng tissues were identified,and a total of 17,645 lnc RNAs,245 circ RNAs,and 299 mi RNAs were obtained from all tissue samples.Analysis showed that 1553 lnc RNAs,16 circ RNAs,and 107 mi RNAs were differentially expressed in the diseased tissues.Then,enrichment analysis of the target genes of differentially expressed nc RNAs revealed that differentially expressed lnc RNAs,circ RNAs,and mi RNAs were involved in fatty acid-related regulation,indicating that alterations in fatty acidrelated pathways may play a key role in ginseng rusty root symptom.Differentially expressed lnc RNAs might be involved in the regulation of ginseng tissue homeostasis.In addition,the differentially expressed nc RNAs also play critical regulatory roles in transcriptional translation processes,primary metabolism such as starch and sucrose,and secondary metabolism such as alkaloids in diseased tissues.Finally,we integrated the correlation between nc RNAs and m RNAs,constructed the corresponding interaction networks,discovered nc RNAs that might play critical roles in diseased tissues,and found several mi RNAs ath-mi R396b-5p,ath-mi R156a-5p and athmi R195 a,were at the core of the network.These mi RNAs are presumed to play a crucial role in the formation of rusty roots.(4)Study on metabolic patterns of diseased tissue in rusty root symptomUsing untargeted metabolomics,we analyzed the differences in metabolic levels in diseased tissues of ginseng compared to healthy tissues.We found significant changes in the metabolic patterns of multiple metabolites in diseased tissues.These differential metabolites are closely related to plant stress resistance and include organic acids,alkaloids,alcohols,and lipids.The levels of three hormones,methyl jasmonate,salicylic acid,and abscisic acid,were also significantly up-regulated in diseased tissues,regulating physiological processes related to ginseng stress resistance.Correlation analysis of metabolomic and transcriptomic data revealed that the phenylpropanoid biosynthesis pathway was significant in ginseng response to rusty root.Finally,ginseng response mechanisms to rusty root were constructed by combining differentially expressed genes,differentially expressed nc RNAs,and differentially accumulated metabolites.(5)Study on the cause of tissue coloration in rusty root symptomThe compounds associated with the rusty root tissue coloration were mainly phenolic acids and flavonoids.The accumulation of five phenolic acids,including cinnamic acid and benzoic acid,and seven flavonoids,including phloretin-2’-O-glucoside and 4’,5,6,7,8-pentamethoxyflavone,and the enrichment of elements such as Al,Fe,and Mg in rusty root tissues may be key factors in the coloration of diseased tissues.It was speculated that the causes of rusty root tissue coloration: Degradation of the soil microecology caused a stress response in ginseng roots with up-regulated expression of several key enzyme genes in the phenylpropanoid biosynthesis pathway,resulting in a significant accumulation of phenolic compounds.These compounds are readily complex with ions to form colored complexes.Some of the flavonoids themselves are yellow,and especially anthocyanins are the main pigment substances in the plant.Simultaneously,to resist external stress,ginseng increased lignin accumulation and formed lignin polymers containing complex and pigment substances in the epidermis,leading to the rusty root phenomenon.Combined with the transcriptome data,the up-regulated expression of three phenylalanine ammonia-lyase genes(Pg＿S1239.2,Pg＿S0954.1,and Pg＿S2256.14),cinnamate 4-hydroxylase gene(Pg＿S1367.7),and one 4-coumarate: Co A ligase gene(Pg＿S4060.1)were screened and may be closely related to ginseng tissue coloration. |
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