Study On The Effect And Mechanism Of Combined Application Of NPK Fertilizers On Growth And Quality Of Atractylodes Chinensis

Objective:Atractylodis Rhizoma,as a traditional Chinese medicine,is widely used in clinical practice,especially in the diagnosis,treatment and prevention of atypical pneumonia（SARS）and new coronavirus pneumonia（COVID-19）.In recent years,the demand for Atractylodes has been increasing day by day,and the storage of Atractylodes lancea is insufficient,and Atractylodes chinensis has become the main source of Atractylodis Rhizoma medicinal materials.Therefore,in this study,A.chinensis was used as the research object,and the orthogonal experimental design was used to study the effect and regulation mechanism of NPK formula fertilization on the growth and quality formation of A.chinensis.Preliminary reveal the law of fertilizer requirement,growth and molecular regulation mechanism of active ingredients synthesis of A.chinensis,provides fertilization guidance for large-scale,standardized and ecological cultivation of A.chinensis,and provides theoretical basis for further improving the content of effective components and ensuring the quality of medicinal materials.Methods:The annual seedlings of A.chinensis were used as experimental materials and the field experiment was conducted.The changes of fertilizer requirement,photosynthesis[net photosynthetic rate（Pn）,stomatal conductance（Gs）,transpiration rate（Tr）,intercellular CO₂concentration（Ci）],growth indexes（plant height,leaf number,stem and leaf fresh weight,longest fibrous root length,fibrous root fresh weight,rhizome fresh weight）and effective components（atractydin,atractylone,β-eudesmol and atractylenolide II）of A.chinensis under combined application of N,P and K were studied,and the fertilization schemes were evaluated by membership function.At the same time,the activities of acetyl coenzyme A carboxylase（ACC）,3-hydroxy-3-methylglutaryl coenzyme A reductase（HMGR）and farnesyl pyrophosphate synthase（FPPS）in rhizomes of A.chinensis were determined by kit method（double antibody sandwich method）.Real-time quantitative PCR（q RT-PCR）was used to determine the expression of ACC,HMGR and FPPS genes in A.chinensis,and comprehensive analysis was carried out to explore the molecular regulation mechanism of the synthesis of effective components of A.chinensis.Results:1.The research on the fertilizer requirement law of A.chinensis in different growth periods under the combined application of N,P and K shows that the demand of A.chinensis for nitrogen,phosphorous and potassium after transplanting showed a downward-rising-decreasing inverted"N"trend.When transplanting at the seedling stage（late May）of A.chinensis,the content of available nitrogen（N）in the soil should be27.45-70.66 mg·kg^-1;after the first topdressing in the vegetative growth period（late June）,the N content should be within 29.21-79.67 mg·kg^-1;after the second top dressing in fruit period（late August）,the N content shall not be less than 23.66 mg·kg^-1.The content of available phosphorus（P）in the soil shall not be less than 99.33 mg·kg^-1when transplanting.The content of available potassium（K）in the soil during transplanting should be between20.79-45.20 mg·kg^-1;after the first topdressing（vegetative growth period）,the content of K should be between 28.69-65.42 mg·kg^-1;After the second top dressing（fruit stage）,the K content shall not be less than 25.34 mg·kg^-1.From the consumption of N,P and K,it can be found that the T8 fertilization scheme is beneficial to the absorption of nitrogen,the T6fertilization scheme is beneficial to the absorption of phosphorus,and the T5 and T7schemes can promote the absorption of potassium.So the synergistic effect of N,P and K is different under different ratios.2.According to the research on the effects of combined application of nitrogen,phosphorus and potassium on photosynthesis of A.chinensis in different growth periods,it can be seen that the photosynthetic characteristics Pn,Gs and Tr of A.chinensis under different combinations of nitrogen,phosphorus and potassium have basically the same changing trends in each growth period,showing"M"type changes.The change of Ci during each growth period showed a"W"-shaped trend.The Pn,Gs and Tr of A.chinensis in the T3 and T6 fertilization schemes were significantly higher than those of other treatment groups,indicating that high phosphorus treatment is beneficial to the progress of photosynthesis of A.chinensis.The plant height of A.chinensis increased significantly from 0-120 days after transplanting,and the number of leaves and the fresh weight of stems and leaves increased significantly from 0-60 days after transplanting,indicating that after basal fertilizer was applied in late May（seedling period）,late June（vegetative growth period）and the rationality of top dressing in late August（fruit period）.T3 and T6 fertilization schemes of A.chinensis longest fibrous root length,fibrous root fresh weight and rhizome fresh weight are significantly higher than other treatment groups,indicating that high-phosphorus fertilization treatment is beneficial to the growth and development of the underground part of A.chinensis.At the same time,from the change trend of rhizome fresh weight,it can be seen that the yield change of A.chinensis conforms to the law of diminishing returns,and the influence of N,P and K on the yield of A.chinensis has a high interactive effect.3.Through content determination,it can be seen that atractydin,atractylone,β-eudesmol and atractylenolide II all decreased to varying degrees after transplanting 30-120 days,and showed an upward trend after 120-150 days.Combined with the change of fresh weight of rhizome,it was obtained that the best harvesting period of A.chinensis is in late October.The membership function analysis showed that the optimal fertilization scheme for high yield and good quality of A.chinensis was（N₂P₃K₁）N 180kg·hm^-2,P₂O₅225 kg·hm^-2and K₂O 105 kg·hm^-2.4.The activities and gene expression levels of key biosynthetic enzymes ACC,HMGR,FPPS in the rhizome of A.chinensis were further determined.The results showed that the key enzyme activities and gene expression levels of ACC,HMGR and FPPS were significantly different under different NPK combinations.During the growth and development of A.chinensis,T3 and T6 fertilization programs have a greater effect on the key enzymes ACC,FPPS activity and gene expression.Under the T4 fertilization program,the key enzyme HMGR activity and gene expression were the highest in the long development period.The key enzyme ACC activity and gene expression are extremely significantly positively correlated with the atractydin content,indicating that the key enzyme ACC has a greater contribution to the synthesis of atractydin in the rhizome of A.chinensis.The positive correlation between FPPS and the contents of atractylone,β-eudesmol and atractylenolide II indicated that FPPS played a major regulatory role in the synthesis of sesquiterpenes.The activity and gene expression of key enzymes ACC,HMGR and FPPS were cross-related with the contents of atractydin,atractylone,β-eudesmol and atractylenolide II,indicating that the biosynthesis of a single component requires the participation of multiple enzymes and genes.Conclusion:Reasonable combined application of nitrogen,phosphorus and potassium is the guarantee of high yield and high quality of A.chinensis.The optimal fertilization scheme in this planting area was T6 N fertilizer（N）180 kg·hm^-2,P fertilizer（P₂O₅）225kg·hm^-2,K fertilizer（K₂O）105 kg·hm^-2,that is,"heavy application of P fertilizer,appropriate application of N and K fertilizer".Consumption at different developmental stages:（1）Seedling stage N:53.90 mg·kg^-1,P:61.26 mg·kg^-1,K:24.82 mg·kg^-1;（2）Vegetative growth stage N:28.61 mg·kg^-1;P:33.15 mg·kg^-1,K:22.72 mg·kg^-1;（3）Flowering N:25.22mg·kg^-1,P:31.46 mg·kg^-1,K:19.93 mg·kg^-1;（4）First fruit Stage N:33.90 mg·kg^-1,P:30.56mg·kg^-1,K:20.37 mg·kg^-1;（5）Full fruit stage N:12.77 mg·kg^-1,P:11.92 mg·kg^-1,K:13.69mg·kg^-1.Correlation studies showed that the key enzyme ACC was closely related to the synthesis of atractydin in the rhizome of A.chinensis,and the key enzyme FPPS played an important regulatory role in the synthesis of atractylone,β-eudesmol and atractylenolide II.The biosynthesis of a single component requires the participation of a variety of enzymes and genes.In summary,reasonable fertilizer application can effectively promote the expression of key enzyme genes ACC,HMGR and FPPS and the increase of enzyme activity,promote the generation of related precursors such as acetyl-Co A and farnesyl diphosphate,and further positively regulate the biosynthesis of polyacetylenes and sesquiterpenes in the rhizome of A.chinensis.