To clarify the species, pathogenicity, and distribution of the pathogens causing the root rot of Atractylodes lancea in Hubei province, the tissue separation method was used to isolate the pathogens from root rot samples in the main planting areas of A. lancea in Hubei. Based on the preliminary identification of the Fusarium genus by the internal transcribed spacer(ITS) sequence, three housekeeping genes, EF1/EF2, Btu-F-FO1/Btu-F-RO1, and FF1/FR1, were amplified and sequenced. Subsequently, a phylogenetic tree was constructed based on these TEF gene sequences to classify the pathogens. The pathogenicity of these strains was determined using the root irrigation method. A total of 194 pathogen strains were isolated using the tissue separation method. Molecular identification using the three housekeeping genes identified the pathogens as F. solani, F. oxysporum, F. commune, F. equiseti, F. tricinctum, F. redolens, F. fujikuroi, F. avenaceum, F. acuminatum, and F. incarnatum. Among them, F. solani and F. oxysporum were the dominant strains, widely distributed in multiple regions, with F. solani accounting for approximately 54% of the total isolated strains and F. oxysporum accounting for approximately 34%. Other strains accounted for a relatively small proportion, totaling approximately 12%. The results of pathogenicity determination showed that there were certain differences in pathogenicity among strains. The analysis of the pathogenicity differentiation of the widely distributed F. solani and F. oxysporum strains revealed that these dominant strains in Hubei were mainly highly pathogenic. This study determined the species, pathogenicity, and distribution of the pathogens causing the root rot of A. lancea in Hubei province. The results provide a scientific basis for further understanding the root rot of A. lancea and its epidemic occurrence and scientifically preventing and controlling this disease.
Plant Diseases/microbiology* ; Atractylodes/microbiology* ; Phylogeny ; Plant Roots/microbiology* ; Fusarium/classification* ; China ; Virulence ; Fungal Proteins/genetics*

Atractylodes lancea is a perennial herbaceous plant of Asteraceae, with rhizomes for medical use. However, A. lancea plants from different habitats have great variability, and the germplasm resources of A. lancea are unclear and mixed during production. Therefore, it is urgent to protect new varieties of A. lancea. The distinctness, uniformity, and stability(DUS) testing of new plant varieties is the foundation of plant variety protection, and the DUS testing guidelines are the technical basis for variety approval agencies to conduct DUS testing. In this study, the phenotypic traits of 94 germplasm accessions of A. lancea were investigated considering the breeding and variety characteristics of A. lancea in China. The traits were classified and described, and 24 traits were preliminarily determined, including 20 basic traits that must be tested and four traits selected to be tested. The 20 basic traits included 3 quality traits, 5 false quality traits, and 12 quantitative traits, corresponding to 1 plant traits, 2 stem traits, 8 leaf traits, 6 flower traits, and 3 seed traits. The measurement ranges and coefficients of variation of eight quantitative traits were determined, on the basis of which the grading criteria and codes of the traits were determined and assigned. The guidelines has guiding significance for the trait evaluation, utilization, and breeding of new varieties of A. lancea.
Atractylodes/growth & development* ; China ; Phenotype ; Guidelines as Topic ; Plant Breeding

A high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) method was established for the simultaneous determination of 498 farm chemical residues in Atractylodis Macrocephalae Rhizoma. Furthermore, the established method was used to determine the residues in 30 batches of Atractylodis Macrocephalae Rhizoma samples from different habitats. The samples were extracted with acetonitrile containing 1% glacial acetic acid, and the extract was purified by dispersive solid-phase extraction with sorbents of magnesium sulfate, primary secondary amine(PSA), C_(18), silica gel, and graphitized carbon black(GCB). The prepared samples were then analyzed by HPLC-MS/MS, and the internal standard method was used to quantify the residues. The experimental results showed that the 498 farm chemicals presented good linear relationship within the range of 5-400 ng·mL~(-1), with correction coefficients greater than 0.990. Within the linear ranges, the recovery of 495 farm chemicals(except daimuron, chinomethionat, and emamectin benzoate) at three spiked levels(0.05, 0.10, and 0.20 mg·kg~(-1)) was in the range of 61.18%-132.1%, with the RSD of 0.24%-15%. A total of 16 farm chemicals were detected in 30 batches of samples. Among them, difenoconazole and tebuconazole showed higher detection rates, and the detection rate of difenoconazole was 76.7%. The residues of 4 batches of samples exceeded the limits of quantitation of 33 banned farm chemicals stipulated in the Chinese Pharmacopoeia. The theoretical maximum residue limits of the farm chemicals except banned farm cheimicals were used as the judgment standard of safety risks, under which the detected residues of clothianidin, difenoconazole, and pirimiphos-methyl exceeded the theoretical maximum residue limits. The new method established in this paper is simple and reliable, and it can thus be used for qualitative and quantitative analyses of farm chemical residues in Atractylodis Macrocephalae Rhizoma.
Tandem Mass Spectrometry/methods* ; Chromatography, High Pressure Liquid/methods* ; Atractylodes/chemistry* ; Rhizome/chemistry* ; Drugs, Chinese Herbal/analysis* ; Pesticide Residues/analysis* ; Liquid Chromatography-Mass Spectrometry

Microbial detection and control of traditional Chinese medicine(TCM) decoction pieces are crucial for the quality control of TCM preparations. It is also a key area of research in the measurement technology and equipment development for TCM manufacturing. Guided by TCM manufacturing measurement methodologies, this study presented a design of a novel portable microbial detection device, using Atractylodis Macrocephalae Rhizoma decoction pieces as a demonstration. Immunomagnetic separation technology was employed for specific isolation and labeling of target microorganisms. Enzymatic signal amplification was utilized to convert weak biological signals into colorimetric signals, constructing an optical biosensor. A self-developed smartphone APP was further applied to analyze the colorimetric signals and quantify target concentrations. A portable and automated detection system based on Arduino microcontroller was developed to automatically perform target microbial separation/extraction, as well as mimetic enzyme labeling and catalytic reactions. The developed equipment specifically focuses on the rapid and quantitative microbial analysis of TCM active pharmaceutical ingredients, intermediates in TCM manufacturing, and final TCM products. Experimental results demonstrate that the equipment could detect Salmonella in samples within 2 h, with a detection limit as low as 5.1 × 10~3 CFU·mL~(-1). The equipment enables the rapid detection of microorganisms in TCM decoction pieces, providing a potential technical solution for on-site rapid screening of microbial contamination indicators in TCM. It has broad application prospects in measurement technology for TCM manufacturing and offers strong technical support for the modernization, industrialization, and intelligent development of TCM.
Drugs, Chinese Herbal/analysis* ; Atractylodes/microbiology* ; Rhizome/microbiology* ; Biosensing Techniques/methods* ; Medicine, Chinese Traditional ; Colorimetry/instrumentation* ; Quality Control

The traditional Chinese medicine Atractlodis Rhizoma is the dried rhizome of the Asteraceae herbal plant Atractylodes lancea, and it has the functions of drying dampness and strengthening the spleen, removing wind and dissipating cold, and brightening the eyes. The sesquiterpenoids in A. lancea are the main ingredients of its pharmacological activities in clinical practice, including atractylone, β-eudesmol, and hinesol, which possess anti-inflammation, antibacterial, antiviral, and hepatoprotective effects. This study focused on the biosynthesis of sesquiterpenoids in A. lancea, summarized the proportion of the main active ingredients in A. lancea from the genuine region and the non-genuine region, elaborated on the research progress of genes related to biosynthesis pathways, and systematically sorted out the biotic and abiotic factors affecting their biosynthesis, so as to provide a theoretical basis for further research on the biosynthetic mechanism of sesquiterpenoids in A. lancea and development of high-quality medicinal materials of A. lancea.
Atractylodes/metabolism* ; Sesquiterpenes/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Biosynthetic Pathways

This study investigated the choroplast genome sequence of wild Atractylodes lancea from Yuexi in Anhui province by high-throughput sequencing, followed by characterization of the genome structure, which laid a foundation for the species identification, analysis of genetic diversity, and resource conservation of A. lancea. To be specific, the total genomic DNA was extracted from the leaves of A. lancea with the improved CTAB method. The chloroplast genome of A. lancea was sequenced by the high-throughput sequencing technology, followed by assembling by metaSPAdes and annotation by CPGAVAS2. Bioiformatics methods were employed for the analysis of simple sequence repeats(SSRs), inverted repeat(IR) border, codon bias, and phylogeny. The results showed that the whole chloroplast genome of A. lancea was 153 178 bp, with an 84 226 bp large single copy(LSC) and a 18 658 bp small single copy(SSC) separated by a pair of IRs(25 147 bp). The genome had the GC content of 37.7% and 124 genes: 87 protein-coding genes, 8 rRNA genes, and 29 tRNA genes. It had 26 287 codons and encoded 20 amino acids. Phylogenetic analysis showed that Atractylodes species clustered into one clade and that A. lancea had close genetic relationship with A. koreana. This study established a method for sequencing the chloroplast genome of A. lancea and enriched the genetic resources of Compositae. The findings are expected to lay a foundation for species identification, analysis of genetic diversity, and resource conservation of A. lancea.
Phylogeny ; Atractylodes/genetics* ; Genome, Chloroplast ; Whole Genome Sequencing ; Microsatellite Repeats ; Lamiales

We explored the correlations between the color difference values [ΔL~*(lightness), Δa~*(red-green), Δb~*(yellow-blue)] and the content of four active components(including sesquiterpenoids and polyacetylenes) in the powder of Atractylodes lancea and A. chinensis, aiming to provide reference for the quality evaluation of Atractylodis Rhizoma and establish a qualitative model that can distinguish between A. lancea and A. chinensis based on the chromatic values. The tristimulus values(L~*, a~*, and b~*) of 23 batches of A. lancea and A. chinensis were measured by a color difference meter. The content of atractylenolide Ⅱ, β-eudesmol, atractylodin, and atractylone in the 23 batches of samples were measured by high performance liquid chromatography(HPLC). Principal component analysis(PCA) and partial least squares-discriminant analysis(PLS-DA) were performed to establish the qualitative models for distinguishing between A. lancea and A. chinensis. SPSS was employed to analyze the correlations between the tristimulus values and the content of the four index components. The results showed that the established PCA and PLS-DA models can divide the A. lancea and A. chinensis samples into two regions, and the tristimulus values of A. lancea and A. chinensis were positively correlated with the content of β-eudesmol and atractylodin. Therefore, the PCA and PLS-DA models can successfully identify A. lancea and A. chinensis, and the appearance color can be used to quickly predict the internal quality of Atractylodis Rhizoma. This study provides a reference for the quality evaluation of Atractylodis Rhizoma and the modern research on the color of Chinese medicinal materials.
Atractylodes ; Sesquiterpenes, Eudesmane ; Drugs, Chinese Herbal ; Rhizome ; Excipients

This study established the fingerprint and combined it with chemical pattern recognition to evaluate the quality of Atractylodes chinensis samples from different producing areas and then employed the quantitative analysis of multi-components by single marker(QAMS) method to verify the feasibility and applicability of the established method in the quality evaluation of A. chinensis. The fingerprints of A. chinensis samples were constructed via high performance liquid chromatography(HPLC) to evaluate the inter-batch consistency. With the quality control component atractylodin as the internal reference, the relative correction factors(RCFs) were established for atractylenolide Ⅰ, atractylenolide Ⅲ, and β-eudesmol and the content of the four components was calculated. The external standard method was used to verify the accuracy of QAMS method. The quality of A. chinensis was further evaluated by similarity analysis, clustering analysis, and principal component analysis. The fingerprints of 13 batches of samples were calibrated with 21 common peaks, and 4 common peaks were identified with the similarities all above 0.9. The RCFs established with atractylodin as the internal reference represented good reproducibility under different experimental conditions. Specifically, the RCFs of atractylenolide Ⅰ, atractylenolide Ⅲ, and β-eudesmol in A. chinensis were 2.091, 4.253, and 6.010, respectively. QAMS and ESM showed no significant difference in the results, indicating that the QAMS method established in this study was stable and reliable. Thus, HPLC fingerprint combined with QAMS can be used for the quality evaluation of A. chinensis, providing a basis for comprehensive and rapid quality evaluation of A. chinensis.
Atractylodes ; Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal/chemistry* ; Quality Control ; Reproducibility of Results

Myloid beta(Aβ) is produced by cleavage of amyloid precursor protein(APP), which is a main reason for Alzheimer's disease(AD) occurrence and development. This study preliminarily investigated the mechanism of Atractylodes macrocephala(AM) against AD based on LKB1-AMPK-TFEB pathway. The effect of AM on memory ability of AD transgenic Caenorhabditis elegans CL2241 was detected, and then the APP plasmid was transiently transferred to mouse neuroblastoma(N2 a) cells in vitro. The mice were divided into the blank control group, APP group(model group), positive control group(100 μmol·L~(-1) rapamycin), and AM low-, medium-and high-dose groups(100, 200 and 300 μg·mL~(-1)). The content of Aβ_(1-42) in cell medium, the protein level of APP, the fluorescence intensity of APP, the transcriptional activity of transcription factor EB(TFEB), the activity of lysosomes in autophagy, and autophagy flux were determined by enzyme-linked immunosorbent assay(ELISA), Western blot, fluorescence microscope, luciferase reporter gene assay, RLuc-LC3 wt/RLuc-LC3 G120 A, and mRFP-GFP-LC3, respectively. The protein expression of TFEB, LC3Ⅱ, LC3Ⅰ, LAMP2, Beclin1, LKB1, p-AMPK and p-ACC was detected by Western blot. Immunofluorescence and reverse transcription-polymerase chain reaction(RT-PCR) were used to detect the fluorescence intensity of TFEB and the mRNA expression of TFEB and downstream target genes, respectively. The results showed that AM reduced the chemotactic index of transgenic C. elegans CL2241, and decreased the content of Aβ in the supernatant of cell culture medium at different concentrations. In addition, AM lowered the protein level of APP and the fluorescence intensity of APP in a dose-dependent manner. Transcriptional activity of TFEB and fluorescence intensity of mRFP-GFP-LC3 plasmid were enhanced after AM treatment, and the value of RLuc-LC3 wt/RLuc-LC3 G120 A was reduced. AM promoted the protein levels of TFEB, LAMP2 and Beclin1 at different concentrations, and increased the protein expression ratio of LC3Ⅱ/LC3Ⅰ in a dose-dependent manner. Immunofluorescence results revealed that AM improved the fluorescence intensity and nuclear expression of TFEB, and RT-PCR results indicated that AM of various concentrations elevated the mRNA expression of TFEB in APP transfected N2 a cells and promoted the transcription level of LAMP2 in a dose-dependent manner, and high-concentration AM also increased the mRNA levels of LC3 and P62. The protein levels of LKB1, p-AMPK and p-ACC were elevated by AM of different concentrations. In summary, AM regulating lysophagy and degrading APP are related to the activation of LKB1-AMPK-TFEB pathway.
AMP-Activated Protein Kinases/metabolism* ; Alzheimer Disease/drug therapy* ; Amyloid beta-Peptides/metabolism* ; Amyloid beta-Protein Precursor/metabolism* ; Animals ; Atractylodes/chemistry* ; Autophagy/drug effects* ; Beclin-1/pharmacology* ; Caenorhabditis elegans/metabolism* ; Macroautophagy ; Mice ; RNA, Messenger ; Sirolimus/pharmacology*

This study compared the transcriptome of Atractylodes lancea rhizome at different development stages and explored genes encoding the key enzymes of the sesquiterpenoid biosynthesis pathway. Specifically, Illumina NovaSeq 6000 was employed for sequencing the cDNA libraries of A. lancea rhizome samples at the growth stage(SZ), flowering stage(KH), and harvesting stage(CS), respectively. Finally, a total of 388 201 748 clean reads were obtained, and 16 925, 8 616, and 13 702 differentially expressed genes(DEGs) were identified between SZ and KH, KH and CS, and SZ and CS, separately. Among them, 53 genes were involved in the sesquiterpenoid biosynthesis pathways: 9 encoding 6 enzymes of the mevalonic acid(MVA) pathway, 15 encoding 7 enzymes of the 2-C-methyl-D-erythritol-4-phosphate(MEP) pathway, and 29 of sesquiterpenoid and triterpenoid biosynthesis pathway. Weighted gene co-expression network analysis(WGCNA) yielded 12 genes related to sesquiterpenoid biosynthesis for the SZ, 1 gene for the KH, and 1 gene for CS, and several candidate genes for sesquiterpenoid biosynthesis were discovered based on the co-expression network. This study laid a solid foundation for further research on the sesquiterpenoid biosynthesis pathway, analysis of the regulation mechanism, and mechanism for the accumulation of sesquiterpenoids in A. lancea.
Atractylodes/genetics* ; Mevalonic Acid/metabolism* ; Rhizome/genetics* ; Sesquiterpenes/metabolism* ; Transcriptome ; Triterpenes/metabolism*