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Atractylodes macrocephala Koidz. (AM), an herb of traditional Chinese medicine, is well-known for anti-oxidant, anti-tumor and immune regulation potential. However, it is low bioactive compound content that restricts the application of this species. Elicitation is considered as an effective method to enhance biomass and bioactive compound in plants. Our precious study found that polysaccharide of Chrysanthemun indicum L. could promote plant growth by triggering plant defense. In the present study, polysaccharide of Chrysanthemun indicum L. is used to stimulate the accumulation of biomass and bioactive compound with different concentration in Atractylodes macrocephala Koidz. during pot, plot and field experiments. The results suggested that polysaccharide of Chrysanthemun indicum L. could significantly enhance the accumulation of biomass, atractylenolides and polysacchrides. Moreover, 2 mg/mL is determined and verified to be the appropriate concentration during field experiments. In addition, RT-qPCR revealed that CIP-induced terpenoid synthesis in AM mainly depended on mevalonate (MVA) pathway. This is the first report on the discovery of polysaccharide of Chrysanthemun indicum L. for the enhanced accumulation of biaomass and bioactive compound and the use of its for agricultural production., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This research evaluates the elicitors activity and structure characterization of four Chrysanthemum indicum polysaccharides (CIPs) which were isolated from C. indicum, obtained CIP1, CIP2, CIP3, CIP4. Results demonstrated that there was a distinct difference in inducibility and CIP3 was significantly stronger than other CIPs through bioactivity-tests. Taking CIP3 with total carbohydrate content 91.93 % as a representative, its structure was elucidated as a relative molecular weight of 8. 741 × 10 3 g/mol and mainly composed of xylose, galacturonic acid, galactose and glucuronic acid. Through GC, IR and NMR, CIP3 was determined to possess a backbone comprised of T-α-d-GalpA, 1,4-α-d-GlcpA, 1,2-α-d-Xylp, 1,3-α-l-Rhap, 1,2,4-α-l-Rhap and sidechains comprised of 1,3-β-d-Galp, 1,6-α-d-Galp, T-α-Glcp, 1,3-β-d-Glcp, 1,4-α-d-Glcp, 1,3,4-α-d-Manp, T-α-l-Fucp. Further results indicated that CIP3 with active sidechains could significantly increase the expression of defense genes in Atractylodes macrocephala Koidz (AM). It is believed that the sidechains of CIP3 were necessary to its elicitor activity via bioactivity tests., (Copyright © 2021. Published by Elsevier Ltd.)

Plant-derived elicitor is a new type of plant vaccine developed in the contemporary era, and it has safe and broad application prospects in organic agriculture. Research on defense mechanisms triggered by elicitor has become a hot topic in recent years. The Chrysanthemum indicum polysaccharide (CIP) obtained by separation and purification from Chrysanthemum indicum was used as an elicitor in this work. This elicitor has been shown to be effective in Atractylodes macrocephala Koidz (A. macrocephala) against Sclerotium rolfsii sacc (S. rolfsii) infection and soil-borne diseases. However, the mechanism of induced disease resistance has not been elucidated. In this research, we study the CIP-induced A. macrocephala defense response from the level of signal molecules and the defensive enzyme gene expression. Several defense responses to CIP treatment have been found in A. macrocephala, including early hydrogen peroxide (H 2 O 2 ) production, accumulation of salicylic acid (SA) and increased phytoalexin (PA) content. In addition, CIP significantly increased the activity of related defense enzymes in A. macrocephala. RT-qPCR analysis showed that defense-related genes such as polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) were up-regulated after CIP treatment. To obtain the sequence of the defense enzyme gene, we are the first to provide a public and comprehensive A. macrocephala database by transcriptome sequencing. These results together demonstrate that CIP triggers defense responses in A. macrocephala. Our research not only provides further research on immune mechanism between plant and elicitor, but also sheds new light on strategy for biocontrol in the future., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest, (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Oxygenous terpenoids are active components of many medicinal plants. However, current studies that have focused on enzymatic oxidation reactions cannot comprehensively clarify the mechanisms of oxygenous terpenoid synthesis and diversity. This study shows that an endophytic bacterium can trigger the generation of reactive oxygen species (ROS) that directly increase oxygenous sesquiterpenoid content and diversity in Atractylodes lancea. A. lancea is a famous but endangered Chinese medicinal plant that contains abundant oxygenous sesquiterpenoids. Geo-authentic A. lancea produces a wider range and a greater abundance of oxygenous sesquiterpenoids than the cultivated herb. Our previous studies have shown the mechanisms behind endophytic promotion of the production of sesquiterpenoid hydrocarbon scaffolds; however, how endophytes promote the formation of oxygenous sesquiterpenoids and their diversity is unclear. After colonization by Pseudomonas fluorescens ALEB7B, oxidative burst and oxygenous sesquiterpenoid accumulation in A. lancea occur synchronously. Treatment with exogenous hydrogen peroxide (H2O2) or singlet oxygen induces oxidative burst and promotes oxygenous sesquiterpenoid accumulation in planta. Conversely, pretreatment of plantlets with the ROS scavenger ascorbic acid significantly inhibits the oxidative burst and oxygenous sesquiterpenoid accumulation induced by P. fluorescens ALEB7B. Further in vitro oxidation experiments show that several oxygenous sesquiterpenoids can be obtained from direct oxidation caused by H2O2 or singlet oxygen. In summary, this study demonstrates that endophytic bacterium-triggered ROS can directly oxidize oxygen-free sesquiterpenoids and increase the oxygenous sesquiterpenoid content and diversity in A. lancea, providing a novel explanation of the mechanisms of oxygenous terpenoid synthesis in planta and an essential complementarity to enzymatic oxidation reactions., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Fungal endophytes have been isolated from almost every plant, infecting their hosts without causing visible disease symptoms, and yet have still proved to be involved in plant secondary metabolites accumulation. To decipher the possible physiological mechanisms of the endophytic fungus-host interaction, the role of protein phosphorylation and the relationship between endophytic fungus-induced kinase activity and nitric oxide (NO) and brassinolide (BL) in endophyte-enhanced volatile oil accumulation in Atractylodes lancea plantlets were investigated using pharmacological and biochemical approaches. Inoculation with the endophytic fungus Gilmaniella sp. AL12 enhanced the activities of total protein phosphorylation, Ca²⁺-dependent protein kinase, and volatile oil accumulation in A. lancea plantlets. The upregulation of protein kinase activity could be blocked by the BL inhibitor brassinazole. Furthermore, pretreatments with the NO-specific scavenger cPTIO significantly reduced the increased activities of protein kinases in A. lancea plantlets inoculated with endophytic fungus. Pretreatments with different protein kinase inhibitors also reduced fungus-induced NO production and volatile oil accumulation, but had barely no effect on the BL level. These data suggest that protein phosphorylation is required for endophyte-induced volatile oil production in A. lancea plantlets, and that crosstalk between protein phosphorylation and the NO pathway may occur and act as a downstream signaling event of the BL pathway., (© 2013 Institute of Botany, Chinese Academy of Sciences.)

Objective: To determine maleic hydrazide (MH) residues and discuss its influence on the quality of Atractylodes macrocephala., Methods: At the bud stage, A. macrocephala different concentration of MH. Then MH residues,the contents of sugar and lactone were determined by HPLC and UV. The quality of A. macrocephala was comprehensively evaluated by independent sample t test and principal component analysis., Results: The range of MH residues was 0.3-2.2 mg/kg. The results of independent sample t test revealed that the trend of the contents of lactone was low-high-low with the increase of MH, and the effect of MH on the content of sugar was barely obvious. Meanwhile, principal component analysis showed that comprehensive evaluation on the quality of A. macrocephala was the best when MH with 75 or 100 times water was applied., Conclusion: Proper concentration MH is applied to ensure low concentration MH residues and improve yield and quality of A. macrocephala.

This study hypothesized that the positive or negative effects of exogenous abscisic acid (ABA) on oxidative stress caused by lead were dose dependent. The effects of different levels of ABA (2.5, 5, and 10 mg L(-1)) on lead toxicity in the leaves of Atractylodes macrocephala were studied by investigating plant growth, soluble sugars, proteins, lipid peroxidation, and antioxidative enzymes. Excess Pb inhibited root dry weight, root length, and the number of lateral roots, but increased shoot growth. In addition, lead stress significantly decreased the levels of chlorophyll pigments, protein, and activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD). Different levels of ABA significantly increased SOD, CAT, POD, and APX activities, but decreased the level of hydrogen peroxide and malondialdehyde in nonstressed plants. Exogenous application of 2.5 mg L(-1) ABA detoxified the stress-generated damages caused by Pb and also enhanced plant growth, soluble sugars, proteins, and all four antioxidant enzyme activities but reduced Pb uptake of lead-stressed plant compared to lead treatment alone. However, the toxic effects of Pb were further increased by the applications of 5 and 10 mg L(-1) ABA. The levels of antioxidants caused by a low concentration of exogenous ABA might be responsible for minimizing the Pb-induced toxicity in A. macrocephala.

Background: Jasmonic acid (JA) is a well-characterized signaling molecule in plant defense responses. However, its relationships with other signal molecules in secondary metabolite production induced by endophytic fungus are largely unknown. Atractylodes lancea (Asteraceae) is a traditional Chinese medicinal plant that produces antimicrobial volatiles oils. We incubated plantlets of A. lancea with the fungus Gilmaniella sp. AL12. to research how JA interacted with other signal molecules in volatile oil production., Results: Fungal inoculation increased JA generation and volatile oil accumulation. To investigate whether JA is required for volatile oil production, plantlets were treated with JA inhibitors ibuprofen (IBU) and nordihydroguaiaretic acid. The inhibitors suppressed both JA and volatile oil production, but fungal inoculation could still induce volatile oils. Plantlets were further treated with the nitric oxide (NO)-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), the H2O2 inhibitors diphenylene iodonium (DPI) and catalase (CAT), and the salicylic acid (SA) biosynthesis inhibitors paclobutrazol and 2-aminoindan-2-phosphonic acid. With fungal inoculation, IBU did not inhibit NO production, and JA generation was significantly suppressed by cPTIO, showing that JA may act as a downstream signal of the NO pathway. Exogenous H2O2 could reverse the inhibitory effects of cPTIO on JA generation, indicating that NO mediates JA induction by the fungus through H2O2-dependent pathways. With fungal inoculation, the H2O2 scavenger DPI/CAT could inhibit JA generation, but IBU could not inhibit H2O2 production, implying that H2O2 directly mediated JA generation. Finally, JA generation was enhanced when SA production was suppressed, and vice versa., Conclusions: Jasmonic acid acts as a downstream signaling molecule in NO- and H2O2-mediated volatile oil accumulation induced by endophytic fungus and has a complementary interaction with the SA signaling pathway.

Objective: To study the pollen morphological characteristics, viability test and storage character of the endangered plant Atractylodes lancea., Method: Pollen grains morphologies of A. lancea were observed by scanning electron microscope. The optimum culture medium and viability determination methods were screened out by liquid culture and dyeing methods, and then the pollen germination capacities in different storage conditions were detected., Result: The pollen grains are quasi-spherical, with tricolpate and spinous sculpture. The optimal culture medium was ME3 + 16% PEG4000 + 10% sucrose, in which the pollen germination capacity reached to 62.1%, while the other three dyeing methods were not able to be applied to detecting the pollen viability of A. lancea. The low storage temperature could significantly prolong the storage time of pollen of A. lancea. At -80 degrees C, pollen viability could be maintained for 60 days., Conclusion: Liquid culture method is suitable for the determination of pollen germination of A. lancea, and the rate of pollen germination is closely related to the storage time and temperature. At last, this study provides a foundation for the artificial pollination and cultivating in wildness of A. lancea.

Objective: To study the residue of in roots of Atractylodes macrocephalal and in soil., Method: Samples were extracted with methanol. The extracts were cleaned up by liquid-liquid extraction and detected by HPLC., Result: Repeatability and accuracy of the method was verified by fortified recovery at 0.01, 0.05, 0.1, 0.2 mg x kg(-1) levels. Average recovery were 86.1%-98.3% and RSD were 1.0%-6.5% in root and soil. A. macrocephala was treated with two dosage of carbendazim during growing. Results of field test showed that the half lives of carbendazim were 6.51-7.98 d in cultivated soil, 4.51-6.50 d in roots, separately. After sample was preliminarily processed, the residue of dried samples was 0.042-0.433 mg x kg(-1), higher than the fresh samples., Conclusion: If 0.2 mg x kg(-1) is recommended as the MRL (maximum residues limited) of carbendazim in the roots of A. macrocephala, it is suggested that the dose of 0.675 kg a.i. x hm(-1) carbendazim is sprayed twice a year, and carbendazim should not be used within 21 days before the harvest.

Atractylenolide III is an organic product of the herb plant Atractylodes ovata that is used as an anti-inflammatory. This compound has very limited solubility in water, but we succeeded in transforming it using a Chlamydomonas cell disruptant containing 9.1% dimethyl sulfoxide. Two types of metabolites were confirmed after 3 h of incubation by gas chromatography, besides the non-modified substrate. Nuclear magnetic resonance and gas chromatography-mass spectrometry analyses indicated that one of the metabolites had two hydroxyl groups whereas atractylenolide III had one hydroxyl group, but no metabolite was obtained when atractylenolide III was added directly to the Chlamydomonas culture medium for 10 d.

The field plot experiments were designed with L9(3(4)) orthogonal comparison to study the effects of Atractylodes macrocephala seeds coated with SCF on field diseases. The results showed that the treatment 2 was the best SCF to control field diseases. The treatment of seed-coating with thiophanate methyl reduced the percentage of field diseases significantly.