Genetic and environmental factors influencing the contents of essential oil compounds in Atractylodes lancea.

Rhizomes of Atractylodes lancea are used in traditional Japanese medicine (Kampo) and Chinese medicine to treat numerous diseases and disorders because they contain many pharmacologically active compounds. The major active compounds in A. lancea are essential oil compounds such as ß-eudesmol, hinesol, atractylon, and atractylodin. The contents of the compounds in A. lancea exhibit high variability depending on their habitat. We cultivated clonal lines of A. lancea in different years (2016, 2017) and different locations (Hokkaido, Ibaraki) to investigate the influence of genetic and environmental factors on the contents of major compounds, namely, ß-eudesmol, hinesol, atractylon, and atractylodin. Broad sense heritability of ß-eudesmol, hinesol, atractylon, and atractylodin contents were 0.84, 0.77, 0.86, and 0.87, respectively. The effects of interannual variability on the contents of the compounds were lower than those of genotype. In addition, the cultivated environmental factors were assessed by different locations, and the correlations between Hokkaido and Ibaraki grown plants based on ß-eudesmol, hinesol, atractylon, and atractylodin contents were 0.94, 0.94, 1.00, and 0.83, respectively. The results suggest that the contents of ß-eudesmol, hinesol, atractylon, and atractylodin in A. lancea are largely influenced by genetic factors, and clonal propagation could be an effective strategy for obtaining populations with high contents of essential oil compounds. Furthermore, the contents of ß-eudesmol, hinesol, atractylon, and atractylodin in A. lancea exhibited few correlations with rhizome yields. A. lancea cultivars with not only high contents of essential oil compounds but also high rhizome yield could be developed through selective breeding.

A DOC coagulant, gypsum treatment can simultaneously reduce As, Cd and Pb uptake by medicinal plants grown in contaminated soil.

The efficiency of gypsum, as a dissolved organic carbon (DOC) coagulator, for the simultaneous immobilization of two heavy metals (Cd and Pb) and one metalloid (As) in agricultural soils near an abandoned mining site was examined. The agricultural soil was defined as long-term contaminated as As (1540mgkg-1), Cd (55mgkg-1) and Pb (1283mgkg-1) concentrations exceeded the Korean guideline values for As (25mgkg-1), Cd (4mgkg-1), and Pb (200mgkg-1). Gypsum was incorporated into the contaminated soil at 3% (w/w). In comparison two commonly using immobilizing agents (lime and compost), together with a mixture (lime+gypsum) were also included in the pot trial for the cultivation of two medical plants (A. gigas and A. macrocephala) and to evaluate the effectiveness of gypsum on As, Cd and Pb immobilization. The results showed that even though pH change-induced immobilizing agents such as lime were more effective than gypsum at immobilizing Cd and Pb, addition of gypsum also effectively reduced heavy metal phytoavailability as indicated by decreases in the concentration of Cd and Pb in medicinal plants. Furthermore, gypsum and gypsum+ lime were also most effective in reducing As concentrations in both plants studied. This was mainly attributed to significant decreases in soil DOC (48-64%) when gypsum and gypsum+lime were applied to the soil. Consequently, it was concluded that enhanced DOC coagulation with gypsum, could be considered as a promising technique for the immobilization of both metals (Cd and Pb) and metalloids (As) in agricultural soils.

Reactive oxygen species and hormone signaling cascades in endophytic bacterium induced essential oil accumulation in Atractylodes lancea.

MAIN CONCLUSION: Pseudomonas fluorescens induces gibberellin and ethylene signaling via hydrogen peroxide in planta . Ethylene activates abscisic acid signaling. Hormones increase sesquiterpenoid biosynthesis gene expression and enzyme activity, inducing essential oil accumulation. Atractylodes lancea is a famous Chinese medicinal plant, whose main active components are essential oils. Wild A. lancea has become endangered due to habitat destruction and over-exploitation. Although cultivation can ensure production of the medicinal material, the essential oil content in cultivated A. lancea is significantly lower than that in the wild herb. The application of microbes as elicitors has become an effective strategy to increase essential oil accumulation in cultivated A. lancea. Our previous study identified an endophytic bacterium, Pseudomonas fluorescens ALEB7B, which can increase essential oil accumulation in A. lancea more efficiently than other endophytes; however, the underlying mechanisms remain unknown (Physiol Plantarum 153:30-42, 2015; Appl Environ Microb 82:1577-1585, 2016). This study demonstrates that P. fluorescens ALEB7B firstly induces hydrogen peroxide (H2O2) signaling in A. lancea, which then simultaneously activates gibberellin (GA) and ethylene (ET) signaling. Subsequently, ET activates abscisic acid (ABA) signaling. GA and ABA signaling increase expression of HMGR and DXR, which encode key enzymes involved in sesquiterpenoid biosynthesis, leading to increased levels of the corresponding enzymes and then an accumulation of essential oils. Specific reactive oxygen species and hormone signaling cascades induced by P. fluorescens ALEB7B may contribute to high-efficiency essential oil accumulation in A. lancea. Illustrating the regulation mechanisms underlying P. fluorescens ALEB7B-induced essential oil accumulation not only provides the theoretical basis for the inducible synthesis of terpenoids in many medicinal plants, but also further reveals the complex and diverse interactions among different plants and their endophytes.

Volatiles released by endophytic Pseudomonas fluorescens promoting the growth and volatile oil accumulation in Atractylodes lancea.

Atractylodes lancea is a well-known, but endangered, Chinese medicinal plant whose volatile oils are its main active components. As the volatile oil content in cultivated A. lancea is much lower than that in the wild herb, the application of microbes or related elicitors to promote growth and volatile oil accumulation in the cultivated herb is an important area of research. This study demonstrates that the endophytic bacterium Pseudomonas fluorescens ALEB7B isolated from the geo-authentic A. lancea can release several nitrogenous volatiles, such as formamide and N,N-dimethyl-formamide, which significantly promote the growth of non-infected A. lancea. Moreover, the main bacterial volatile benzaldehyde significantly promotes volatile oil accumulation in non-infected A. lancea via activating plant defense responses. Notably, the bacterial nitrogenous volatiles cannot be detected in the A. lancea - Pseudomonas fluorescens symbiont while the benzaldehyde can be detected, indicating the nitrogenous volatiles or their precursors may have been consumed by the host plant. This study firstly demonstrates that the interaction between plant and endophytic bacterium is not limited to the commonly known physical contact, extending the ecological functions of endophyte in the phytosphere and deepening the understandings about the symbiotic interaction.

CRYOPRESERVATION OF IN VITRO-GROWN SHOOT TIPS OF CHINESE MEDICINAL PLANT Atractylodes macrocephala KOIDZ. USING A DROPLET-VITRIFICATION METHOD.

BACKGROUND: Atractylodes macrocephala Koidz. is an important medicinal species from China that has been used for thousands of years for its special pharmacological antioxidant, hepatoprotective, anti-inflammatory, anti-allergic, antithrombotic, antiviral, and anticarcinogenic activities. OBJECTIVE: The aim of this research was to develop an efficient droplet-vitrification protocol for A. macrocephala shoot tips which could be used as a strategy for long-term conservation within gene banks. MATERIALS AND METHODS: The duration of preculture, loading, and PVS2 steps, as well as the recovery medium formulation, were optimized to achieve high levels of survival and regrowth for A. macrocephala shoot tips after liquid nitrogen exposure. RESULTS: Survival and regrowth levels after cryopreservation in the cultivar 'Baizhu' were as high as 76% and 62%, respectively. Thermal analysis using differential scanning calorimetry suggested that the PVS2 treatment plays a critical role for successful cryopreservation. CONCLUSION: The droplet-vitrification method established in this study could be used to cryopreserve A. macrocephala.

[Effects of Lime on Seedling Growth,Yield and Volatile Constituents of Atractylodes lancea].

OBJECTIVE: To investigate the effects of different amounts of lime on yield and quality of Atractylodes lancea, and to provide reference for the herb growing site soil improvement and self-poisoning ease. METHODS: Add different gradients of lime, and then measure their growth targets, yield and four kinds of volatile constituents content(hinesol, atractylone, ß-eudesmol and atractylodin). Volatile constituents yield per plant was calculated. RESULTS: Adding 160 g/m2 lime had a significant role in promoting the growth and yield of herb; Adding 80 g/m2 lime was conducive to the volatile constituents production, and adding lime decreased the atractylone and atractylodin content, while increased the hinesol and ß-eudesmol content; Adding 160 g/m2 lime promoted the volatile constituents yield per plant. CONCLUSION: Adding lime plays a role of neutralize soil pH, antibacteria and prevention incognita, and has a certain degree of ease autotoxicity and obstacle,and then promotes the yield and volatile constituents production of Atractylodes lancea.

[Effects of different microhabitas on growth and four kinds of volatile oil components of Atractylodes lancea].

To investigate the suitable site environment for Atractylodes lancea, field trials in different niches was carried out, and the seedling growth, biomass and volatile oil components in different microhabitas was studied. The study found that the survival rate, plant height, volatile oil content of those which growing under the bamboo were significantly higher than those exposed in the open field. The survival rate understory was (76 ± 15.1) %, plant height understory was (77 ± 14. 8) cm and the summation of the four kinds of volatile oil content understory reached up to 4.09%; The same evaluation values of these indicators of the four faces in the open field respectively: survival rate is 30%, plant height was (77 ± 14.8) cm and the summation of the four kinds of volatile oil content was 2.24%. But, the yield of the understory (41 ± 22.3) g was significantly lower than those four faces in the open field (104.5 g) on the contrary. For the four open field towards, the yield of the east facing, which organic matter and other nutritional conditions were better than others, was significantly higher than those in the other facings. A. lancea was found to be an anti-poor and shading-like or growing in east facing slope herb through the correlation analysis of light, temperature, soil and leaf nutrients with seedling growth, biomass and volatile oil components. It also reminds us that the understory herb with high survival rate, low incidence, low management costs, and high medicinal ingredients, although it's production is not so high, but it can be improved by increased organic fertilizer. So the ecological planting patterns which can intercropping herbs with the forest was proposed.

[Research on climatic factors of ecology suitability regionalization of atractylodis].

Through study on the correlation between atractylodis lactones ingredient content and climatic factors, we research regionalization from climatic of five main producing provinces of the country, in order to provide a scientific basis for atractylodis' conscious cultivation. By sampling from 40 origins which from five main producing provinces of the country, we use SPSS to analysis variation of atractylodis lactones ingredient content in different conditions of climatic factors and the effect of each factors. Then according to the relationship between atractylodis lactones ingredient content and climatic factors, we use ArcGIS to conduct ecological suitability regionalization based on climatic factors. The most suitable climatic condition for cultivation of atractylodis: the wettest month precipitation 220-230 mm, the warmest average temperature 25 degrees C, the average temperature of driest season 10 degrees C.

Drought degree constrains the beneficial effects of a fungal endophyte on Atractylodes lancea.

AIMS: Plants, fungal endophytes (FEs) and the changing environment interact with each other forming an interlaced network. This study evaluates nonadditive and interactive effects of the FE Acremonium strictum and drought treatment on Atractylodes lancea plantlets. METHODS AND RESULTS: By applying FEs (meristem cultures of At. lancea, fungal inoculation of Ac. strictum and plantlet acclimatization) and drought treatment (regular watering, mild drought, severe drought), a research system of At. lancea ramets under different treatments was established. During 12 days of drought treatment, the plantlets' physiological responses and basic growth traits were measured and analysed. Although drought and FE presence affected plantlet traits to differing degrees, the interactive effects of the two were more pronounced. In particular under mild drought treatment, the FE conferred drought tolerance to plantlets by enhancing leaf soluble sugars, proteins, proline and antioxidant enzyme activity; decreasing the degree of plasmalemma oxidation; and increasing the host's abscisic acid level and root:shoot ratio. When exposed to regular watering or severe drought, these effects were not significant. CONCLUSIONS: Plant traits plasticity was conferred by dual effects of drought stress and FEs, and these factors are interactive. Although FEs can help plants cope with drought stress, the beneficial effects are strictly constrained by drought degree. SIGNIFICANCE AND IMPACT OF THE STUDY: During finite environmental stress, FEs can benefit plants, and for this reason, they may alleviate the effects of climate change on plants. However, because the benefits of FEs are highly context dependent, the role of FEs in a changing background should be re-assessed.

[Research on topographic factors of ecology suitability regionalization of Atractylodis macrocephala].

Through study on the correlation between Atractylodis macrocephala lactones ingredient content and topographic factors, we researched regionalization from topography of five main producing provinces of the country, in order to provide a scientific basis for A. macrocephala reasonable cultivation. By sampling from 40 origins of five main producing provinces of the country, the variation of A. macrocephala lactones ingredient content in different conditions of topographic factors and the effect of altitude, slope and aspect was analyzed by SPSS. Then according to the relationship between A. macrocephala lactones ingredient content and topographic factors, the ecological suitability regionalization was conducted by using ArcGIS based on topographic factors. It is suitable for growth of A. macrocephala in the hilly and mountainous areas of southern whose A. macrocephala lactones ingredient content is in high levels. It is unsuitable for growth of A. macrocephala in Northern plain areas, but we can cultivate A. macrocephala in the hilly and mountainous areas of Northern. The most suitable topographic condition for cultivation of A. macrocephala : altitude 200 meters above, slope 3.00-4.99 degrees.

[Determination of maleic hydrazide residues and its influence on the quality of Atractylodes macrocephala].

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.

[Pollen morphological characteristics, viability test and storage of endangered medicinal plant Atractylodes lancea].

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.

[Effect of endophytic fungal elicitors on growth and atractylodin accumulation of cell suspension cultures of Atractylodes lancea].

OBJECTIVE: To investigate the effects of endophytic fungal elicitors on the growth and atractylodin accumulation of cell suspension cultures of Atractylodes lancea. METHOD: The endophytic fungal elicitors were added to the medium with different concentrations and culture period. Their effects on biomass, atractylodin content and relevant enzyme activities in suspension cultured cells were studied. RESULT: The cell growth was not affected by elicitors at low concentration and obviously inhibited at high concentration. Inhibition rate reached 46.7% by 100 mg L(-1) elicitor. In addition, six strains from A. lancea, among which Rhizoctonia SP1 activity was higher, had distinctly promoted the accumulation of atractylodin. Atractylodin biosynthesis was notably promoted by 20-60 mg L(-1) Rhizoctonia SP1 elicitor. When 40 mg L(-1) Rhizoctonia SP1 elicitor was added in the medium at the 12 day, the maximum content of atractylodin was 28.06 microg L(-1) at the 21 day with 48.3% higher than that of the control and PPO, POD and CAT activities remarkably increased. CONCLUSION: Adding the endophytic elicitors to the medium is able to be effective approaches to enhance atractylodin yield in the suspension culture cell of A. lancea.

[Analysis odor of Rhizoma Aatractylodis Macrocephalae based on electronic nose].

OBJECTIVE: To appraise rationally the quality between wild and cultivated Rhizoma Atractylodis Macrocephalae. METHODS: Evaporability composition of Rhizoma Atractylodis Macrocephalae was sampled from headspace of emanating, and then response values was obtained. Principal component analysis (PCA) and discriminant factor analysis (DFA) are used to combine the optimum feature parameters, and statistical quality control analysis (SQC). RESULTS: The odor of wild Atractylodis Macrocephalae Rhizoma was diffenrent from that of the cultivated. The odor of Atractylodis Macrocephalae Rhizoma cultivated in Zhejiang was similar to the cultivated in Anhui which transplanted from Zhejiang and wild Atractylodis Macrocephalae Rhizoma which cultivated in the environment with full of sunlight. The odor of Atractylodis Macrocephalae Rhizoma cultivated in Hubei and Jiangxi were diffenrent to the odor of others. CONCLUSION: The quality of semiwild cultivated Atractylodis Macrocephalae Rhizoma was the similarest to the wild. The electronic nose combined with PCA, DFA and SQC can be applied to identify the quality of Traditional Chinese Medicine.

[Study on the soft-wood cutting of Atractylodes macrocephala].

OBJECTIVE: The research analyzed the effect of different elements on soft-wood cutting of Atractylodes macrocephala. METHODS: The experiment was studied by L9 (3(4)) orthogonal design with 3 factors and 3 levels. The research confirmed the best project of soft-wood cutting of Atractylodes macrocephala by using variance analysis and line grading law. RESULTS: The maximum livability and rooting rate were both 86.67% when it was treated with GuoGuangShengGen of 2,000 mg/L, 20 s, the ratio of turf to perlite was 1:3 and the matrix was the most favourable rate of soft-wood cutting of Atractylodes macrocephala. CONCLUSION: This method can improve Atractylodes macrocephala cutting suruival rate and provide the theoretical basis for cutting.

[Biomass structure analysis of Atractylodes lancea in different ecological environments].

OBJECTIVE: To study the ecological environments of Atractylodes lancea by biomass structural analysis. METHOD: Through the scientific investigation in Maoshan, the sampling spots were set up, the relation between growth and ecological environments was researched and the ecological environments of A. lancea were divided as following: the vegetation layer, the shrub layer, the shrub-weed layer and the weed layer. The ramet biomass, height, leaves and coverage of A. lancea were studied. RESULT: The several factors (ramet biomass, height, leaves and coverage) showed the regular change. Among maximum, minimum and average, the shrub layer was the biggest, the shrub and weed layer was the second biggest and the vegetation layer and the weed layer was the least. CONCLUSION: A. lancea tends to distribute in the shrub layer and the shrub-weed layer.

[Analysis in distribution of Atractylodes lancea at Maoshan regions].

OBJECTIVE: To Investigate the distribution of Atractylodes lancea at Maoshan regions. METHODS: To combine the plot sampling with GIS technology in the analysis of distribution and its factors. RESULTS: The biomass of Atractylodes lancea was related to the growth of Ouercus serrata var. brevipetiolata, slope and humidity. The distribution of Atractylodes lancea which was less in north Maoshen region, most in south region, least in middle region. CONCLUSION: The main factor of distributing sintuation is the human beings. The leading factors in the biomass of Atractylodes lancea are Ouercus serrata var. brevipetiolata and slope.

[Remote sensing resource monitoring on Atractylodes lancea].

Remote sensing technology was used for investigation of the resources of Atractylodes lancea. Firstly, the general situation of Jiangshu Maoshan and A. lancea in Maoshan was introduced; Secondly, the methods of remote sensing on the resource of the wild drugs were explained. Thirdly, the TM images were interpret according to the differences of the objects reflex spectrum, and growth environments in Damao mountain, Ermao mountain and Xiaomao mountain were divided into different sub-areas according to the results of the field investigations. Finally, the resource of A. lancea in Jiangshu Maoshan was estimated.

[Construction and application of special analysis database of geoherbs based on 3S technology].

In this paper,the structures, data sources, data codes of "the spacial analysis database of geoherbs" based 3S technology are introduced, and the essential functions of the database, such as data management, remote sensing, spacial interpolation, spacial statistics, spacial analysis and developing are described. At last, two examples for database usage are given, the one is classification and calculating of NDVI index of remote sensing image in geoherbal area of Atractylodes lancea, the other one is adaptation analysis of A. lancea. These indicate that "the spacial analysis database of geoherbs" has bright prospect in spacial analysis of geoherbs.

[Change of microbial community in rhizoma sphere of cultivated Atractylodes lancea].

OBJECTIVE: To find the development rules of microbial community in rhizoma sphere of the cultivated Atractylodes lancea. METHOD: Total bacteria, fungi and actinomyces were counted by CFU x g(-1) though dilution plate method. And genomic DNA of microbes were extracted and amplified by primers of E. coli's 27f and 1492r to get the 16S rDNA, then the restriction endonuclease Hinf was used to digest the 16S rDNA. RESULT: Total bacteria, fungi and actinomyces in 2-year old soil were lower than in 1-year old soil, they decreased 46. 14%, 49. 25%, 31.88% respectively and made the ratio of themselves changed. At the same time, all the 8 soil samples got fine 16S rDNA bands, which were about 1500 bp. And the main bands of most of the samples were found at 1000 bp, but the weak bands of each were different although most bands in the same year samples were more similar than in different year ones. CONCLUSION: It is indicated that the change of soil microbial community may has some relation to the continous cropping barrier of A. lancea.