Rapid temporal changes in root colonization by arbuscular mycorrhizal fungi and fine root endophytes, not dark septate endophytes, track plant activity and environment in an alpine ecosystem.

Fungal root endophytes play an important role in plant nutrition, helping plants acquire nutrients in exchange for photosynthates. We sought to characterize the progression of root colonization by arbuscular mycorrhizal fungi (AMF), dark septate endophytes (DSE), and fine root endophytes (FRE) over an alpine growing season, and to understand the role of the host plant and environment in driving colonization levels. We sampled four forbs on a regular schedule from June 26th-September 11th from a moist meadow (3535 m a.s.l) on Niwot Ridge, Rocky Mountain Front Range, CO, USA. We quantified the degree of root colonization by storage structures, exchange structures, and hyphae of all three groups of fungi. AMF and FRE percent colonization fluctuated significantly over time, while DSE did not. All AMF structures changed over time, and the degree of change in vesicles differed by plant species. FRE hyphae, AMF arbuscules and AMF vesicles peaked late in the season as plants produced seeds. AMF hyphae levels started high, decreased, and then increased within 20 days, highlighting the dynamic nature of plant-fungal interactions. Overall, our results show that AMF and FRE, not DSE, root colonization rapidly changes over the course of a growing season and these changes are driven by plant phenology and seasonal changes in the environment.

Subcellular distribution and chemical form of phosphorus involved in alleviating phosphorus toxicity of the phosphorus-accumulator Polygonum hydropiper.

Polygonum hydropiper is a dominant plant species in Shifang phosphorus (P) mine area and is a promising P-accumulator used for P-phytoextraction. To date, little information is available on the physiological response involved in alleviating P toxicity of P. hydropiper under high P. A pot experiment was carried out to investigate growth, P subcellular distribution, chemical forms in two ecotypes of P. hydropiper under high levels (1, 4, and 8 mmol P L-1) of inorganic P (Pi) and organic P (Po), supplied as KH2PO4 and myo-inositol hexaphosphoric acid dodecasodium salt, respectively. The mining ecotype (ME) showed a greater ability to tolerate high P than the non-mining ecotype (NME), as shown by its superior growth with undamaged leaf anatomical structure. The ME showed 1.3-2.2 times greater shoot P accumulation than the NME. More than 93% of P accumulated in tissue cell wall and soluble fraction. The increasing P treatments increased all tissue P forms, especially Pi form. The ME showed significantly higher ester P, nucleic P and insoluble P in tissues than the NME at 8 mmol L-1; however, it demonstrated lower Pi, expect for roots at 5 weeks. The percentages of Pi and nucleic P in roots of the ME were higher than other P forms, and the percentages of nucleic P dominated in the leaves. Probably, the combination of preferential distribution of P in cell wall and soluble fraction in tissues and storage of P in low activity as nucleic P in leaves allows the ME to adapt high P.

Anti-inflammatory and anticholinesterase activity of six flavonoids isolated from Polygonum and Dorstenia species.

This study was aimed at investigating the anti-inflammatory and anticholinesterase activity of six naturally occurring flavonoids: (-) pinostrobin (1), 2',4'-dihydroxy-3',6'-dimethoxychalcone (2), 6-8-diprenyleriodictyol (3), isobavachalcone (4), 4-hydroxylonchocarpin (5) and 6-prenylapigenin (6). These compounds were isolated from Dorstenia and Polygonum species used traditionally to treat pain. The anti-inflammatory activity was determined by using the Griess assay and the 15-lipoxygenase inhibitory activity was determined with the ferrous oxidation-xylenol orange assay. Acetylcholinesterase inhibition was determined by the Ellman's method. At the lowest concentration tested (3.12 µg/ml), compounds 2, 3 and 4 had significant NO inhibitory activity with 90.71, 84.65 and 79.57 % inhibition respectively compared to the positive control quercetin (67.93 %). At this concentration there was no significant cytotoxicity against macrophages with 91.67, 72.86 and 70.86 % cell viability respectively, compared to 73.1 % for quercetin. Compound 4 had the most potent lipoxygenase inhibitory activity (IC50 of 25.92 µg/ml). With the exception of (-) pinostrobin (1), all the flavonoids had selective anticholinesterase activity with IC50 values ranging between 5.93 and 8.76 µg/ml compared to the IC50 4.94 µg/ml of eserine the positive control. These results indicate that the studied flavonoids especially isobavachalcone are potential anti-inflammatory natural products that may have the potential to be developed as therapeutic agents against inflammatory conditions and even Alzheimer's disease.

Floral development at multiple spatial scales in Polygonum jucundum (Polygonaceae), a distylous species with broadly open flowers.

Distyly, a special polymorph, has evolved in many groups of angiosperms and has attracted attention since Darwin's time. Development studies on distylous taxa have helped us to understand the evolutionary process of this polymorph, but most of these studies focus on species with narrowly tubular corolla. Here, we studied the floral development of Polygonum jucundum, a distylous species with broadly open flowers, at multiple spatial scales. Results showed that the difference in stigma height between flowers of the two morphs was caused by differences in style growth throughout the entire floral development process. The observed difference in anther heights between the two morphs was because the filaments grew faster in short-styled (SS) than in long-styled (LS) flowers in the later stages of floral development. In addition, the longer styles in LS flowers than in SS flowers was because of faster cell division in the early stages of floral development. However, SS flowers had longer filaments than LS flowers primarily because of greater cell elongation. These results indicate that floral development in P. jucundum differs from that of distylous taxa with floral tubes shown in previous studies. Further, we conclude that the presence of distyly in species with open flowers is a result of convergent evolution.

Influence of swine manure on growth, P uptake and activities of acid phosphatase and phytase of Polygonum hydropiper.

Excessive application of animal manure to the farmland results in enrichment of P in the soil. Phytoremediation is a promising strategy for extracting excess P from manure impacted soil. P uptake characteristics of a mining ecotype (ME) and a non-mining ecotype (NME) of Polygonum hydropiper were investigated in this study by adopting soil culture containing various concentrations of swine manure (0-200 g swine manure kg(-1) soil). A peak value in the biomass of P. hydropiper was determined in 100 g kg(-1) soil. Significant increase of P content in tissues of two ecotypes was noticed with an increase in swine manure concentration. Maximum P accumulation in shoots and roots was observed at the concentration of 100 g kg(-1) soil, however, the ME accumulated more P as compared to the NME. The ME showed a lower plant effective number and a higher P extraction ratio compared to the NME. Both acid phosphatase and phytase activities of P. hydropiper were obviously enhanced under swine manure impacted soil compared with control, while those of ME higher than the NMEs. Therefore, the two ecotypes of P. hydropiper can accumulate P from soil amended with swine manure and establishes the foundation for phytoremediation.

[Optinization of rapid propagation technique and induction and identification of autotetraploid of Polygonum multiflorum].

OBJECTIVE: To establish and optimize the rapid propagation system of Polygonum multiflorum, as well as explore method for induction and identification of autotetraploid. METHOD: Propagation medium was optimized by orthogonal test. The buds were immersed in colchicine solution with different concentrations for different time to select induction conditions for autotetraploid of P. multiflorum. RESULT: The most appropriate propagation medium was MS medium supplemented with 1.0 mg x L(-1) 6-BA, 0.3 mg x L(-1) NAA, and 0.4 mg x L(-1) PP333. That the buds were soaked in 0.2% colchicine solution for 30 h, or soaked in 0.3% colchicine solution for 18 h, was optimal condition to induce autopolyploid of P. multiflorum with induction rate as high as 16.7%. CONCLUSION: Rapid propagation of P. multiflorum could be achieved by tissue culture. Furthermore, colchicine was an effective inducer of polyploidy, and 25 tetraploid lines were obtained through chromosome identification. The experiment laid a foundation for the wild resource conservation, superior varieties breeding of P. multiflorum.

[Study on characteristic HPLC fingerprint of Polygonum orientale inflorescence].

OBJECTIVE: To establish a characteristic HPLC fingerprint of Polygonum orientale inflorescence, and to provide reference for its quality evaluation. METHODS: Taxifolin was used as reference. HPLC analysis was carried out with Diamonsil C18 column (200 mm x 4.6 mm, 5 microm) using acetonitrile -0.1% phosphoric acid(gradient elution)as mobile phase at flow rate of 1.0 mL/min. The detection wavelength was set at 280 nm and the column temperature was 30 degrees C. RESULTS: Eighteen common peaks were pointed out from the HPLC fingerprint of Polygonum orientale inflorescence from 12 different habitats. Among of them,four common peaks were identified as taxifolin, catechin, gallic acid and 3,3'-dimethyl ellagic acid-4-O-beta-D-glucoside. Analyzed by "Similarity Evaluation for Chromatographic Fingerprint of Traditional Chinese Medicine" software, the HPLC fingerprint similarities of 12 samples were more than 0.9. CONCLUSION: This method is repeatable and exclusive. It can be used for identification and quality control of Polygonum orientale inflorescence.

[Habitat survey and biological characteristics study of Polygonum multiflorum germplasms in Guizhou region].

OBJECTIVE: To provide reference in selecting premium provenance and improve the cultivation techniques of Polygonum multiflorum. METHODS: Field survey, routine field-observation and sampling fixed plant for analysis in lab were adopted. RESULTS: The growing adaptability of Polygonum multiflorum was very strong, which growed flourishly in the condition with adequate light, ample rainfall, rich heat and fertile soil; Along with the lower of latitude, the vegetative period was prolonged and reproductive stage was delayed, which prolonged the time of roots' nutrition acquisition. Time for root shoot ratio increasing continuously of low latitude germplasms was higher than that of higher latitude germplasms. CONCLUSION: Polygonum multiflorum germplasms have different biological characteristics because of different regions and habitats, which can provide useful reference for selecting premium provenance and adjusting measures to local conditions in different areas.

[Impact of 2, 3-dihydrocyclopentacoumarins to growth of transgenic hairy roots of Polygonum multiflorum and accumulation of stilbene glucosides].

OBJECTIVE: To investigate the influence of 2,3-dihydrocyclopenta coumarins to the growth of transgenic hairy root of Polygonum multiflorum and the accumulation of active ingredient stilbene glucoside in the culture. METHOD: In different culture time, 2,3-dihydrocyclopenta coumarin compounds were administrated to the hairy roots. The yield of stilbene glucoside and the growth of the hairy roots were determined after some period of co-culture. Through the investigation of the different concentrations of the 2,3-dihydrocyclopentacoumarin, and sub-culture times, the optimal experimental conditions were established. RESULT: The optimal administration time of the 2,3-dihydrocyclopentacoumarin was on the fourth day, the optimal concentration of 2,3-dihydrocyclopentacoumarin was 0.025 g x L(-1). Under this condition, the growth of the hairy root greatly increased. Comparing with control group, the accumulation of stilbene glucoside was increased about four times. CONCLUSION: 2, 3-dihydrocyclopentacoumarins could promote the hairy hair root growth and stimulate the stilbene glucoside accumulation.

Determination of the volatile fraction of Polygonum bistorta L. at different growing stages and evaluation of its antimicrobial activity against two major honeybee (Apis mellifera) pathogens.

The composition of the volatile fraction of Polygonum bistorta L. (also known as bistort or snakeroot) was investigated. Fresh aerial parts of this plant species were collected in the Western Italian Alps during the summer at three different phenological stages, namely vegetative, flowering, and fruiting, and steam-distilled in a Clevenger-type apparatus. The oils accounted for 0.004 to 0.010% of the fresh plant material, and their compositions were determined by GC/FID and GC/MS. The composition of the oils during the vegetative period varied both in quantity and quality; several classes of compounds were found with a predominance of alcohols in the vegetative phase, terpenes and linear-chained saturated hydrocarbons in the flowering phase, while saturated aliphatic acids and their methyl esters were predominant in fruiting phase. The most abundant compounds were 3-methylbut-3-en-1-ol in the vegetative phase, linalool in the flowering phase, and dodecanoic acid and its methyl ester in the fruiting phase. The obtained essential oils were then tested against two major bee pathogens, i.e., Paenibacillus larvae and Melissococcus plutonius, and against a reference bacterial species, Bacillus subtilis. Data were compared to those obtained with reference standards used against those pathogens such as the essential oils obtained from leaves and bark of Cinnamomum zeylanicum (cinnamon), and the antibiotic oxytetracyclin.

[HPLC fingerprint chromatogram of Polygonum multiflorum from Guizhou].

OBJECTIVE: To establish the fingerprint of Polygonum multiflorum from Guizhou and provide a standard for its quality control. METHODS: HPLC analysis was performed on Agillent ZABAX-C18 (4.6 mm x 250 mm, 5 microm), gradient eluted composed of acetonitrile-0.4% water solution of phosphoric acid. Column temperature was set at 25 degrees C and the flow rate was 1 mL/min. The detection wavelength was 280 nm and the analysis time was 60 min. RESULTS: 9 common peaks were identified. The RSD of the relative retention time and the relative peak area were less than 3% in analyzing its precision, stability and repeatability of the common peaks, and the similarity of the 16 batches of sample was more than 0.9. CONCLUSION: The method is simple and reliable, and it can provide a standard and guidance for quality control of Polygonum multiflorum.

[Water cultured propagation of Polygonum multiflorum and dynamic changes of physiological and biochemical characteristics during adventitious roots formation].

Water cultured propagation technology of Polygonum multiflorum was investigated with Rooting Powder No. 2 (ABT 2) comparison experiments, and the dynamic changes of endogenous hormones including indole acetic acid (IAA), abscisic acid (ABA), zeatin riboside (ZRs) contents and activities of indoleacetic acid oxidase (IAAO), polyphenol oxidase (PPO) were analyzed during rooting period. The results showed that rooting percentage of softwood cutting with 50 mg x L(-1) ABT2 and 10 mg x L(-1) ABT2 + 0.2% Urea + 0.2% KH2PO4 treatments was 94%, rooting percentage of softwood cuttings of control was 46% only. The adventitious rooting displayed three distinct phases i. e. root-inducing, root formation and root-elongating phases. The dynamic changes of contents of endogenous plant hormones (IAA, ABA, ZRs) and activities of IAAO, PPO tested were tightly related to the rooting process of soft-wood cuttings in P. multiflorum. During root-inducing phase the contents of IAA, ABA and ZRs decreased sharply, whereas ZRs content and IAAO activity kept higher level. IAA content reached the peak and PPO activity increased obviously during root formation phase, while IAAO activities and ABA, ZRs contents declined to minimum. During root-elongating phase PPO, IAAO activities were higher and IAA, ABA, ZRs contents kept steady. During rooting ABT2 (50 mg x L(-1)) treatment increased the content of IAA and PPO activity in cuttings, while the opposite result occurred in contents of ZRs, ABA and IAAO activity.

[HPLC fingerprint analysis of acetoacetate extraction of Polygonum orientale].

OBJECTIVE: To establish the HPLC fingerprint analysis method for the quality control of acetoacetate extraction of Polygonum orientale. METHODS: Agilent C18 (4.6 mm x 250 mm, 5 microm); DAD detector; Mobile phase: methyl alcohol-water gradient elution at a flow rate of 1.0 mL/min, and at a column temperature of 30 degrees C. RESULTS: 26 peaks were selected as the common fingerprint peaks. The relative standard deviations for relative retention values were less than 3% in the precision and repeated test. CONCLUSION: The repetition, stability and precision of the method is good and it can be used to control the quality of Polygonum orientale.

[Determination of 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucopyranoside and anthraquinon simultaneously in Polygonum multiflori of different growing stage and gathering periods by HPLC].

OBJECTIVE: A HPLC method was established for the determination of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucopyranoside and anthraquinon simultaneously in Polygonum multiflori. The contents variation from different growing stages in P. multiflori were observed. METHOD: The HPLC method was used to measure the contents of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucopyranoside and anthraquinon simultaneously in 14 years old P. multiflori which were harvested in July to December. Chromatographic conditions: The chromatography column was C18 with a mobile phase composed of methanol-0.1% water phosphoric acid. The detection wavelength was 320 nm and 254 nm respectively, and the flow rate was 1.0 mL x min(-1). RESULT: In a certain range, 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucopyranoside, emodin and physcion showed good linearity, precision, reproducibility, stability and average recovery. CONCLUSION: The developed method is sensitive, accurate. The contents of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucopyranoside and anthraquinon in P. multiflorum rapidly accumulated in one to two years, and in three year that was the highest. The samples collected in November accumulate much higher amounts of 2, 3, 5, 4'-tetrahydroxystilbene-2-O-beta-D-glucopyranoside and anthraquinon than those which were collected in other months.

[Germination characters of Polygonum capitatum seed].

OBJECTIVE: To study the influence of the Polygonum capitatum seed germination by different temperatures, cultivating methods and salt stresses, which can provide reference for seed germination inspection and field seeding. METHODS: Cultivating seeds in Petri dishes and count germination rate. RESULTS: The highest germination rate occurs at 15 degrees C; In salt stress condition, with the increase of the salt concentration seed germination rate declines and under the condition of low salt concentration germination rate shows no obvious difference with the controlling group; at the same temperature, germination rate is higher when cultivated with light than under dark, and seeds cultured in the filters have higher germination rate than those which cultured on the filter. CONCLUSION: Germination experiment should carry out at 15 degrees C, with light and between paper. Seeds germinate potential should calculate after the germination experiment progressing 20 d. When planting, salt concentration of the planting seedling field can not above 1 - 2 g/L, otherwise the germination will be affected.

Factors influencing the production of stilbenes by the knotweed, Reynoutria x bohemica.

BACKGROUND: Japanese knotweed, Reynoutria japonica, is known for its high growth rate, even on adverse substrates, and for containing organic substances that are beneficial to human health. Its hybrid, Reynoutria x bohemica, was described in the Czech Republic in 1983 and has been widespread ever since. We examined whether Reynoutria x bohemica as a medicinal plant providing stilbenes and emodin, can be cultivated in spoil bank substrates and hence in the coalmine spoil banks changed into arable fields. We designed a pot experiment and a field experiment to assess the effects of various factors on the growth efficiency of Reynoutria x bohemica on clayish substrates and on the production of stilbenes and emodin in this plant. RESULTS: In the pot experiment, plants were grown on different substrates that varied in organic matter and nutrient content, namely the content of nitrogen and phosphorus. Nitrogen was also introduced into the substrates by melilot, a leguminous plant with nitrogen-fixing rhizobia. Melilot served as a donor of mycorrhizal fungi to knotweed, which did not form any mycorrhiza when grown alone. As expected, the production of knotweed biomass was highest on high-nutrient substrates, namely compost. However, the concentration of the organic constituents studied was higher in plants grown on clayish low-nutrient substrates in the presence of melilot. The content of resveratrol including that of its derivatives, resveratrolosid, piceatannol, piceid and astringin, was significantly higher in the presence of melilot on clay, loess and clayCS. Nitrogen supplied to knotweed by melilot was correlated with the ratio of resveratrol to resveratrol glucosides, indicating that knotweed bestowed some of its glucose production upon covering part of the energy demanded for nitrogen fixation by melilot's rhizobia, and that there is an exchange of organic substances between these two plant species. The three-year field experiment confirmed the ability of Reynoutria x bohemica to grow on vast coalmine spoil banks. The production of this species reached 2.6 t of dry mass per hectare. CONCLUSIONS: Relationships between nitrogen, phosphorus, emodin, and belowground knotweed biomass belong to the most interesting results of this study. Compared with melilot absence, its presence increased the number of significant relationships by introducing those of resveratrol and its derivatives, and phosphorus and nitrogen. Knotweed phosphorus was predominantly taken up from the substrate and was negatively correlated with the content of resveratrol and resveratrol derivatives, while knotweed nitrogen was mainly supplied by melilot rhizobia and was positively correlated with the content of resveratrol and resveratrol derivatives.

Biotransformation of thymol by hairy roots of transgenic Polygonum multiflorum.

OBJECTIVE: The exogenous substrate, thymol, was firstly biotransformed by using suspension hairy roots of transgenic Polygonum multflorum, and its biotransformed situation was also investigated. METHODS: After five days co-cultivated period, the transformed product was isolated by Thin Layer Chromatograph and Column Chromatograph, with the structure elucidated by physic-chemical methods and spectra data. Meanwhile, the time course of biotransformation (T-C) for thymol was also measured by HPLC to illuminate its bio-transformed situation. RESULTS: The glycosylated product, namely DMP, was isolated and purified, which structure was determined as 5-methyl-2-(1-methylethyl) phenyl- beta-D-glucopyranoside. And the distribution of DMP in the medium or culture was varied in different co-cultivated periods, and for five days co-cultivated period, it mainly existed in the medium. CONCLUSION: The hairy roots of Polygonum multiflorum were able to convert the aromatic exogenous substrate, thymol, into its glycoside. Furthermore, the time course indicated the relationship between DMP and co-cultivated period.

[Biotransformation of furannoligularenone by hairy root cultures of Polygonum multiflorum].

OBJECTIVE: To investigate the biotransformation of furannoligularenone by hairy roots of Polygonum multiflorum. METHODS: Furannoligularenone was added to the medium of hairy roots of P. multiflorum after precultured for 9 days, then they were co-cultured. The products were isolated and identified on the basis of their physical chemical properties and spectroscopic data. The T-C curve of biotransformation was investigated with HPLC. RESULTS: The hairy roots of P. multiflorum transformed the substrate to two products, 3-oxo-eremophila-1,7(11)-dien-12,8-olide(II) and 3-oxo-8-hydroxy-eremophila-1,7(11)-dien-12,8-olide(III). After co-cultured for 3 days, the mole conversion ratio of the substrate reached the highest (27.2%). CONCLUSION: It's possible to biotransform furannoligularenone by hairy roots of P. multiflorum.

[Preliminary study on TLC fingerprint of radix polygoni multiflori from different areas].

OBJECTIVE: To establish thin-layer chromatography (TLC) fingerprint of Radix Polygoni Multiflori collected from different areas. METHODS: Quantitative analysis was carried out using TLC scanning method, two mixed solvents of petroleum ether (60-90 degrees C)-ethylacetate-formic acid (15:5:1) and chloroform-methanol-water [6.5: 2.25: 0.42, pH = 4.0 (HAc-NaAc buffer)] were employed as the mobile phase. RESULTS: The TLC chromatograms were constituted 14 characteristic common peaks. The plots based on the height of common peaks can be conveniently used to evaluate the quality of different area samples. CONCLUSION: The TLC fingerprints can be used to classify and identify Radix Polygoni Multiflori rapidly and effectively.