Resource allocation strategies among vegetative growth, sexual reproduction, asexual reproduction and defense during growing season of Aconitum kusnezoffii Reichb.

Natural plants must actively allocate their limited resources for survival and reproduction. Although vegetative growth, sexual reproduction, asexual reproduction and defense are all basic processes in the life cycle of plants, the strategies used to allocate resources between these processes are poorly understood. These processes are conspicuous in naturally grown Aconitum kusnezoffii Reichb., which makes it a suitable study subject. Here, the morphology, dry matter, total organic carbon, total nitrogen and aconitum alkaloid levels of shoot, principal root (PR) and lateral roots were measured throughout the growing season. Then, transcriptome and metabolite content analyses were performed. We found that vegetative growth began first. After vegetative growth ceased, sexual development began. Flower organ development was accompanied by increased photosynthesis and the PR consumed temporarily stored resources after flower formation. Asexual propagule development initiated earlier than sexual reproduction and kept accumulating resources after that. Development was slow before flower formation, mainly manifesting as increasing length; then, after flower formation it accelerated via enhanced material transport and accumulation. Defense compounds were maintained at low levels before flowering. In particular, the turnover of defense compounds was enhanced before and after flower bud emergence, providing resources for other processes. After flower formation, defense compounds were accumulated. The pattern found herein provides a vivid example for further studies on resource allocation strategies. The exciting finding that the PR, as a more direct storage site for photosynthate, is a buffer unit for resources, and that defense compounds can be reused for other processes, suggests a need to explore potential mechanisms.

Biocontrol of Root Diseases and Growth Promotion of the Tuberous Plant Aconitum carmichaelii Induced by Actinomycetes Are Related to Shifts in the Rhizosphere Microbiota.

Soil Actinomycetes have been used as biocontrol agents against soil-borne plant diseases, yet little is known about their effects on the structure of the rhizosphere microbiota and the long-term effects on crop yield and disease intensity after the application of Actinomycetes is stopped. Here, we conducted 3-year plot experiments to investigate the roles of two Actinomycetes strains (Streptomyces pactum Act12 and Streptomyces rochei D74) in the biocontrol of soil-borne root diseases and growth promotion of monkhood (Aconitum carmichaelii). We also examined their long-term effects after soil application of a mixed Actinomycetes preparation (spore powder) was completed. High-throughput sequencing was used to analyze shifts in the rhizosphere microbiota. The antifungal activity and root colonization ability of the two Actinomycetes were also tested. Disease severity of southern blight and root rot decreased following application of the Actinomycetes preparation, whereas biomass yield of tubers increased compared with the control group. Significant effects of disease control and plant growth promotion were also observed after application was stopped. The Actinomycetes preparation induced marked increases in the abundance of beneficial microbes and decreases in the abundance of harmful microbes in rhizosphere soil. Adding cell-free culture filtrates of both strains Act12 and D74 inhibited the growth of fungal pathogens capable of causing southern blight (Sclerotium rolfsii) and root rot (Fusarium oxysporum) in A. carmichaelii. A GFP-labeled strain was used to show that D74 can colonize roots of A. carmichaelii. In conclusion, a preparation of two Actinomycetes plays a role in the biocontrol of root diseases and growth promotion of A. carmichaelii by inhibiting pathogen growth and shaping the rhizosphere microbiota.

Biocontrol effects of Penicillium griseofulvum against monkshood (Aconitum carmichaelii Debx.) root diseases caused by Sclerotium rolfsiii and Fusarium spp.

AIMS: The aims of this study were to investigate the biocontrol effects of Penicillium griseofulvum strain CF3 and its mechanisms against soil-borne root pathogens (Fusarium oxysporum and Sclerotium rolfsii) of the medical plant Aconitum carmichaelii Debx. METHODS AND RESULTS: The effects of P. griseofulvum strain CF3 were evaluated with regard to the hyphal growth of S. rolfsii and F. oxysporum, the sclerotial formation and germination of S. rolfsii and its expression of sclerotia-formation-related genes. A field experiment was conducted to explore how strain CF3 controls the severity of soil-borne diseases, promotes the growth of A. carmichaelii plants and mediates shifts in the culturable rhizosphere microbial populations. The results showed that treatment with a cell-free culture filtrate of strain CF3 considerably inhibited the hyphal growth of both S. rolfsii and F. oxysporum, in addition to limiting the sclerotial formation and germination of S. rolfsii. Three genes related to sclerotial formation (ArsclR, ArnsdD1 and ArnsdD2) were predicted in S. rolfsii and their expression was found suppressed by the CF3 treatment. Field application of the CF3 biocontrol agent in a powder form (1·9 × 1010 conidia per gram of substrate) reduced soil-borne disease severity by 15·0%. The shoot and root growth of A. carmichaelii plants was promoted by 61·6 and 83·1% respectively, as the biocontrol strain massively colonized the rhizosphere soil. The CF3 treatment also markedly reduced the density of some known species harmful to plants while increasing the density of some beneficial species in the rhizosphere soil. SIGNIFICANCE AND IMPACT OF THE STUDY: Genes related to sclerotia formation of S. rolfsii are predicted for the first time and their expression patterns in the presence of P. griseofulvum strain CF3 are evaluated. This comprehensive study provides a candidate fungal biocontrol strain and reveals its potential mechanisms against S. rolfsii and F. oxysporum in A. carmichaelii plants.

[Resources and usage status of Tibetan medicinal plant "Bangga"].

The study is aimed to clarify the actual original plant, find out the usage status and the resource distribution of the Tibetan medicinal plant "Bangga". By using the way of the literatures survey, interview and investigation, it found out that the actual original plant of the Tibetan medicinal plant "Bangga" were the whole dried plant or the aerial part of Aconitum tanguticum or A. naviculare of Ranunculaceae, among which A. tanguticummainly distributed in Sichuan, Gansu, Qinghai, Tibet (Qamdo), and A. naviculare mainly distributed in Tibet. Sichuan, Gansu, Qinghai and other Tibetan areas mainly used the resources of A. tanguticum, Tibet (except the Qamdo area) mainly uses the A. naviculare, which resource was imminent in danger. Other species described in the literature were not used. It showed that the use of herbs related to their resources, it is recommended to strengthen the protection and guide the market.

[Extraction and purification technologies of total flavonoids from Aconitum tanguticum].

OBJECTIVE: To optimize the extraction and purification technologies of total flavonoids from Aconitum tanguticum whole plant. METHODS: With the content of total flavonoids as index, the optimum extraction conditions for the concentration, volume of alcohol, extracting time and times were selected by orthogonal optimized; Comparing the adsorption quantity (mg/g) and resolution (%), four kinds of macroporous adsorption resins including D101, AB-8, X-5 and XAD-16 were investigated for the enrichment ability of total flavonoids from Aconitum tanguticum; Concentration and pH value of sample, sampling amount, elution solvent and loading and elution velocity for the optimum adsorption resin were determined. RESULTS: The content of total flavonoids in Aconitum tanguticum was about 4.39%; The optimum extraction technique was 70% alcohol reflux extraction for three times,each time for one hour, the ratio of material and liquid was 1:10 (w/v); The optimum purification technology was: using XAD-16 macroporous resin, the initial concentration of total flavonoids of Aconitum tanguticum was 8 mg/mL, the sampling amount was 112 mg/g dry resin, the pH value was 5, the loading velocity was 3 mL/min, the elution solvent was 70% ethanol and the elution velocity was 5 mL/min. Under the optimum conditions, the average content of total flavonoids was raised from 4.39% to 46.19%. CONCLUSION: The optimum extraction and purification technologies for total flavonoids of Aconitum tanguticum were suitable for industrial production for its simplicity and responsibility.

nrDNA ITS sequence based SCAR marker to authenticate Aconitum heterophyllum and Cyperus rotundus in Ayurvedic raw drug source and prepared herbal products.

To authenticate Ayurvedic medicinal plants Ativisha (Aconitum heterophyllum) and Musta (Cyperus rotundus) at the raw drug source and in prepared herbal products, nrDNA ITS sequence based SCAR markers were designed and validated spp.-specific SCAR primers gave amplicon of 415 bp and 134 bp, respectively, in authentic species. The SCAR primers (Cyr-FP and Cyr-RP) could identify tissue sample containing 750 µg to 4.76 mg/100mg of Musta in complex mixtures of DNA extracted from commercial herbal drugs. Ativisha could not be identified through SCAR markers suggesting that authentic species may not been used to prepare herbal drugs despite its being labelled as one of the ingredients in formulations. Analysis of individual tubers of Ativisha and Musta assures the presence of admixtures in raw drug trade of Ativisha, indicates the need to monitor the basic raw material supply and concludes, supplying plant materials through cultivation to manufacturing industries can minimize the risks of adulteration.

Protein changes during ethanol induced seed germination in Aconitum heterophyllum.

Aconitum heterophyllum is a high altitude medicinal plant that has become endangered due to overexploitation for their aconitins. The most effective, conventional propagation method for any plant species is by seed. However, in Aconitum seed germination is erratic, and seedling survival is low. In the present study results have been discussed on the possible implication of ethanol treatment on removal of barriers on radical emergence in terms of protein changes. Eighty seven percent of seed germination was achieved in Aconitum with ethanol treatment. Comparative 2-DE analysis of ethanol treated and untreated seed protein profiles in Phase II of germination revealed 40 differentially expressed proteins. Twenty-seven out of 40 proteins were induced, 5 were increased and 8 were repressed. Mass spectrometry and subsequent identification confirmed that these proteins were involved in metabolism, DNA regulation, stress tolerance and plasmamembrane/cell wall biosynthesis/extension processes. These protein changes might be responsible for physiological and physical changes, respectively, resulted in increase in germination percentage. Further, characterization of these proteins will be of great help in understanding the molecular mechanism lying behind enhanced germination in response to ethanol treatment.

[Difference evaluation of three kinds of root of Aconitum carmichaelii in Sichuan based on UPLC analysis of six alkaloids and chemometrics].

An ultra performance liquid chromatography (UPLC) method was established and validated to simultaneously determine the contents of six aconitum alkaloids in mother, daughter and fibrous roots of 19 batches of Aconitum carmichaelii from Sichuan province. The separation of the six alkaloids was achieved on a ACQUITY UPLC BEH C18 (2.1 mm x 100 mm, 1.7 microm) column at 40 degrees C with a mobile phase consisting of acetonitrile in 30 mmol x L(-1) ammonium acetate buffer solution (adjusted to pH 10.0 with aqueous ammonia) in gradient mode. The data and plots showed that the six aconitum alkaloids have different distributions. Four aconitum alkaloids were almost same in mother and daughter root except benzoylmesaconine and mesaconitine, while the fibrous root differed from the other two roots. The comparisons of significant differences of six aconitum alkaloids between the mother and daughter roots definitely demonstrated that benzoylmesaconine and mesaconitine were the representative components. The 38 detecting samples were classified as two clusters by hierarchical clustering analysis (HCA) and principle component analysis (PCA), the results indicated that the mother root was different from the daughter root on chemical material basis. The study might contribute to the reasonable clinical application of A. carmichaelii.

[Effects of spraying uniconazole on plant morphology and total alkaloid content of Aconitum carmichaeli].

OBJECTIVE: To study the effects of spraying uniconazole on plant morphology and total alkaloid content of Aconitum carmichaeli for providing theory base for establishment of planting measure. METHODS: The mainly morphological character and total alkaloid content of Radix Aconiti Lateralis Praeparata were studied under different uniconazole spraying time and density. RESULTS: Unieonazole could decrease plant height and leaf area, increase stem diameter, leaves and fibril roots, and the amount of dry matter. The difference in total alkaloid was not significant. CONCLUSION: In this experiment,Spraying uniconazole has no significant effect on total alkaloid content of Aconitum carmichaeli. It is better for increasing yield per plant to applied 120 mg/L unieonazole at sub-root enlargement stage than sub-root formation stage, and it is better to applied the same density at another stage for adjusting the plant morphology.

[Effects of micronutrient fertilizer application on yield and quality of Aconitum carmichaeli].

OBJECTIVE: To study the effects of Fe, Zn, B and Mn fertilizer with different ratio on the yield and quality of Aconitum carmichaeli. METHOD: Field experiment with the uniform design was applied, the yield and the contents of the total alkaloids and diester-alkaloids were measured. RESULT: Fe, Zn, B and Mn fertilizer of appropriate ratio could promote the growth of vegetative organs, increase the biomass, the content of alkaloids and the yield of Aconite significantly. Fe, Zn fertilizer of highly concentrated ratio increased the proportion of first sub-roots, but inhibited the growth of other vegetative organs, the number of roots was less than that with other treatments, so it was not conducive to the formation of production. High concentration of Mn was not conducive to the growth of underground of Aconite, its number of sub-roots was fewer, but the number of third sub-roots was more than that with other treatments, the yield was low. The yield treated with low concentration of B was 10% higher than that with high concentration, and the high concentration of B was not conducive to increase the content of the alkaloids. Among these treatments, The fourth treatment was the optimal combination, of which the volume of sub-roots was the largest and the most homogeneous, the growth of the vegetative organs was better and the accumulation of dry matters was more, the yield of this treatment was 10,754.7 kg x hm(-2), which was increased by 14.9%, and the content of alkaloid was increased by 13.9%. CONCLUSION: The ratio of 4 is the best treatment for high yield and quality cultivation of Aconite.

The relationship between growth of the aerial part and alkaloid content variation in cultivated Aconitum carmichaeli Debeaux.

Processed root of aconite, Aconitum carmichaeli Debeaux--known as bushi in Japan--is indispensable for treating diseases among elderly persons in Japanese and Chinese traditional medicine. Its active component is bushi diester alkaloid (BDA), which consists of aconitine (ACO), mesaconitine (MES), hypaconitine (HYP), and jesaconitine (JES). Since an overdose of BDA results in severe side effects, the BDA content should be within safe limits. However, the BDA content of raw aconite root, even that produced by standard cultivation procedures, varies greatly. In this study, to clarify the cause of BDA variation, we examined the weight and BDA content of each part of cultivated A. carmichaeli: the aerial part, the mother tuberous root (MT), the daughter tuberous root (DT), and the rootlet (RL). We found the following positive relationships: between aerial part weight and DT weight, aerial part weight and BDA content in stem of apex, and BDA content in stem of apex and total BDA of DT attached to the plant. Furthermore, DT belonging to a higher weight group showed less BDA content variation. In addition, BDA of DT and those of MT and RL differ in both content and composition. In conclusion, it was suggested that the weight or the size of the aerial part was a good marker for monitoring BDA content and its variation in the tuberous root, and it was found to be desirable to prevent mixing MT and RL at harvest.

[Investigation and collection development pattern for genuineness of Aconitum carmichali].

OBJECTIVE: To study the collection the development pattern for genuineness of Aconitum carmichalii, which lays a good basis for the sustainable utilization of A. carmichalii resources. METHOD: We adopted the combined methods of investigation of herbal literatures, researching of origins in A. carmichalii and consultation with the experts, identified the development pattern of A. carmichalii. From genuine producing areas and its genuineness. RESULT: The genuine producing area of A. carmichali is Jiangyou district of Sichuan province, genuine medicinal materials of A. carmichalii is mainly oriented by production techniques. It has cultivation techniques, unique and exquisite processing, which have trim root delicately and remove top complexly. CONCLUSION: A. carmichalii from Jiangyou is famous genuine medicinal materials in Sichuan Province. It should strengthen the inheritance and creative research for cultivation techniques, unique and exquisite processing, ensure the safety and effect in medication.

[Evaluation of the accumulation of dry substance and active componentsevaluation of the accumulation of dry substance and active components of Radix Aconiti Lateralis Praeparata].

OBJECTIVE: To study the accumulation rules of dry substance and active components in the Radix Aconiti Lateralis Praeparata for providing the basic evidences to collection time. METHODS: The Roots were collected periodically and their fresh weight and dry weight were measured. The contents of total alkaloids and three diester aconitum alkaloids aconitine, hypaconitine and mesaconitine were determined by titration and HPLC respectively. RESULTS: During the stage of root inflation, the content of total alkaloids was at a certain level, while the content of diester aconitum alkaloids were increased gradually. The total amount of alkaloid and diester aconitom alkaloids were increased rapidly from the last third of April to the middle third of June, and then maintained at relative constant levels and were decreased from the first third of July. CONCLUSION: It is more preferable to collect the Radix Aconiti Lateralis Praeparata during the period from the last third of June to first third of July.

Female reproductive success decreases with display size in monkshood, Aconitum kusnezoffii (Ranunculaceae).

BACKGROUND AND AIMS: Reduction in female fitness in large clones can occur as a result of increased geitonogamous self-fertilization and its influence through inbreeding depression. This possibility was investigated in the self-compatible, bee-pollinated perennial herb Aconitum kusnezoffii which varies in clone size. METHODS: Field investigations were conducted on pollinator behaviour, flowering phenology and variation in seed set. The effects of self-pollination following controlled self- and cross-pollination were also examined. Selfing rates of differently sized clones were assessed using allozyme markers. KEY RESULTS: High rates of geitonogamous pollination were associated with large display size. Female fitness at the ramet level decreased with clone size. Fruit and seed set under cross-pollination were significantly higher than those under self-pollination. The pre-dispersal inbreeding depression was estimated as 0.502 based on the difference in seed set per flower between self- and cross-pollinated flowers. Selfing rates of differently sized clones did not differ. CONCLUSIONS: It is concluded that in A. kusnezoffii the negative effects of self-pollination causing reduced female fertility with clone size arise primarily from a strong early-acting inbreeding depression leading to the abortion of selfed embryos prior to seed maturation.

Allee effects within small populations of Aconitum napellus ssp. lusitanicum, a protected subspecies in northern France.

Plants growing at low density can suffer from Allee effects as a result of pollen limitation. Previous studies of Allee effects have focused on the effects of variation among populations in size or density on reproduction. Here, the effects of plant distribution within populations on fitness components are explored in a rare plant, Aconitum napellus ssp. lusitanicum, and ecological and genetic mechanisms underlying these effects are identified. To detect pollen limitation, seed production was compared under natural versus hand-supplemented pollinations on inflorescences of different sizes in natural patches differing both in flower density and in isolation from other patches. Germination rate and juvenile survival of seeds produced in low- and high-density patches were also compared. Pollen-supplemented flowers always produced more seeds than open-pollinated flowers, especially among small plants and plants growing at low density. Offspring produced in low-density patches exhibited lower fitness that those produced in high-density patches. This could have been caused by post-fertilization mechanisms, including inbreeding depression or differential maternal resource allocation. These results show that Allee effects on fitness components (ecological and genetic Allee effects) occur within A. napellus populations at different spatial scales. The spatial distribution of plants seems to be a crucial factor affecting reproductive output and fitness.

[Study of application fertilizer on Aconitum carmichaeli].

OBJECTIVE: To study N, P, K and organic fertilizer on the action of the yield and quality of Aconitum carmichaeli. METHODS: Four factors quadratic regression rotation combination design and agricultural biological statistics were applicated. RESULTS: The amount and proportion of N, P, K and organic fertilizer had evident influence on the yield and quality of Radix Aconiti Lateralis Preparata. The regression equation really reflected the fact. CONCLUSION: The application of N 13.04 - 15.13 kg, P 38.70 - 44.34 kg, K 22.50 - 26.46kg and organic fertilizer 1900 - 2200 kg per mu can gain high yield (> or =550 kg/mu) at great possibilities in the experiment.

[Establishment of plantlet regeneration system of medicinal plant Aconitum coreanum].

OBJECTIVE: To set up the rapid propagation and plantlet regeneration system of medicinal plant Aconitum coreanum (Levi.) Rpaics. METHOD: Adventitious buds and regenerated plantlet were obtained by shoot tip culture through different media and combination of plant growth regulators. RESULT: MS medium was the optimal medium for differentiation and proliferation of shoot tip. MS medium with 6-BA 2 to approximately 5 mg/L was found to be most suitable for the induction of bud. MS with 6-BA 2 mg/L + GA3 1 to approximately 5 mg/L or adenine 1 to approximately 5 mg/L was best combination for the growth and proliferation of adventitious buds, and MS with IBA 0.5 to approximately 1 mg/L for the induction of root. CONCLUSION: Tip culture may be used as a useful method to get regenerated plantlet of Aconitum coreanum under certain conditions.