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ObjectiveTo investigate the effects of the inoculation of potassium-solubilizing bacteria on the rhizosphere soil microenvironment of Paris polyphylla var. yunnanensis. MethodThe effects of different potassium-solubilizing bacteria on the physical and chemical properties， microbial community structure， and soil enzyme activity in the rhizosphere soil of P. polyphylla var. yunnanensis were investigated by pot planting at room temperature. The correlation of various indexes was analyzed. ResultThe inoculation with different potassium-solubilizing bacteria could significantly affect the physical and chemical properties of rhizosphere soil of P. polyphylla var. yunnanensis. The mass fractions of available nitrogen， available phosphorus， and available potassium were 24.5-90.5 mg·kg-1， 2.53-25.9 mg·kg-1， and 132-312 mg·kg-1， respectively， and the soil pH was 7.08-7.75， which were in line with the optimal ranges of P. polyphylla var. yunnanensis planting. The inoculation of different potassium-solubilizing bacteria could affect the number of bacteria， actinomycetes， and fungi in rhizosphere soil to varying degrees. The transformation of soil from "fungal type" to "bacterial type" marks the improvement of soil fertility. It also affected the enzyme activity of rhizosphere soil， and the activities of neutral phosphatase， protease， and polyphenol oxidase showed an increasing trend. The correlation analysis showed that the number of bacteria was negatively correlated with the number of fungi （r=-0.856， P<0.01）， positively correlated with the number of actinomycetes， the content of available nitrogen and available potassium， and negatively correlated with soil pH. ConclusionThe inoculation of potassium-solubilizing bacteria can effectively improve the content of available potassium， available nitrogen， available phosphorus， and other nutrients in the rhizosphere soil of P. polyphylla var. yunnanensis， improve soil fertility， alleviate the continuous cropping obstacles of P. polyphylla var. yunnanensis， and lay a theoretical foundation for the green and sustainable development of P. polyphylla var. yunnanensis.
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ObjectiveTo study the effect of organophosphate-solubilizing bacteria and compound bacteria on the photosynthesis and physiological and biochemical characteristics of leaves of Paris polyphylla var. yunnanensis， and to provide a reference for selecting suitable bacterial fertilizers in artificial cultivation of this medicinal species. MethodPot experiment was carried out indoor and the following groups were designed： control （CK）， inoculation with Bacillus mycoides （S1）， inoculation with B. wiedmannii （S2）， inoculation with B. proteolyticus （S3）， inoculation with B. mycoides and B. wiedmannii （S4）， inoculation with B. mycoides and B. proteolyticus （S5）， inoculation with B. wiedmannii and B. proteolyticus （S6）， and inoculation with B. mycoides， B. wiedmannii and B. proteolyticus （S7）. Then， the growth and development， photosynthesis， and various physiological and biochemical indexes of the leaves of this species were observed. ResultCompared with CK， the treatment groups showed decrease in content of malondialdehyde in the leaves （P<0.05）， particularly S7 （content was only about 1/3 that of the CK）. The leaf area， photosynthetic parameters， photosynthetic pigment content， soluble sugar content， soluble protein content， and activity of superoxide dismutase （SOD）， peroxidase （POD）， and catalase （CAT） in leaves of the treatment groups were all improved. Among them， the leaf area， soluble sugar content， and soluble protein content were the highest in S7， which were 2.8， 2.1， and 2.2 times that of the CK， respectively. SOD activity peaked in S6 （2.9 times higher than that in the CK） and the highest activity of POD and CAT was detected in S5 （1.5 times and 2.1 times， respectively higher than that in the CK）. ConclusionInoculation with different organophosphate-solubilizing bacteria or compound bacteria can promote the growth and development of P. polyphylla var. yunnanensis and improve its resistance to stresses. The combination of B. mycoides and B. proteolyticus and the combination of the three achieved the have the best effect. This study provides a reference for the selection of bacterial fertilizers for artificial cultivation of P. polyphylla var. yunnanensis.
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ObjectiveThe effect of inoculation with different organophosphate-resolving bacteria or compound bacteria on the quality of Paris polyphylla var. yunnanensis medicinal materials and rhizosphere soil fertility were studied to provide a reference for the development and application of biological bacterial fertilizer in artificial cultivation of P. polyphylla var. yunnanensis. MethodThe three dominant species of organophosphate-solubilizing bacteria were inoculated separately and in combination in sterilized soil by single-factor indoor pot planting， and no inoculation was used as the control group. The effect of inoculation of organophosphate-solubilizing bacteria on total saponins content in rhizomes of P. polyphylla var. yunnanensis， as well as microbial numbers， enzyme activities and nutrient contents in rhizosphere soil were analyzed. ResultIn the seven treatments inoculated with organophosphate-solubilizing bacteria， the total saponin content in the rhizomes of Paris polyphylla var. yunnanensis was increased by 16.42%， 3.83%， 16.86%， 33.69%， 2.11%， 13.44%， and 28.83%， respectively， compared with the control. Inoculation with organophosphate-solubilizing bacteria increased the number of soil microorganisms to varying degrees， and the effects of S6 and S7 treatments were the most significant. Inoculation with organophosphate-solubilizing bacteria improved the enzyme activity， and the effect of S7 treatment was the most significant. The activities of acid phosphatase， neutral phosphatase， alkaline phosphatase， protease， invertase and catalase were 49.96% and 104.67% ， 110.17%， 99.61%， 26.26%， 11.29% higher than those of the control， respectively. Inoculation with organophosphate-solubilizing bacteria reduced the pH of the rhizosphere soil and increased the content of soil available nutrients. Under the S7 treatment， the contents of alkaline hydrolyzable nitrogen， available phosphorus and available potassium in the rhizosphere soil of P. polyphylla var. yunnanensis were 181.46%， 51.64% and 42.62% higher than those of the control， respectively. Correlation analysis showed that there was a significant positive correlation between total saponins and phosphatase activities， a significant positive correlation between soil microorganisms and soil enzyme activities， and a very significant positive correlation between soil nutrients. ConclusionInoculation of different organophosphate-resolving bacteria or compound bacteria can improve the quality and rhizosphere soil fertility of P. polyphylla var. yunnanensis. Among them， the mixed inoculation of three kinds of bacteria and the mixed inoculation of B. mycoides and B. wiedmannii have the best effect.
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ObjectiveTo investigate the effects of inoculation of potassium-solubilizing bacteria on the physiological characteristics of Paris polyphylla var. yunnanensis. MethodThe effects of different potassium-solubilizing bacteria on leaf area，photosynthetic pigment content， and active component indexes of P. polyphylla var. yunnanensis were investigated by pot planting at room temperature. ResultThe results showed that the inoculation of potassium-solubilizing bacteria could increase the leaf area of P. polyphylla var. yunnanensis. The largest average leaf area was observed in the S3 group，reaching 10.497 cm2，with a maximum increase of 11.0% compared with that of the CK group. The inoculation of potassium-solubilizing bacteria could increase the content of photosynthetic pigments in leaves. Among them，the increase in chlorophyll a content was significant （P<0.05），which was 80.6% higher than that of the CK group. The chlorophyll a/b values in all treatment groups were higher than that of the CK group. The activities of superoxide dismutase（SOD），catalase（CAT） and peroxidase（POD） in the leaves of P. polyphylla var. yunnanensis increased by 88.4%，33.8%， and 30.6%，respectively， as compared with that in the CK group. The inoculation of potassium-solubilizing bacteria could promote the accumulation of osmoregulation substances such as soluble sugar and soluble protein in leaf cells of P. polyphylla var. yunnanensis，with a maximum increase of 55.3% and 70.5% respectively compared with that of the CK group. Meanwhile，it also reduced the content of malondialdehyde（MDA），with a maximum decrease of 34.1% compared with that of the CK group. The results of correlation analysis showed that there were some correlations between different chemical components in P. polyphylla var. yunnanensis leaves. ConclusionThe inoculation of potassium-solubilizing bacteria can effectively promote the growth and development of P. polyphylla var. yunnanensis and improve its survival ability in adversity. It is of practical scientific significance and potential application value for improving the survival rate of P. polyphylla var. yunnanensis by artificial planting.
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ObjectiveTo study the effects of different plant growth-promoting rhizobacteria （PGPR） on the growth of Paris polyphylla var. yunnanensis seedlings and the quality of its medicinal parts， in order to provide reference for the cultivation of high-quality P. polyphylla var. yunnanensis. MethodThe pot culture experiment at room temperature and the single-factor completely random design were employed for exploring the effects of five PGPR on physiological characteristics and inorganic elements of P. polyphylla var. yunnanensis. ResultThe results showed that the exogenous inoculation of different PGPR promoted the growth and development of P. polyphylla var. yunnanensis to varying degrees， delayed the senescence of leaves， and improved the medicinal value of new and old rhizomes. Compared with the non-inoculated control， the exogenous inoculation of compound microbial fertilizer （FH） and microbial agent Sanju Guanjin liquid （SJ） enhanced the root vigor， increased the content of photosynthetic pigments and the activities of anti-oxidant enzymes ［superoxide dismutase （SOD）， catalase （CAT）， and peroxidase （POD）］， and reduced the content of malondialdehyde （MDA） in leaves. Their inhibition rates against MDA were 10.46%-39.62% and 20.99%-53.12%， respectively. With the growth of P. polyphylla var. yunnanensis， the inhibition rate against MDA gradually increased， which effectively delayed the senescence of P. polyphylla var. yunnanensis leaves. In addition， the exogenous inoculation of different PGPR promoted the accumulation of nutrient elements in new and old rhizomes， lowered the heavy metal content to varying degrees， and improved the medicinal value of P. polyphylla var. yunnanensis rhizomes. ConclusionFH and SJ have exhibited the best promoting effect on the growth of P. polyphylla var. yunnanensis seedlings and also the best regulatory effect on the medicinal value of P. polyphylla var. yunnanensis rhizomes， which has provided reference for the application and promotion of PGPR in the growth of P. polyphylla var. yunnanensis.
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In this study, a method was established for in-situ visualization of metabolite distribution in the rhizome of Paris polyphylla var. yunnanensis. To be specific, through matrix-assisted laser desorption/ionization-mass spectrometry imaging(MALDI-MSI), the spatial locations of steroidal saponins, amino acids, organic acids, phytosterols, phytoecdysones, nucleosides, and esters in rhizome of the medicinal plant were directly analyzed, and six unknown compounds with differential distribution in rhizome tissues were identified. The specific procedure is as follows: preparation of rhizome tissue section, matrix screening and optimization, and MALDI-MSI analysis. The results showed that the steroidal saponins were mainly distributed in the central, amino acids in epidermis and cortex, low-molecular-weight organic acids in central epidermis, phytosterols in the epidermis and lateral cortex, the phytoecdysones in epidermis and cortex, nucleosides(uneven distribution) in epidermis and cortex, growth hormones around the epidermis and cortex, particularly outside the cortex, and esters in cortex with unobvious difference among different tissues. In this study, the spatial distribution of meta-bolites in the rhizome of P. polyphylla var. yunnanensis was characterized for the first time. The result can serve as a reference for identifying and extracting endogenous metabolites of P. polyphylla var. yunnanensis, exploring the synthesis and metabolism mechanisms of the metabolites, and evaluating the quality of medicinal materials.
Subject(s)
Liliaceae/chemistry , Melanthiaceae , Rhizome/chemistry , Saponins/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
In this study, liquid chromatography-mass spectrometry(LC-MS) and high performance liquid chromatography(HPLC) were employed for qualitative and quantitative analysis of the steroidal saponins in rhizomes of Paris polyphylla var. yunnanensis from three different habitats cultured in vitro, in an attempt to explore whether the rhizomes of the medicinal herb cultured in vitro can synthesize the steroidal saponins, including polyphyllinsⅠ, Ⅱ, and Ⅶ, the quality markers specified in Chinese Pharmacopoeia(2020 edition). A total of 20 steroidal saponins were identified in the rhizomes from Changxin, Yunlong(S1), Fengyi, Dali(S2), and Niujie, Eryuan(S3): parisyunnanoside A and parisyunnanoside D or E, proto-polyphyllin Ⅱ, polyphyllins G and H, polyphyllinsⅠ, Ⅱ, Ⅴ, Ⅵ, and Ⅶ, dioscin, gracillin, prosapogenin A, Tg, isomer of Th, saponin Th, reclinatoside, proto-pairs D, pseudoproto-dioscin, and 23-O-glc-(23S,25R)-spirost-5-en-3β,23α,27-triol-3-O-rha-(1→2)-[ara(1→4)]-glc or 27-O-glc-(23S,25R)-spirost-5-en-3β,27α-diol-3-O-rha-(1→2)-[ara(1→4)]-glc. Among them, polyphyllinsⅠ, Ⅱ, and Ⅶ were detected in the rhizomes from S1, with the mass fraction of 0.109 1%, 0.165 2%, and 0.051 03%, respectively(total 0.325 3%). Polyphyllins Ⅱ and Ⅶ were identified in the rhizomes from S2 with the respective mass fraction of 0.192 2% and 0.074 23% and total content of 0.266 5%. Moreover, polyphyllins Ⅱ and Ⅶ were also found in the rhizomes from S3, which had the mass fraction of 0.207 7% and 0.186 9%, separately, with the total content of 0.394 6%. Thus, steroidal saponins, including the quality makers polyphyllins Ⅰ, Ⅱ, and Ⅶ recorded in Chinese Pharmacopoeia(2020 edition) can be synthesized in rhizomes of Paris polyphylla var. yunnanensis cultured in vitro, but their total content fails to meet the standard(0.60% in Chinese Pharmacopoeia). Therefore, in vitro culture of the Paris polyphylla var. yunnanensis is feasible, but the culture conditions need to be further improved.
Subject(s)
Chromatography, High Pressure Liquid , Liliaceae , Melanthiaceae , Rhizome , SaponinsABSTRACT
The study aiming at exploring the potassium-dissolving capacity of rhizosphere potassium-dissolving bacteria from diffe-rent sources and screen the strains with high potassium-dissolving ability, so as to lay a theoretical foundation for cultivation and quality improvement of Paris polyphylla var. yunnanensis sources. The rhizosphere soil of 10 wild and transplanted species from Yunnan, Sichuan and Guizhou provinces was used as the research object. Potassium-dissolving bacteria were isolated and purified, and their potassium-dissolving capacity was determined by flame spectrophotometry, and identified by physiological, biochemical and molecular biological methods. Twenty-six potassium-dissolving bacteria were purified and 13 were obtained from wild and transplanted strains respectively. It was found through the determination of potassium-dissolving capacity that the potassium-dissolving capacity of 26 strains was significantly different, and the mass concentration of K~+ in the fermentation broth were 1.04-2.75 mg·L~(-1), the mcentration of potassium were 0.01-1.82 mg·L~(-1). The strains were identified as Bacillus, Agrobacterium rhizome and Staphylococcus by physiological, biochemical and 16 S rDNA molecular methods, among them Bacillus amylolyticus(4 strains) was the dominant bacterium of Bacillus. The physiology and biochemistry of rhizosphere potassium-dissolving bacteria in P. polyphylla var. yunnanensis rhizosphere were diffe-rent, and the living environment were different, so the potassium-dissolving capacity also changed. Strain Y4-1 with the highest potassium decomposability was Bacillus amylolytic with a potassium increase of 1.82 mg·L~(-1). The potassium-dissolving ability and the distribution of potassium-dissolving bacteria were different in various habitats. The screening of potassium-dissolving bacteria provided a new strain for the preparation of microbial fertilizer. It is expected that B. amyloidococcus Y4-1 can be used as an ideal strain to cultivate mycorrhizal seedlings of P. polyphylla var. yunnanensis.
Subject(s)
China , Liliaceae , Paenibacillus , Potassium , Rhizosphere , SoilABSTRACT
Objective:To clone the full-length glycosyltransferase genes （<italic>PpUGT</italic>1，<italic>PpUGT</italic>7） related to saponins biosynthesis in <italic>Paris polyphylla</italic> var. <italic>yunnanensis</italic>，and perform bioinformatics analysis，relative expression analysis and prokaryotic expression analysis. Method:Total RNA was isolated from <italic>P. polyphylla </italic>var. <italic>yunnanensis </italic>with use of the Eastep<sup>®</sup> Super Total RNA Extraction Kit and converted to cDNA. Specific primers were designed according to the transcriptome data to clone the full-length gene. Relevant software was then used for bioinformatic analysis of the protein sequences. The relative gene expression levels were detected by real-time fluorescent quantitative polymerase chain reaction （Real-time PCR） and the prokaryotic expression vectors were built to heterologously express recombinant protein in <italic>Escherichia coli.</italic> Result:The open reading frame （ORF） of <italic>PpUGT</italic>1 was 1 827 bp，encoding 608 amino acids，and was predicted as a steroid glycosyltransferase；the ORF of <italic>PpUGT</italic>7 was 1 380 bp，encoding 459 amino acids，and was predicted as a triterpenoid glycosyltransferase. The calculated relative molecular mass of two proteins were 67.6 kDa and 51.3 kDa respectively，and both of them were hydrophilic proteins，no transmembrane domain，no signal peptides，both showing high similarity and conservativeness with homologous sequences. The results of Real-time PCR showed that the expression level of <italic>PpUGT</italic>1 was root>leaf>flower>stem；the expression level of <italic>PpUGT</italic>7 was stem>leaf>flower>root. In addition，PpUGTs proteins were expressed in <italic>E. coli</italic>. in a soluble form. Conclusion:The genes of <italic>PpUGT</italic>1 and <italic>PpUGT</italic>7 were cloned successfully. Real-time PCR showed the genes were expressed differently in different plant organs， and their recombinant proteins were successfully expressed in <italic>Escherichia coli</italic>. This study lays a foundation for functional characterization of PpUGTs and analysis of the biosynthesis pathway of saponins in <italic>Paris polyphylla </italic>var. <italic>yunnanensis</italic>.
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Objective:To study the effects of different arbuscular mycorrhizal（AM） fungi combinations on the growth and quality of<italic> Paris polyphylla</italic> var. <italic>yunnanensis</italic> seedlings，in order to provide reference for the cultivation of high quality <italic>P. polyphylla</italic> var. <italic>yunnanensis</italic>. Method:Through the room temperature pot inoculation test method，nine groups of different AM fungi combinations were inoculated into sterilized soil，and the control group was not inoculated. The effects of different AM fungi combinations on root infection rate，photosynthetic parameters，physiological indexes and chemical components of <italic>P. polyphylla</italic> var. <italic>yunnanensis</italic> seedlings were observed. Result:The spore density in rhizosphere soil，the infection rate and intensity of AM fungi in roots，and the activities of succinate dehydrogenase and alkaline phosphatase in roots of <italic>P. polyphylla</italic> var. <italic>yunnanensis</italic> were significantly increased by inoculating the combination of exogenous AM fungi. Compared with the control group，the contents of chlorophyll，malondialdehyde，soluble sugar，soluble protein content and the activities of antioxidant enzymes in leaves of <italic>P. polyphylla</italic> var. <italic>yunnanensis</italic> were increased in different AM fungi treatment groups. The content of total saponin increased from T1 to T2，decreased rapidly from T2 to T4，and increased at T5. Conclusion:Different mixed AM fungus can form a good symbiotic relationship with the roots of <italic>P. polyphylla</italic> var. <italic>yunnanensis</italic>，improve the mycorrhizal vitality，enhance the resistance of <italic>P. polyphylla</italic> var. <italic>yunnanensis</italic> to adverse environment，promote the growth and development of <italic>P. polyphylla</italic> var. <italic>yunnanensis</italic> and improve the quality of rhizome. According to the comprehensive inoculation effect，the combination of S5，S8 and S9 AM fungus has the best effect，which provides reference value for the application of mycorrhizal biotechnology of <italic>P. polyphylla</italic> var. <italic>yunnanensis</italic>.
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Objective:To explore the effect of natural decay of exogenously added fibrous roots on the growth and development of <italic>Paris polyphylla</italic> var. <italic>yunnanensis </italic>and its medicinal quality. Method:The effects of natural decay of fibrous roots at different amounts on mycorrhizal infection rate， physiological and biochemical indexes， and saponin contents of <italic>P. polyphylla</italic> var. <italic>yunnanensis </italic>were studied in pot culture experiments at room temperature. Result:The results showed that the infection rate of arbuscular mycorrhizal （AM） fungi in the root of <italic>P. polyphylla</italic> var. <italic>yunnanensis </italic>was not significantly affected by different fibrous root treatments， but there were significant differences in infection intensity. The photosynthetic pigment content in the leaves declined significantly with the increase in fibrous root amount， and the total chlorophyll was decreased by 78.7% at most. The contents of soluble protein， soluble sugar and malondialdehyde in the leaves of <italic>P. polyphylla</italic> var. <italic>yunnanensis </italic>showed an overall upward trend. The activities of the three protective enzymes varied. The peroxidase and malondialdehyde were reduced by 181.6% and 200.0% at most. In the root system of <italic>P. polyphylla </italic>var. <italic>yunnanensis</italic>， the contents of the above-mentioned six components decreased to varying degrees， with the largest reductions of peroxidase and malondialdehyde reaching 44.6% and 69.7%. Different fibrous root treatments resulted in a decrease in active component content of <italic>P. polyphylla </italic>var. <italic>yunnanensis</italic>. The total content of the four saponins was decreased by 58.9% at most， and the total saponin content by 46.9%. Conclusion:The natural decay of fibrous roots affects the soil microbial environment of root system， reduces the photosynthetic pigment content in leaves， and destroys the stability of cells， thus interfering with the growth and development of <italic>P. polyphylla </italic>var. <italic>yunnanensis</italic>， reducing its medicinal components， and causing continuous cropping obstacles.
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The wild resources of Paris polyphylla var. yunnanensis, a secondary endangered medicinal plant, are severely scarce. Introduction and cultivation can alleviate market demand. To screen phosphatolytic bacteria in the rhizosphere soil of P. polyphylla var. yunnanensis and provide data support for the development of high-efficiency microbial fertilizer, in this study, the dilution plate coating method was used to isolate and screen the phosphorus solubilizing bacteria with the ability of mineralizing organic phosphorus from the rhizosphere soil of wild and transplanted varieties of P. polyphylla var. yunnanensis in 10 different locations in Yunnan, Sichuan and Guizhou. After separation and purification, the phosphatolytic capacity was analyzed by qualitative and quantitative analysis. Combined with physiological and biochemical experiments, the strains were identified using 16 S rDNA sequencing analysis. Forty one strains were selected from the rhizosphere soil of P. polyphylla var. yunnanensis from 10 different habitats. Among them, 21 strains were obtained from the rhizosphere soil of the wild variety P. polyphylla var. yunnanensis and 20 strains were obtained from the rhizosphere soil of the transplanted variety. And significance analysis found that 41 organophosphate solubilizing strains had significant differences in their ability to solubilize phosphorus. The amount of phosphate solubilizing was 0.08-67.61 mg·L~(-1), the pH value was between 4.27 and 6.82. The phosphatolytic amount of strain Y3-5 was 67.61 mg·L~(-1), and the phosphorus increase amount was 57.57 mg·L~(-1). All 41 strains were identified as Gram-positive Bacillus. Combining physiological characteristic and phylogenetic trees, Bacillus mobilis Y3-5 was finally selected as the candidate rhizosphere phosphatolytic bacteria of P. polyphylla var. yunnanensis. The distribution of phosphorus solubilizing bacteria in the rhizosphere soil of P. polyphylla var. yunnanensis was different, and there were significant diffe-rences in phosphorus solubility. Organophosphate-dissolving strain Y3-5 is expected to be a candidate strain of P. polyphylla var. yunnanensis microbial fertilizer.
Subject(s)
Bacillus , Bacteria/genetics , China , Liliaceae , PhylogenyABSTRACT
Objective: To investigate the relationship between basic nutrient, the activity of soil enzyme and the quality of rhizome in the rhizosphere soil of wild and transplanted Paris polyphylla var. yunnanensis, and select the primal soil factors affecting the active components of P. polyphylla var. yunnanensis, and provide the reference for the rational fertilization of P. polyphylla var. yunnanensis. Methods: A total of 33 samples of rhizome and soil collected from P. polyphylla var. yunnanensis in Yunnan, Guizhou and Sichuan were used as materials. The basic nutrient, the activity of soil enzyme and the quality of rhizome of the samples were measured. On this basis, the correlation between the quality of rhizome and soil factor was analyzed. The primal soil factors affecting the quality of rhizome of P. polyphylla var. yunnanensis were also selected by using the method of stepwise regression analysis. Results: There were some differences in the contents of the total saponins, the total polysaccharides and the total flavonoids between the samples of wild and transplanted P. polyphylla var. yunnanensis, and there were also regional characteristics in quality between them. In all the rhizosphere soil samples of P. polyphylla var. yunnanensis, the pH of the soil was moderate, the basic nutrient was sufficient and rich, and the activity of soil enzyme was high, which were suitable for the growth and development of P. polyphylla var. yunnanensis. And the soil fertility quality of the wild P. polyphylla var. yunnanensis was better than the transplanted. The content of total saponins in the wild P. polyphylla var. yunnanensis was significantly positively correlated with the activity of polyphenol oxidase, and the content of total polysaccharides was significantly negatively correlated with the pH of the rhizosphere soil and the activity of urease. The content of total saponins in the transplanted P. polyphylla var. yunnanensis was negatively correlated with the content of available potassium in rhizosphere soil, the content of total flavonoids was significantly positively correlated with the activity of sucrase, and the content of total polysaccharides was significantly positively correlated with the activity of alkaline phosphatase. Conclusion Based on the traditional evaluation, the quality of the transplanted P. polyphylla var. yunnanensis samples is equal to the wild samples, they can be used as the same in the market. The quality of P. polyphylla var. yunnanensis is mainly affected by the soil factors. In the process of protecting wild P. polyphylla var. yunnanensis and cultivating P. polyphylla var. yunnanensis, it is need to be careful to increase and decrease the content and the ratio of soil nutrient according to the actual conditions.
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@#Six steroidal saponins were isolated from the n-butanol extract of 90% ethanol extract of the aerial parts of Paris polyphylla var. yunnanensis by silica gel for column chromatography, MCI column chromatography and preparative high performance liquid chromatography (HPLC). According to the physicochemical properties and spectral data, they were identified as 26-O-β-D-glucopyranosyl-kryptogenin-3-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosy-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (1), dioseptemloside G (2), polyphylloside III (3), chonglouoside SL-19 (4), protodioscin (5), chonglouoside SL-5 (6). Among these, compound 1 was a new compound, and compound 2 was obtained from Paris plants for the first time. The platelet aggregation activities and cytotoxicities of the above compounds were evaluated. The results showed that none of the isolated compounds showed significant platelet aggregation activity; compound 2 and 4 exhibited strong cytotoxicity against human colon adenocarcinoma cell line HT29.
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Objective:To study the effects of different arbuscular mycorrhizal （AM） fungi combinations on rhizosphere soil physicochemical properties and microbial quantity in the seedlings of Paris polyphylla var. yunnanensis at different inoculation periods，so as to lay a foundation for cultivating high-quality P. polyphylla var. yunnanensis. Method:The spore density，infection rate，nutrient and enzyme activity in the soil around the roots of P. polyphylla var. yunnanensis seedlings under different AM fungi combinations and different inoculation periods were analyzed by the greenhouse pot inoculation trials and soil agrochemical analysis methods. Result:The infection rate of different AM fungi treatment groups was more than 80% in different inoculation periods，and the spore density was higher than control （CK） group in some periods. It reflected that the relationship between AM fungi and roots of Paris polyphylla seedlings was favorable. The content of nitrogen in the soil decreased，but the content of available P，available K and soil pH increased. The soil nutrients in the cultivar one-year seedlings and wild seedlings were higher. The total number of soil microorganisms showed that bacteria>actinomycetes>fungi. The cultivar two-year seedlings with AM fungi combinations of S3，S5 and S8 had better soil structure and higher biomass carbon content. The growth rates of phosphatase and protease activity were higher in the soil，but catalase activity was lowest. In the treatment groups S2，S3，S4，S5 and S6，the soil enzyme activities of the wild seedlings and one-year-old seedlings were best. Conclusion:Different AM fungal treatment groups and different inoculation periods had certain effects on rhizosphere soil physicochemical properties and microbial quantity in the seedlings of P. polyphylla var. yunnanensis， which provided a technical basis for the cultivation of P. polyphylla var. yunnanensis.
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Objective:To investigate the effects of arbuscular mycorrhiza（AM） fungi inoculation on the rhizosphere soil of Paris polyphylla var. yunnanensis under field conditions，so as to provide a reference for the standardized cultivation and development of high-quality varieties of P. polyphylla var. yunnanensis. Method:The effects of inoculation with mycorrhizal fungi on the rhizosphere soil structure of P. polyphylla var. yunnanensis were observed through a combination of small-scale field inoculation test and laboratory analysis. Soil indexes were determined by soil agrochemical methods. Result:The treatment groups inoculated with exogenous AM fungi showed a regulatory effect on the infection rate and intensity of AM fungi infection in the root system of P. polyphylla var. yunnanensis. After treatment with AM fungi，the soil pH was basically not affected，and the contents of organic matter，available nitrogen，available phosphorus，and available potassium increased. Moreover，the number of fungi decreased，the number of bacteria and actinomycetes increased，and soil enzyme activities increased. The results of correlation analysis showed that there was a significant positive correlation among the soil physical and chemical indexes，especially the bacterial number and the three types of phosphatases showed extremely significant correlation （r=0.849，0.800，0.804，P<0.01）. Conclusion:The application of the two mixed fungicides could increase the number of microorganisms and enzyme activities in the rhizosphere of P. polyphylla var. yunnanensis， and there was a certain synergy effect among the soil factors. Among the three field trials，the effects in Anshun，Guizhou and Wanzhou，Chongqing were more ideal，which provided a theoretical and practical basis for large-scale promotion of P. polyphylla var. yunnanensis in the field.
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Objective:To investigate the effect of different arbuscular mycorrhizal （AM） fungi combinations on the rhizospheric environment of Paris polyphylla var. yunnanensis. Method:The different combinations of 12 arbuscular mycorrhizal （AM） fungi species were inoculated to the seedlings P. polyphylla var. yunnanensis planted in the sterilized soil under the condition of room temperature to investigate their infection abilities and effects on the root activity，soil nutrient contents，enzyme activities and microbial community structure of P. polyphylla var. yunnanensis rhizospheric environment. Result:The inoculation of exogenous AM fungi can regulate the spore densities and infection rate of P. polyphylla var. yunnanensis rhizosphere AM to improve the root activity， the exogenous AM fungi can also regulate the nutrient contents in the rhizosphere soil，increase the contents of total glomalin and easily extracted glomalin，increase the abilities of P. polyphylla var. yunnanensis to absorb the available N，P and K，and increase the enzyme activities in the rhizosphere soil， improve the microbial community structure， and improve the rhizospheric environment of P. polyphylla var. yunnanensis by increasing the bacteria/fungi and bacteria/actinomycetes quantity ratios and reducing the fungi/actinomycetes quantity ratio. Conclusion:Different AM fungal treatment groups had certain effects on the physicochemical properties and microbial community structure of the rhizosphere soil of P.polyphylla var. yunnanensis，which provided a technical basis for the cultivation of P. polyphylla var. yunnanensis.
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Objective:To clarify the effect of the arbuscular mycorrhizal （AM） fungi on the rhizosphere soil nutrient content，AM fungi infection rate and total rhizome saponins content of Paris polyphylla var. yunnanensis under symbiosis culture. Method:The changes in the root AM fungi infection rate，rhizosphere soil nutrient content，total rhizome saponins content of P. polyphylla var. yunnanensis and the relationship of the rhizosphere soil factors，the infection rate and the total rhizome saponins content after AM fungi inoculation were analyzed by the method of combining room temperature pot inoculation and data analysis. Result:As compared with the CK group，the root AM fungi infection rate of the AM inoculation group was significantly enhanced （P<0.05），the content of easily extractable glomalin，total glomalin，and total nitrogen increased significantly，while available potassium content and pH significantly decreased. After inoculation with AM fungi，the contents of total phosphorus，available phosphorus，available nitrogen，ammonium nitrogen，nitrate nitrogen，available potassium，and organic matter in the rhizosphere soil of P. polyphylla var. yunnanensis showed significant differences as compared with the CK group. The soil nutrient status was improved，and the total saponin content in the rhizome of P. polyphylla var. yunnanensis was increased. Conclusion:Inoculation with AM fungi can improve the rhizosphere soil nutrient status of P. polyphylla var. yunnanensis，promote the nutrient transformation in the rhizosphere soil，promote the growth of P. polyphylla var. yunnanensis，and improve the quality of medicinal herbs.
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Objective: Saponin is the primary effective component in Paris polyphylla var. yunnanensis. The aims of this study are to obtain the full-length cDNA sequence of a key gene (i.e. MVD) in the saponin synthesis pathway and to analyze its bioinformatics and expression in different tissues. Methods Conserved fragments of MVD cDNA in P. polyphylla var. yunnanensis were obtained by homologous cloning, and its full-length cDNA sequence was acquired by RACE, which was temporarily named PpMVD. Bioinformatics analysis was then conducted and the expression of PpMVD in different tissues of P. polyphylla var. yunnanensis was detected by real-time fluorescence quantitative PCR. Results The total length of PpMVD cDNA was 1 583 bp, and the length of ORF was 1 269 bp, which encoding 422 amino acids. The molecular weight of this protein was 46 820, isoelectric point was 5.69, and the instability index was 45.40. It contained 159 alpha-helix (accounted for 37.68%), 19 β-pleated sheet (accounted for 4.50%), 69 extended chains (accounted for 16.35%), 175 irregular curly (accounted for 41.47%) in the secondary structure of PpMVD protein which had no transmembrane helical region and signal peptide. This gene contained a conserved structural domain of MVD1-Superfamily which belonged to GHMP kinase protein family. The qRT-PCR results showed that the expression of PpMVD gene was significantly different in seed, leaves, stems, and tubers, i.e. tubers > leaf > seed > stem. The highest expression wasobserved in tuber, which was about 3.3 times of that in stem. Conclusion In this study, we first cloned the full-length PpMVD cDNA sequence, and found that it was highly expressed in tuber, which was the major medicinal organ of P. polyphylla var. yunnanensis. This study laid a foundation for the further study on the effect of PpMVD gene on the saponin biosynthesis, and facilitated the efficient and sustainable utilization of P. polyphylla var. yunnanensis.
ABSTRACT
Objective: To study the effects of different inoculation periods on seedling growth and steroidal saponin content of Paris polyphylla var. yunnanensis, in order to lay a foundation for cultivating and domesticating high-quality Paris polyphylla var. yunnanensis. Methods: The content of steroidal saponins I, II, VI, and VII in the different vaccination periods of Paris polyphylla var. yunnanensis was determined by HPLC. Meanwhile, trypan blue, UV spectrophotometry and so on were adopted to explore colonization rate, colonization intensity, physiological and biochemical indexes, rhizome biomass of the roots of Paris polyphylla var. yunnanensis. Results: Infection rate of AM fungi was higher in different inoculation periods, activities of protective enzymes, photosynthetic pigments and soluble sugars were increased, soluble protein was not changed, content of MDA was decreased, stress resistance of the seedlings of Paris polyphylla var. yunnanensis was improved, and the growth and development were good. Quality of the cultivar one-year seedlings (recovered in August 2015, T7) was relatively low, and cultivar one-year seedlings (harvested in June or July 2015, T5 and T6) and cultivar two years seedlings (recovered in August 2015, T8) were the best. S2, S3 and S6 treatment groups had better effect in different AM fungi mixed groups. Conclusion: Seedling growth and steroidal saponin content of Paris polyphylla var. yunnanensis inoculated by different inoculation periods had certain effects.