Bacterial Diversity in Roots, Stems, and Leaves of Chinese Medicinal Plant Paris polyphylla var. yunnanensis.

The root of Paris polyphylla var. yunnanensis, a famous and endangered traditional Chinese herb, has a significant medicinal value. The aim of this study was to analyze the composition and functional characteristics of bacterial endophytes in roots, stems, and leaves of P. polyphylla var. yunnanensis. The 16S rRNA gene sequencing and functional prediction of bacterial endophytes in roots, stems, and leaves of P. polyphylla var. yunnanensis were conducted. The Chao and Shannon indices of the bacteria in roots were significantly higher than those in stems and leaves. The dominant endophyte phyla were Cyanobacteria, Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. The main genera detected in roots were unclassified Cyanobacteria, Rhizobium, Flavobacterium, and Sphingobium; the main genera in stems were norank_c__Cyanobacteria, Bacillus, and Pseudomonas; the main genera in leaves were norank_c__Cyanobacteria and Rhizobium. The microbiota in roots was particularly enriched in functional categories "extracellular structures" and "cytoskeleton" compared with stems and leaves (p < 0.05). Our study reveals the structural and functional characteristics of the endophytic bacteria in roots, stems, and leaves of P. polyphylla var. yunnanensis, which aids in the scientific understanding of this plant.The root of Paris polyphylla var. yunnanensis, a famous and endangered traditional Chinese herb, has a significant medicinal value. The aim of this study was to analyze the composition and functional characteristics of bacterial endophytes in roots, stems, and leaves of P. polyphylla var. yunnanensis. The 16S rRNA gene sequencing and functional prediction of bacterial endophytes in roots, stems, and leaves of P. polyphylla var. yunnanensis were conducted. The Chao and Shannon indices of the bacteria in roots were significantly higher than those in stems and leaves. The dominant endophyte phyla were Cyanobacteria, Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. The main genera detected in roots were unclassified Cyanobacteria, Rhizobium, Flavobacterium, and Sphingobium; the main genera in stems were norank_c__Cyanobacteria, Bacillus, and Pseudomonas; the main genera in leaves were norank_c__Cyanobacteria and Rhizobium. The microbiota in roots was particularly enriched in functional categories "extracellular structures" and "cytoskeleton" compared with stems and leaves (p < 0.05). Our study reveals the structural and functional characteristics of the endophytic bacteria in roots, stems, and leaves of P. polyphylla var. yunnanensis, which aids in the scientific understanding of this plant.

Metabolites from Penicillium sp. Associated with Paris polyphylla.

One new gentisyl alcohol derivative and-seven known compounds were isolated from the culture of Penicillium sp. Their chemical structures were.elucidated by extensive spectroscopic analysis. Compounds 1, 4, and 6 inhibited Bacillus, subtilis, Staphylococcus aureus, and Escherichia coli with MICs of 32-128 µg/mL.

A new agent developed by biotransformation of polyphyllin VII inhibits chemoresistance in breast cancer.

Biotransformation by the endophytes of certain plants changes various compounds, and this 'green' chemistry becomes increasingly important for finding new products with pharmacological activity. In this study, polyphyllin VII (PPL7) was biotransformed by endophytes from the medicinal plant Paris polyphylla Smith, var. yunnanensis. This produced a new compound, ZH-2, with pharmacological activity in vitro and in vivo. ZH-2 was more potent than PPL7 in selectively killing more chemoresistant than chemosensitive breast cancer cells. ZH-2 also re-sensitized chemoresistant breast cancer cells, as evidenced by the improved anti-cancer activity of commonly-used chemotherapeutic agent in vitro, in vivo, and in clinical samples. This anti-chemoresistance effect of ZH-2 was associated with inhibiting the epithelial-mesenchymal transition (EMT) pathway. Taken together, our findings are the first one to link biotransformation with a biomedicine. The results provide insights into developing new pharmacologically-active agents via biotransformation by endophytes.

[Correlation between distribution of rhizospheric microorganisms and contents of steroidal saponins of Paris polyphylla var. yunnanensis].

In this paper, the varying pattern of the amount of rhizospheric microorganisms, including bacteria, actinomycetes and fungus, was observed during the cultivation of Paris polyphylla var. yunnanensis. And the correlations between number of rhizospheric microorganisms and the quality of P. polyphylla var. yunnanensis were also studied. The results showed that the rhizospheric microorganism source of P. polyphylla var. yunnanensis was rich. The distribution of rhizospheric microorganisms (soil bacteria, fungus, actinomycetes, potassium-solubilizing bacteria, inorganic phosphorus-solubilizing bacteria, organic phosphorus-solubilizing bacteria) collected from different origin places existed significant difference (P < 0.05). The varying pattern for the amount of rhizospheric microorganisms was showed as following: the amount of bacteria > the amount of actinomycetes > the amount of fungus. The medicinal quality of P. polyphylla var. yunnanensis was influenced by their habits, and the increase of cultivation years caused the obvious decrease of the quality of P. polyphylla var. yunnanensis. Therefore, the increase of cultivation years will cause the variation of the soil micro-ecology flora, and decrease the nutrient absorption and the utilization of P. polyphylla var. yunnanensis, which will make the decrease of the medical quality of P. polyphylla var. yunnanensis.

[Photosynthetic parameters and physiological indexes of Paris polyphylla var. yunnanensis influenced by arbuscular mycorrhizal fungi].

Through potted inoculation test at room temperature and indoor analysis, the photosynthetic parameters and physiological and biochemical indexes of Paris polyphylla var. yunnanensis were observed after 28 arbuscular mycorrhizal (AM) fungi were injected into the P. polyphylla var. yunnanensis growing in a sterile soil environment. The results showed that AM fungi established a good symbiosis with P. polyphylla var. yunnanensis. The AM fungi influenced the photosynthetic parameters and physiological and biochemical indexes of P. polyphylla var. yunnanensis. And the influences were varied depending on different AM fungi. The application of AM fungi improved photosynthesis intensity of P. polyphylla var. yunnanensis mesophyll cells, the contents of soluble protein and soluble sugar, protective enzyme activity of P. polyphylla var. yunnanensis leaf, which was beneficial to resist the adverse environment and promote the growth of P. polyphylla var. yunnanensis. Otherwise, there was a certain mutual selectivity between P. polyphylla var. yunnanensis and AM fungi. From the comprehensive effect of inoculation, Racocetra coralloidea, Scutellospora calospora, Claroideoglomus claroideum, S. pellucida and Rhizophagus clarus were the most suitable AM fungi to P. polyphylla var. yunnanensis when P. polyphylla var. yunnanensis was planted in the field.

Microbial diversity in Paris polyphylla var. yunnanensis rhizomes of varying ages.

Endophyte microorganisms live inside plants without causing them any apparent damage. Recently, endophytic microorganisms have attracted attention because they can produce bioactive compounds of biotechnological interest. The endophytic microorganisms in Paris polyphylla var. yunnanensis (Liliaceae) - a species used since antiquity in traditional Chinese medicine - are under scrutiny because they may be responsible for producing the bioactive metabolites associated with the plant. The levels of bioactive metabolites in the rhizomes of P. polyphylla increase with rhizome age. To elucidate the roles played by endophytes in the accumulation of bioactive metabolites, we investigated the community structure and diversity of the endophytic microorganisms in P. polyphylla rhizomes of different ages (4, 6, and 8 years) using 16S rRNA and internal transcribed spacer (ITS) sequence analysis. 16S rDNA amplicon pyrosequencing revealed that the number of operational taxonomic units was lower in the 8-year-old samples than in the other samples. A total of 28 phyla were observed in the P. polyphylla samples and the predominant bacteria were of the Cyanobacteria and Proteobacteria phyla. Moreover, the percentage of Cyanobacteria increased with rhizome age. Similarly, ITS1 amplicon pyrosequencing identified developmental changes in the most abundant fungal classes; some classes were more prevalent in the 8-year-old rhizomes than in younger rhizomes, indicating the importance in secondary metabolism in older rhizomes. Our study showed that endophyte microorganism diversity and prevalence depend on P. polyphylla rhizome age. There was also an indication that some endophyte microorganisms contribute to the higher saponin content in older P. polyphylla specimens.

[Effects of mycorrhizal colonization and medicine quality of Paris polyphylla var. yunnanensis inoculated by different foreign AM fungi species].

After 28 foreign species of AM fungi were inoculated in sterilized soil, the effects of the AM mycorrhizal colonization and the medicine quality of Paris polyphylla var. yunnanensis were observed by combination of inoculation test in pot at room temperature and instrumental analysis. The results showed that, compared with control group (CK), the inoculation of foreign AM fungi in the soil influenced the spore density, mycorrhizal infection rate, and colonization intensity of AM fungi in root system of P. polyphylla var. yunnanensis. The inoculation of foreign AM fungi enhanced the mycorrhiza viability of P. polyphylla var. yunnanensis by increasing the activity of succinic dehydrogenase (SDH) and alkaline phosphatase (ALP) in intraradical hyphae. The content of single steroid saponin in rhizome of P. polyphylla var. yunnanensis showed variation after P. polyphylla var. yunnanensis was inoculated by different foreign species of AM fungi, which was beneficial for increasing the medicine quality; however, the kinds of steroid saponin showed no difference. In a degree, there was a selectivity of symbiosis between P. polyphylla var. yunnanensis and foreign AM fungi. And we found that the Claroideoglomus claroideum and Racocetra coralloidea were best foreign AM fungi species for cultivating P. polyphylla var. yunnanensis under field condition.

[Isolation and diversity analyses of endophytic fungi from Paris polyphylla var. yunnanensis].

The paper is aimed at studying the diversity of endophytic fungi community from Paris polyphylla var. yunnanensis, and to provide a scientific basis for the utilization value of the endophytic fungi as bioactive material resources. In the present study, endophytic fungi were isolated from roots, rhizomes and leaves of wild P. polyphylla var. yunnanensis collected from Baoshan, Heqing county and Songming city of Yunnan province, and identified and classified by morphological methods together with its ITS sequence analysis. Seven and forty-nine strains of endophytic fungi were isolated from P. polyphylla var. yunnanensis. They were identified belonging to 41 genus. In these 41 genus, 3 genus exist in root only, 12 genus only exist in rhizome and 8 genus only exist in leaf. There was difference in endophytic fungi isolated from different sample sites. Endophytic fungi diversity from rhizomes of Heqing site was the highest. Endophytic fungi similarity coefficient was low among different sites and tissues. Based on these results, it is reasonable to propose that endophytic fungi of P. polyphylla var. yannanensis from different tissue and different sample sites has a certain difference which is possibly relate to their different habitats, different structure and composition of each tissue.

[Effects of fungal elicitors on the secondary metabolite steroidal saponin in Paris polyphylla var. yunnanensis].

To compare the effects of inoculated or non-inoculated with arbuscular mycorrhizal (AM) fungi on the steroidal saponin component in root of Paris polyphylla var. yunnanensis. By pot experiments, steroid saponin component in root of P. polyphylla var. yunnanensis was determined and compared by HPLC. The results showed there was difference in the effects of different AM fungal on the secondary metabolite steroid saponin in P. polyphylla var. yunnanensis. After elicitors treatment, AM fungal did not change the chemical backgrounds of P. polyphylla var. yunnanensis, but can improve partly the content of chemical compositions in roots. In conclusion, there was selectivity between AM fungal and P. polyphylla var. yunnanensis. Glomus intraradices was the most appropriate strain for inoculation P. polyphylla var. yunnanensis.

[Study on correlations between total saponins content in rhizome or mycorrhizal infection rate of Pairs polyphylla var. yunnanensis and soil factors].

OBJECTIVE: Through correlation and path analysis between total saponins content in rhizome/mycorrhizal infection rate in roots of Pairs polyphylla var. yunnanensis and soil factors, to make an inquiry into the role of soil factors in the quality formation of P. polyphylla var. yunnanensis. METHOD: Tested total saponins in rhizome, mycorrhizal fungal infection rate in root and physical and chemical properties in rhizosphere soil in 25 different growth areas, and statistically analyzed the relationship between total saponins in rhizome/mycorrhizal infection rate in roots of P. polyphylla var. yunnanensis and soil factors by using correlation and path analysis. RESULT: The symbiosis relationship between AM mycorrhizal and roots of P. polyphylla var. yunnanensis were better established under natural condition, of which the infection ratio between 36.41%, 83.37%. There were significantly positive correlation between total saponins content in rhizome and urease activity or alkaline phosphatase activities or organic matter in soil, but there was significantly negative correlation between total saponins content and bulk density. There was significantly positive correlation between AM infection ratio and alkaline nitrogen. Path analysis indicated that total saponins of rhizome mainly affected by alkaline nitrogen in soil rhizosphere, secondly by soil organic matter and soil urease activity. While the mycorrhizal fungal colonization ratio was mainly affected by soil pH, secondly by alkaline nitrogen, urease activity, and available phospherus in soil. CONCLUSION: There is closed relationship between quality formation of P. polyphylla var. yunnanensis and soil factors. Path analysis is better for reflecting the contribution of soil factors to total saponins and mycorrhizal infection ratio.

Effect of Citrus reticulata and Cymbopogon citratus essential oils on Aspergillus flavus growth and aflatoxin production on Asparagus racemosus.

Essential oils extracted from Citrus reticulata and Cymbopogon citratus were tested in vitro against the toxigenic strain of Aspergillus flavus, isolated from the tuberous roots of Asparagus racemosus, used in preparation of herbal drugs. The essential oils completely inhibited the growth of A. flavus at 750 ppm and also exhibited a broad fungitoxic spectrum against nine additional fungi isolated from the roots. Citrus reticulata and Cymbopogon citratus essential oils completely inhibited aflatoxin B(1) production at 750 and 500 ppm, respectively. During in vivo investigation, the incidence of fungi and aflatoxin B(1) production decreased considerably in essential oil-treated root samples. The findings thus indicate possible exploitation of the essential oils as effective inhibitor of aflatoxin B(1) production and as post-harvest fungitoxicant of traditionally used plant origin for the control of storage fungi. These essential oils may be recommended as plant-based antifungals as well as aflatoxin B(1) suppressors in post-harvest processing of herbal samples.

[Screening and identification of steroidal saponins-producing endophytes from Paris polyphylla var. chinensis Franch].

Two endophytic strains SS01 and SS02 with the potential for producing steroidal saponins were isolated from the underground stems of Paris polyphylla var. chinensis Franch. The TLC comparison indicated that there are 3 sports with similar R(f) between the metabolites of SS01 and the saponins of Paris polyphylla var. chinensis Franch. And there are 2 sports with similar R(f) between the metabolites of SS02 and the saponins of Paris polyphylla var. chinensis The and that biochemical SS01 Franch. characteristics of morphology, physiological belonged showed to Enterobacteriaceae and SS02 belonged to Bacillus sp.. The 16S rDNA of SS01 and SS02 were PCR and sequenced. The accessions of GenBank are AY842143 and AY842144, respectively. The two 16S rDNA phylogenetic trees were constructed by comparing with the published 16S rDNA sequences of the relative bacteria species. In the first phylogenetic tree SS01 and Cedecea davisae DSM 4568 was the closest relative with 98.9% sequence similarity, and in the second phylogenetic tree SS02 and Paenibacillus daejeonensis was the closest relative with 97.7% sequence similarity. According to the phylogenetic analysis they were identified as Cedecea davisae SS01 and Paenibacillus daejeonensis SS02, respectively.

High diversity of endophytic fungi from the pharmaceutical plant, Heterosmilax japonica Kunth revealed by cultivation-independent approach.

Heterosmilax japonica Kunth is well recognized for its diuretic effects in China. However, little is known about its endophytic fungi. In this study, microbial communities inhabiting the stems of H. japonica in spring and summer were investigated by light microscopy and cultivation-independent approaches, such as RFLP analysis and sequencing of rDNA ITS library. Molecular phylogenetic analysis showed that a broad spectrum of fungi, including Mycosphaerella, Phomopsis, Aureobasidium, Cladosporium, Glomerella, Botryosphaeria, Guignardia, is able to colonize the plants internally. Particularly, several rDNA sequences determined in this study like YJ4-61 are not specifically affiliated with any currently documented fungal sequences in the public database. Several sequence types, such as YJ4-9 and YJ4-70, are significantly similar to some uncultured environmental samples. Furthermore, our result also showed that the samples collected in spring harbored more abundant endophytic populations than that in summer, implying a seasonal fluctuation for the endophytes in H. japonica.

Response to high-pressure, low-temperature treatment in vegetables: determination of survival rates of microbial populations using flow cytometry and detection of peroxidase activity using confocal microscopy.

Application of high hydrostatic pressure (200, 300, 350 and 400 MPa) at 5 degrees C for 30 min to different micro-organisms, including Gram-positive and Gram-negative bacteria, moulds and yeasts, proved to be more effective in inactivating these organisms than treatments at 20 degrees C for 10 min and at 10 degrees C for 20 min. Moulds, yeasts, Gram-negative bacteria and Listeria monocytogenes were most sensitive, and their populations were completely inactivated at pressures between 300 and 350 MPa. The same conditions of pressure, temperature, and time were applied to different vegetables (lettuce, tomato, asparagus, spinach, cauliflower and onion), achieving reductions of from 2-4 log units in both viable mesophiles and moulds and yeasts at pressures of between 300 and 400 MPa. Sensory characteristics were unaltered, especially in asparagus, onion, tomato and cauliflower, though slight browning was observed in cauliflower at 350 MPa. Flow cytometry was applied to certain of the microbial populations used in the above experiment before and after the pressurization treatment. The results were indicative of differing percentage survival rates depending on micro-organism type, with higher survival rates for Gram-positive bacteria, except L. monocytogenes, than in the other test micro-organisms. Growth of survivors was undetectable using the plate count method, suggesting that micro-organisms suffering from pressure stress were metabolically inactive though alive. The pressurization treatments did not inactivate the peroxidase responsible for browning in vegetables. Confocal microscopic examination of epidermal tissue from onion showed that the enzyme had been displaced to the cell interior. Use of low temperatures and moderately long pressurization times yielded improved inactivation of micro-organisms and better sensorial characteristics of the vegetables, and should lower industrial costs.