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This study aims to evaluate the in vivo function of Fusarium oxysporum in Glycyrrhiza uralensis by salt tolerance,indoleacetic acid(IAA) production capacity, phosphate-dissolving capacity, and iron carrier production capacity. The stable genetic transformation system of the F. oxysporum was established by Agrobacterium tumefaciens-mediated genetic transformation( ATMT)technology, and the stability and staining efficiency of transformants were detected by the cloning of the marker gene green fluorescent protein(GFP) and the efficiency of β-glucuronidase staining(GUS). Efficient and stable transformants were selected for restaining G. uralensis and evaluating its influence on the growth of the G. uralensis seedlings. The results show that F. oxysporum has good salt tolerance and could still grow on potato glucose agar(PDA) medium containing 7% sodium chloride, but the growth rate slows down with the increase in sodium chloride content in PDA medium. F. oxysporum has the function of producing indoleacetic acid, and the concentration of IAA in its fermentation broth is about 3. 32 mg · m L~(-1). In this study, the genetic transformation system of F. oxysporum is successfully constructed, and the ATMT system is efficient and stable. One transformant with both high staining efficiency and genetic stability is selected, and the restaining rate of the transformant in G. uralensis is 76. 92%, which could significantly improve the main root length of one-month-old G. uralensis seedlings and promote the growth and development of G. uralensis seedlings. The results of this study can lay the foundation for the development of biological bacterial fertilizer and the growth regulation of high-quality G. uralensis.

Drought stress has become the primary severe threat to global agriculture production, including medicinal plants. Plant growth-promoting bacteria (PGPB) and environmentally friendly element silicon (Si) have emerged as effective methods in alleviating drought stress in various plants. Here, the effects of the plant endophytic G5 interaction with Si on regulating nitrogen absorption, assimilation, and metabolism pathways were investigated in the morphophysiological and gene attributes of Glycyrrhiza uralensis exposed to drought. Results showed that G5+Si application improved nitrogen absorption and assimilation by increasing the available nitrogen content in the soil, further improving the nitrogen utilization efficiency. Then, G5+Si triggered the accumulation of the major adjustment substances proline, γ-aminobutyric acid, putrescine, and chlorophyll, which played an important role in contributing to maintaining balance and energy supply in G. uralensis exposed to drought. These findings will provide new ideas for the combined application of PGPR and Si on both soil and plant systems in a drought habitat.

Recently, endorhizospheric microbiota is realized to be able to promote the secondary metabolism in medicinal plants, but the detailed metabolic regulation metabolisms and whether the promotion is influenced by environmental factors are unclear yet. Here, the major flavonoids and endophytic bacterial communities in various Glycyrrhiza uralensis Fisch. roots collected from seven distinct places in northwest China, as well as the edaphic conditions, were characterized and analyzed. It was found that the soil moisture and temperature might modulate the secondary metabolism in G. uralensis roots partially through some endophytes. One rationally isolated endophyte Rhizobium rhizolycopersici GUH21 was proved to promote the accumulation of isoliquiritin and glycyrrhizic acid significantly in roots of the potted G. uralensis under the relatively high-level watering and low temperature. Furthermore, we did the comparative transcriptome analysis of G. uralensis seedling roots in different treatments to investigate the detailed mechanisms of the environment-endophyte-plant interactions and found that the low temperature went hand in hand with the high-level watering to activate the aglycone biosynthesis in G. uralensis, while GUH21 and the high-level watering cooperatively promoted the in planta glucosyl unit production. Our study is of significance for the development of methods to rationally promote the medicinal plant quality. KEY POINTS: • Soil temperature and moisture related to isoliquiritin contents in Glycyrrhiza uralensis Fisch. • Soil temperature and moisture related to the hosts' endophytic bacterial community structures. • The causal relation among abiotic factors-endophytes-host was proved through the pot experiment., (© 2023. The Author(s).)

Glycyrrhiza uralensis Fisch is a widely cultivated traditional Chinese medicine plant. In the present study, culture-independent microbial diversity analysis and functional prediction of rhizosphere microbes associated with wild and cultivated G. uralensis Fisch plant (collected from two locations) were carried. Soil physicochemical parameters were tested to assess their impact on microbial communities. A total of 4428 OTUs belonging to 41 bacterial phyla were identified. In general, cultivated sample sites were dominated by Actinobacteria whereas wild sample sites were dominated by Proteobacteria. The alpha diversity analysis showed the observed species number was higher in cultivated soil samples when compared with wild soil samples. In beta diversity analysis, it was noticed that the weighted-unifrac distance of two cultivated samples was closer although the samples were collected from different regions. Functional annotation based on PICRUST and FAPROTAX showed that the nitrogen metabolism pathway such as nitrate reduction, nitrogen fixation, nitrite ammonification, and nitrite respiration were more abundant in rhizosphere microorganisms of wild G. uralensis Fisch. These results also correlate in redundancy analysis results which show correlation between NO 3- -N and wild samples, which indicated that nitrogen nutrition conditions might be related to the quality of G. uralensis Fisch., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: To decipher the root and microbial interaction, secondary metabolite accumulation in roots and the microbial community's succession model during the plant's growth period demands an in-depth investigation. However, till now, no comprehensive study is available on the succession of endophytic fungi and arbuscular mycorrhizal fungi (AMF) with roots of medicinal licorice plants and the effects of endophytic fungi and AMF on the secondary metabolite accumulation in licorice plant's root., Results: In the current study, interaction between root and microbes in 1-3 years old medicinal licorice plant's root and rhizospheric soil was investigated. Secondary metabolites content in licorice root was determined using high-performance liquid chromatography (HPLC). The composition and diversity of endophytic and AMF in the root and soil were deciphered using high-throughput sequencing technology. During the plant's growth period, as compared to AMF, time and species significantly affected the diversity and richness of endophytic fungi, such as Ascomycota, Basidiomycota, Fusarium, Cladosporium, Sarocladium. The growth period also influenced the AMF diversity, evident by the significant increase in the relative abundance of Glomus and the significant decrease in the relative abundance of Diversispora. It indicated a different succession pattern between the endophytic fungal and AMF communities. Meanwhile, distance-based redundancy analysis and Mantel tests revealed root's water content and secondary metabolites (glycyrrhizic acid, liquiritin, and total flavonoids), which conferred endophytic fungi and AMF diversity. Additionally, plant growth significantly altered soil's physicochemical properties, which influenced the distribution of endophytic fungal and AMF communities., Conclusions: This study indicated a different succession pattern between the endophytic fungal and AMF communities. During the plant's growth period, the contents of three secondary metabolites in roots increased per year, which contributed to the overall differences in composition and distribution of endophytic fungal and AMF communities. The endophytic fungal communities were more sensitive to secondary metabolites than AMF communities. The current study provides novel insights into the interaction between rhizospheric microbes and root exudates.

Background: The dried roots and rhizomes of medicinal licorices are widely used worldwide as a traditional medicinal herb, which are mainly attributed to a variety of bioactive compounds that can be extracted from licorice root. Endophytes and plants form a symbiotic relationship, which is an important source of host secondary metabolites., Results: In this study, we used high-throughput sequencing technology and high-performance liquid chromatography to explore the composition and structure of the endophytic bacterial community and the content of bioactive compounds (glycyrrhizic acid, liquiritin and total flavonoids) in different species of medicinal licorices (Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata) and in different planting years (1-3 years). Our results showed that the contents of the bioactive compounds in the roots of medicinal licorices were not affected by the species, but were significantly affected by the main effect growing year (1-3) (P < 0.05), and with a trend of stable increase in the contents observed with each growing year. In 27 samples, a total of 1,979,531 effective sequences were obtained after quality control, and 2432 effective operational taxonomic units (OTUs) were obtained at 97% identity. The phylum Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, and the genera unified-Rhizobiaceae, Pseudomonas, Novosphingobium, and Pantoea were significantly dominant in the 27 samples. Distance-based redundancy analysis (db-RDA) showed that the content of total flavonoids explained the differences in composition and distribution of endophytic bacterial communities in roots of cultivated medicinal liquorices to the greatest extent. Total soil salt was the most important factor that significantly affected the endophytic bacterial community in soil factors, followed by ammonium nitrogen and nitrate nitrogen. Among the leaf nutrition factors, leaf water content had the most significant effect on the endophytic bacterial community, followed by total phosphorus and total potassium., Conclusions: This study not only provides information on the composition and distribution of endophytic bacteria in the roots of medicinal licorices, but also reveals the influence of abiotic factors on the community of endophytic bacteria and bioactive compounds, which provides a reference for improving the quality of licorice.

Glycyrrhiza uralensis Fisch is threatened by over-development and consumption, and therefore, in urgent need of protection. Elicitation is considered to be an effective strategy to enhance the secondary metabolites in plant cell and organ cultures. Secondary metabolite, signal molecules, and gene expression in adventitious roots were studied by HPLC-ESI-MS n , commercially available kits and qRT-PCR method, respectively. In the present study, with the addition of linolenic acid, linoleic acid, and Pichia pastoris, the highest concentration of metabolites was achieved by P. pastoris treatment. The contents of total flavonoids (7.16 mg/g) and polysaccharide (149.76 mg/g) peaked at 100 mg/L of P. pastoris, which increased by 3.09-fold and 3.28-fold compared with the control, respectively. However, the highest concentration of glycyrrhizic acid (0.62 mg/g) and glycyrrhetinic acid (0.29 mg/g) were obtained in 200 mg/L of P. pastoris and which were 3.89-fold and 2.42-fold more than the control group, respectively. ESI-MS n analysis indicated that licoricesaponine B2, licoricesapoine G2, licoricesaponine J2, ononin, uralenin, gancaonin C were only identified in the P. pastoris treatment group. Furthermore, P. pastoris also enhanced accumulation of salicylic acid, jasmonic acid, nitric oxide and activities of antioxidant enzymes involved in the plant defense response. In addition, the transcriptional activity of genes involved in glycyrrhizic acid biosynthesis was significantly increased under the treatment of P. pastoris. The results provided a scientific evidence for the further exploitation of G. uralensis adventitious roots and clinical medication. PRACTICAL APPLICATIONS: This study provided an effective strategy to enhance metabolites by Pichia pastoris treatment in adventitious roots of G. uralensis. The data provide a scientific evidence for the further exploitation of G. uralensis adventitious roots and clinical medication., (© 2019 Wiley Periodicals, Inc.)

Little is known about the composition, diversity, and geographical distribution of bacterial communities associated with medicinal plants in arid lands. To address this, a collection of 116 endophytic bacteria were isolated from wild populations of the herb Glycyrrhiza uralensis Fisch (licorice) in Xinyuan, Gongliu, and Tekesi of Xinjiang Province, China, and identified based on their 16S rRNA gene sequences. The endophytes were highly diverse, including 20 genera and 35 species. The number of distinct bacterial genera obtained from root tissues was higher (n = 14) compared to stem (n = 9) and leaf (n = 6) tissue. Geographically, the diversity of culturable endophytic genera was higher at the Tekesi (n = 14) and Xinyuan (n = 12) sites than the Gongliu site (n = 4), reflecting the extremely low organic carbon content, high salinity, and low nutrient status of Gongliu soils. The endophytic bacteria exhibited a number of plant growth-promoting activities ex situ, including diazotrophy, phosphate and potassium solubilization, siderophore production, auxin synthesis, and production of hydrolytic enzymes. Twelve endophytes were selected based on their ex situ plant growth-promoting activities for growth chamber assays to test for their ability to promote growth of G. uralensis F. and Triticum aestivum (wheat) plants. Several strains belonging to the genera Bacillus (n = 6) and Achromobacter (n = 1) stimulated total biomass production in both G. uralensis and T. aestivum under low-nutrient conditions. This work is the first report on the isolation and characterization of endophytes associated with G. uralensis F. in arid lands. The results demonstrate the broad diversity of endophytes associated with wild licorice and suggest that some Bacillus strains may be promising candidates for biofertilizers to promote enhanced survival and growth of licorice and other valuable crops in arid environments.

A Gram-positive and non-motile actinobacterium, designated strain EGI 60016 T , was isolated from healthy roots of Glycyrrhiza uralensis F. collected from Xinyuan County, Xinjiang Province, China. The 16S rRNA gene sequence of strain EGI 60016 T was found to show 97.5 and 97.3 % sequence similarities to Nesterenkonia rhizosphaerae EGI 80099 T and Nesternkonia massiliensis NP1 T , respectively. The neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that strain EGI 60016 T formed a distinct clade with N. rhizosphaerae EGI 80099 T and N. massiliensis NP1 T . The polar lipids detected for strain EGI 60016 T were diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, an unidentified glycolipid, an unidentified lipid and an unidentified phospholipid. The DNA G+C content was determined to be 64.1 mol%. Other chemotaxonomic features of strain EGI 60016 T included MK-7, MK-8 and MK-9 as the respiratory quinones, and anteiso-C15 : 0 and anteiso-C17 : 0 as the major fatty acids. Based on the results of the phylogenetic analysis supported by morphological, physiological, chemotaxonomic and other differentiating phenotypic characteristics, strain EGI 60016 T is considered to represent a novel species of the genus Nesterenkonia, for which the name Nesterenkonia endophytica sp. nov. is proposed. The type strain is EGI 60016 T (=CCTCC AB 2017176 T =NBRC 112398 T ).

Liquorice (Glycyrrhiza uralensis) is an important medicinal plant for which there is a huge market demand. It has been reported that arbuscular mycorrhizal (AM) symbiosis and drought stress can stimulate the accumulation of the active ingredients, glycyrrhizin and liquiritin, in liquorice plants, but the potential interactions of AM symbiosis and drought stress remain largely unknown. In the present work, we investigated mycorrhizal effects on plant growth and accumulation of glycyrrhizin and liquiritin in liquorice plants under different water regimes. The results indicated that AM plants generally exhibited better growth and physiological status including stomatal conductance, photosynthesis rate, and water use efficiency compared with non-AM plants. AM inoculation up-regulated the expression of an aquaporin gene PIP and decreased root abscisic acid (ABA) concentrations under drought stress. In general, AM plants displayed lower root carbon (C) and nitrogen (N) concentrations, higher phosphorus (P) concentrations, and therefore, lower C:P and N:P ratios but higher C:N ratio than non-AM plants. On the other hand, AM inoculation increased root glycyrrhizin and liquiritin concentrations, and the mycorrhizal effects were more pronounced under moderate drought stress than under well-watered condition or severe drought stress for glycyrrhizin accumulation. The accumulation of glycyrrhizin and liquiritin in AM plants was consistent with the C:N ratio changes in support of the carbon-nutrient balance hypothesis. Moreover, the glycyrrhizin accumulation was positively correlated with the expression of glycyrrhizin biosynthesis genes SQS1, β-AS, CYP88D6, and CYP72A154. By contrast, no significant interaction of AM inoculation with water treatment was observed for liquiritin accumulation, while we similarly observed a positive correlation between liquiritin accumulation and the expression of a liquiritin biosynthesis gene CHS. These results suggested that AM inoculation in combination with proper water management potentially could improve glycyrrhizin and liquiritin accumulation in liquorice roots and may be practiced to promote liquorice cultivation.

This research explored the effects of protein and polysaccharide in Meyerozyma guilliermondii on active compounds in Glycyrrhiza uralensis Fisch adventitious roots. In this study, a responsive protein LSP1 was purified from the Meyerozyma guilliermondii since the excellent induction. The contents of total flavonoids (3.46 mg · g -1 ), glycyrrhizic acid (0.41 mg · g -1 ), glycyrrhetinic acid (0.41 mg · g -1 ), and polysaccharide (94.49 mg · g -1 ) in adventitious root peaked at LSP1 group, which were 1.6, 3.4, 2.4, 2.0-fold that of control, respectively. Besides, the responsive protein LSP1 significantly activated the defense signaling, mitogen-activated protein kinases and extremely up-regulated the expression of defense-related genes and functional genes involved in glycyrrhizic acid biosynthesis., (© 2017 Wiley Periodicals, Inc.)

This study reports the best culture conditions for roots growth and accumulation of active components by optimizing the parameters. Glycyrrhiza uralensis adventitious roots metabolites were significantly increased after adding Saccharomyces cerevisiae and Meyerozyma guilliermondii. The highest contents of polysaccharide, glycyrrhizic acid, glycyrrhetinic acid, and total flavonoids were obtained in M. guilliermondii group; the content of glycyrrhizic acid was 5.3-fold higher than the control. In control and treatment groups, 12 compounds were identified by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS), among which some new compounds have been detected in elicitor groups including 5,7-dihydroxyflavanone, glycyrrhisoflavanone, licorice saponin J2, uralsaponin B, (3R)-vestitol, and uralenol. Meyerozyma guilliermondii significantly upregulated the expression of the genes such as 3-hydroxy-3-methylglutaryl coenzyme A reductase, farnesyl diphosphate synthase, geranyl diphosphate synthase, squalene synthase, squalene epoxidase, β-amyrin synthase, and CYP88D6 and CYP72A154. Meanwhile, it increased the biosynthesis of signaling molecules (nitric oxide, salicylic acid, and jasmonic acid) in defense mechanism., (© 2016 International Union of Biochemistry and Molecular Biology, Inc.)

Objective: To filtrate the effective fermentation products of endophytes from Glycyrrhiza uralensis by comparing expectorant effects., Methods: Ninety Wistar rats with half males and half females were randomly divided into 9 groups( n =10):including normal control group, model group, positive control group, Glycyrrhiza decoction group, 5 groups of the fermentation products of endophytes(JTZB005,JTZB006,JTZB043,JTZB060,JTZB063). The model of phlegm blocking in lung was induced by inhaling SO 2 for 30 minutes and cold wind for 10 minutes twice per day for ten days. Ten days later, 10 ml/kg normal saline was administrated in normal control group and model group, 0.08 mg/kg Fufang Beimu Lvhuaan tablets was administrated in positive control group, 0.95 g/kg Glycyrrhiza water decoction and the fermentation products of endophytes were respectively administrated in Glycyrrhiza decoction group and 5 groups of the fermentation products of endophytes. All groups were given the corresponding dose by intragastric administration once a day for seven days. The changes of rats' general activities were observed during the experimental time. Two hours after the last gavage, rats were sacrificed. The upper lobe of the right lung was removed for HE and immunohistochemical staining. Pathological of lung tissues and the expressions of aquaporin-1(AQP1)and aquaporin-5(AQP5) in lung tissue were measured. In addition, the lung tissue of middle lobe of right lung were accurately weighed, and 10% homogenate were made. The supernatant was removed by centrifugation at 3 000 r/min for ten minutes. The levels of nitric oxide (NO) was determined by nitric acid reductase method. The content of tumor necrosis factor(TNF-α) and intercellular adhesion molecule 1(ICAM-1) and the concentration of cyclooxygenase-2(COX-2) in lung tissue were measured by enzyme-linked immunosorbent(ELISA) method., Results: After modeling,the levels of NO, TNF-α, ICAM-1, and the concentration of COX-2 in lung tissue were increased significantly, the expressions of AQP1 and AQP5 were decreased significantly ( P <0.01). Compared with the model group, the levels of NO,TNF-α,ICAM-1 and the concentration of COX-2 in lung tissue were decreased significantly, and the expressions of AQP1 and AQP5 were increased significantly in Glycyrrhiza decoction group, JTZB005,JTZB006 and JTZB063 group( P <0.05, P <0.01)., Conclusions: By filtrating, JTZB005、JTZB006、JTZB063 have expectorant effects.

In rhizobial species that nodulate inverted repeat-lacking clade (IRLC) legumes, such as the interaction between Sinorhizobium meliloti and Medicago, bacteroid differentiation is driven by an endoreduplication event that is induced by host nodule-specific cysteine rich (NCR) antimicrobial peptides and requires the participation of the bacterial protein BacA. We have studied bacteroid differentiation of Sinorhizobium fredii HH103 in three host plants: Glycine max, Cajanus cajan and the IRLC legume Glycyrrhiza uralensis. Flow cytometry, microscopy analyses and viability studies of bacteroids as well as confocal microscopy studies carried out in nodules showed that S. fredii HH103 bacteroids, regardless of the host plant, had deoxyribonucleic acid (DNA) contents, cellular sizes and survival rates similar to those of free-living bacteria. Contrary to S. meliloti, S. fredii HH103 showed little or no sensitivity to Medicago NCR247 and NCR335 peptides. Inactivation of S. fredii HH103 bacA neither affected symbiosis with Glycyrrhiza nor increased bacterial sensitivity to Medicago NCRs. Finally, HH103 bacteroids isolated from Glycyrrhiza, but not those isolated from Cajanus or Glycine, showed an altered lipopolysaccharide. Our studies indicate that, in contrast to the S. meliloti-Medicago model symbiosis, bacteroids in the S. fredii HH103-Glycyrrhiza symbiosis do not undergo NCR-induced and bacA-dependent terminal differentiation., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Chinese liquorice (Glycyrrhiza uralensis Fish.) is a salt-tolerant medicinal legume that could be utilized for bioremediation of salt-affected soils. We studied whether co-inoculation of the symbiotic Mesorhizobium sp. strain NWXJ19 or NWXJ31 with the plant growth-promoting Pseudomonas extremorientalis TSAU20 could restore growth, nodulation, and shoot/root nitrogen contents of salt-stressed G. uralensis, which was grown in potting soil and irrigated with 0, 50, and 75 mM NaCl solutions under greenhouse conditions. Irrigation with NaCl solutions clearly retarded the growth of uninoculated liquorice, and the higher the NaCl concentration (75 and 100 mM NaCl), the more adverse is the effect. The two Mesorhizobium strains, added either alone or in combination with P. extremorientalis TSAU20, responded differently to the salt levels used. The strain NWXJ19 was a good symbiont for plants irrigated with 50 mM NaCl, whereas the strain NWXJ31 was more efficient for plants irrigated with water or 75 mM NaCl solution. P. extremorientalis TSAU20 combined with single Mesorhizobium strains alleviated the salt stress of liquorice plants and improved yield and nodule numbers significantly in comparison with single-strain-inoculated liquorice. Both salt stress and inoculation raised the nitrogen content of shoots and roots. The nitrogen contents were at their highest, i.e., 30 and 35 % greater compared to non-stressed uninoculated plants, when plants were inoculated with P. extremorientalis TSAU20 and Mesorhizobium sp. NWXJ31 as well as irrigated with 75 mM NaCl solution. From this study, we conclude that dual inoculation with plant growth-promoting rhizobacteria could be a new approach to improve the tolerance of G. uralensis to salt stress, thereby improving its suitability for the remediation of saline lands.

A novel Gram-staining negative, motile, rod-shaped and aerobic bacterial strain, designated EGI 60010(T), was isolated from healthy roots of Glycyrrhiza uralensis F. collected from Yili County, Xinjiang Province, North-West China. The 16S rRNA gene sequence of strain EGI 60010(T) showed 97.2 % sequence similarities with Ochrobactrum anthropi ATCC 49188(T) and Ochrobactrum cytisi ESC1(T), and 97.1 % with Ochrobactrum lupini LUP21(T). The phylogenetic analysis based on 16S rRNA gene sequences showed that the new isolate clustered with members of the genera Ochrobactrum, and formed a distinct clade in the neighbour-joining tree. Q-10 was identified as the respiratory quinone for strain EGI 60010(T). The major fatty acids were summed feature 8 (C18:1 ω6c and/or C18:1 ω7c), C19:0 cyclo ω8c, summed feature 4 (C17:1 iso I/anteiso B) and C16:0. The polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol and phosphatidylcholine. The DNA G+C content of strain EGI 60010(T) was determined to be 60.4 mol%. The genomic DNA relatedness values determined between strain EGI 60010(T) and the closely related strains O. anthropi JCM 21032(T), O. cytisi CCTCC AB2014258(T) and O. lupini NBRC 102587(T) were 50.3, 50.0 and 41.6 %, respectively. Based on the results of the molecular studies supported by its differentiating phenotypic characteristics, strain EGI 60010(T) was considered to represent a novel species within the genus Ochrobactrum, for which the name Ochrobactrum endophyticum sp. nov., is proposed. The type strain is EGI 60010(T) (=CGMCC 1.15082(T) = KCTC 42485(T) = DSM 29930(T)).

In the present study, the culture conditions for the accumulation of Glycyrrhiza uralensis adventitious root metabolites in balloon-type bubble bioreactors (BTBBs) have been optimized. The results of the culture showed that the best culture conditions were a cone angle of 90° bioreactor and 0.4-0.6-0.4-vvm aeration volume. Aspergillus niger can be used as a fungal elicitor to enhance the production of defense compounds in plants. With the addition of a fungal elicitor (derived from Aspergillus niger), the maximum accumulation of total flavonoids (16.12 mg g(-1)) and glycyrrhetinic acid (0.18 mg g(-1)) occurred at a dose of 400 mg L(-1) of Aspergillus niger resulting in a 3.47-fold and 1.8-fold increase over control roots. However, the highest concentration of polysaccharide (106.06 mg g(-1)) was achieved with a mixture of elicitors (Aspergillus niger and salicylic acid) added to the medium, resulting in a 1.09-fold increase over Aspergillus niger treatment alone. Electrospray ionization tandem mass spectrometry (ESI-MS(n)) analysis was performed, showing that seven compounds were present after treatment with the elicitors, including uralsaponin B, licorice saponin B2, liquiritin, and (3R)-vestitol, only identified in the mixed elicitor treatment group. It has also been found that elicitors (Aspergillus niger and salicylic acid) significantly upregulated the expression of the cinnamate 4-hydroxylase (C4H), β-amyrin synthase (β-AS), squalene epoxidase (SE) and a cytochrome P450 monooxygenase (CYP72A154) genes, which are involved in the biosynthesis of bioactive compounds, and increased superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activity.

In order to obtain the fungicides with minimal impact on efficiency of mycorrhizal symbiosis, the effect of five fungicides including polyoxins, jinggangmycins, thiophanate methylate, chlorothalonil and carbendazim on the growth of medicinal plant and efficiency of mycorrhizal symbiosis were studied. Pot cultured Glycyrrhiza uralensis was treated with different fungicides with the concentration that commonly used in the field. 60 d after treated with fungicides, infection rate, infection density, biomass indexes, photosyn- thetic index and the content of active component were measured. Experimental results showed that carbendazim had the strongest inhibition on mycorrhizal symbiosis effect. Carbendazim significantly inhibited the mycorrhizal infection rate, significantly suppressed the actual photosynthetic efficiency of G. uralensis and the most indicators of biomass. Polyoxins showed the lowest inhibiting affection. Polyoxins had no significant effect on mycorrhizal infection rate, the actual photosynthetic efficiency of G. uralensis and the most indicators of biomass. The other three fungicides also had an inhibitory effect on efficiency of mycorrhizal symbiosis, and the inhibition degrees were all between polyoxins's and carbendazim's. The author considered that fungicide's inhibition degree on mycorrhizal effect might be related with the species of fungicides, so the author suggested that the farmer should try to choose bio-fungicides like polyoxins.

A novel Gram-staining-negative, non-motile, rod-shaped and aerobic bacterial strain, designated EGI 60015(T), was isolated from healthy roots of Glycyrrhiza uralensis F. collected from Yili County, Xinjiang Province, Northwest China. The 16S rRNA gene sequence of strain EGI 60015(T) was found to show 97.6% sequence similarity with Novosphingobium pentaromativorans US6-1(T). The phylogenetic analysis based on 16S rRNA gene sequences showed that the strain formed a clade with N. pentaromativorans US6-1(T) in the neighbor-joining tree. Q-10 was identified as the respiratory quinone of strain EGI 60015(T). The major fatty acids were summed feature 8 (C18:1 ω6c and/or C18:1 ω7c; 55.04%), summed feature 4 (C17:1 anteiso B and/or iso I; 18.34%) and summed feature 3 (C16:1 ω6c and/or C16:1 ω7c; 8.53%). The polar lipids detected were phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and sphingoglycolipids. The DNA G+C content of strain EGI 60015(T) was determined to be 66.6 mol%. The genomic DNA relatedness value between EGI 60015(T) and N. pentaromativorans US6-1(T) (54%) was below the 70% limit for species identification. Based on the result of the molecular studies supported by its morphological, physiological, chemotaxonomic and other differentiating phenotypic characteristics, strain EGI 60015(T) was considered to represent a novel species within the genus Novosphingobium, for which the name Novosphingobium endophyticum sp. nov. is proposed. The type strain is EGI 60015(T) (=CGMCC 1.15095(T) = KCTC 42486(T) = DSM 29948(T)).

A novel endophytic actinomycete, designated strain EGI 60009T, was isolated from the roots of Glycyrrhiza uralensis F. collected from Xinjiang Province, north-west China. The isolate was able to grow in the presence of 0-9% (w/v) NaCl. Strain EGI 60009T had particular morphological properties: the substrate mycelia fragmented into rod-like elements and aerial mycelia differentiated into short spore chains. ll-2, 6-Diaminopimelic acid was the cell-wall diamino acid and rhamnose, galactose and glucose were the cell-wall sugars. MK-9(H4) was the predominant menaquinone. The major fatty acids of strain EGI 60009T were iso-C15 : 0, anteiso-C15 : 0, anteiso-C17 : 0, iso-C17 : 0, iso-C17 : 1 and I/anteiso-C17 : 0 B. Mycolic acids were absent. The DNA G+C content of strain EGI 60009T was 70.4 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain EGI 60009T belongs to the family Jiangellaceae and formed a distinct clade in the phylogenetic tree. 16S rRNA gene sequence similarities between strain EGI 60009T and other members of the genera Jiangella and Haloactinopolyspora were 96.1-96.4 and 95.7-96.0%, respectively. Based on these results and supported by morphological, physiological and chemotaxonomic data and numerous phenotypic differences, a novel species of a new genus, Phytoactinopolyspora endophytica gen. nov., sp. nov., is proposed. The type strain of Phytoactinopolyspora endophytica is EGI 60009T ( = KCTC 29657T = CPCC204078T).

Mesorhizobium tianshanense employs MsiA as canavanine exporter, which is upregulated by MsiR, to successfully form a symbiosis with the legume Glycyrrhiza uralensis. In this research, through gel-shift and bacterial two-hybrid examination, MsiR was found to spontaneously form dimers and bind to msiA promoter without additional canavanine. Six truncated forms of MsiR were constructed, and the conserved helix-turn-helix (HTH), substrate-binding, and surface-loop domains were found essential for MsiR functions. Random mutagenesis was used to study the functional sites of MsiR. Seven point mutants were selected, in which three mutants constitutively induced msiA expression without additional canavanine, two mutants partially changed substrate specificity, and the other two were almost null mutants. Results from the site mutation show that the functional subunits (HTH domain, dimerization interface domains, and C-terminal) are important in the conformation and induction ability of MsiR.

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharide (KPS) biosynthesis, is constituted by the rkpU, rkpAGHIJ, and kpsF3 genes. Two mutants in this region affecting the rkpA (SVQ536) and rkpI (SVQ538) genes were constructed. Polyacrylamide gel electrophoresis and (1)H-NMR analyses did not detect KPS in these mutants. RT-PCR experiments indicated that, most probably, the rkpAGHI genes are cotranscribed. Glycine max cultivars (cvs.) Williams and Peking inoculated with mutants SVQ536 and SVQ538 showed reduced nodulation and symptoms of nitrogen starvation. Many pseudonodules were also formed on the American cv. Williams but not on the Asiatic cv. Peking, suggesting that in the determinate nodule-forming S. fredii-soybean symbiosis, bacterial KPS might be involved in determining cultivar-strain specificity. S. fredii HH103 mutants unable to produce KPS or exopolysaccharide (EPS) also showed reduced symbiotic capacity with Glycyrrhiza uralensis, an indeterminate nodule-forming legume. A HH103 exoA-rkpH double mutant unable to produce KPS and EPS was still able to form some nitrogen-fixing nodules on G. uralensis. Thus, here we describe for the first time a Sinorhizobium mutant strain, which produces neither KPS nor EPS is able to induce the formation of functional nodules in an indeterminate nodule-forming legume.

A filamentous fungi strain, Aspergillus parasiticus Speare BGB, producing beta-glucuronidase was screened to transform glycyrrhizinic acid (GL) in liquorice into 18-beta-glycyrrhetinic acid (GA). Under the following cultivate conditions in shake flask, 1% GL (purity 30%), medium capacity 40% of flask, the initial pH value at 4.5, cultivate temperature of 32 degrees C, inoculum size of 5% and culturing time for 96 h the bioconversion ratio of GL into GA could reach 95%. A variety of parameters of submerged state fermentation, including the growth characteristics of A. parasiticus Speare BGB, the change amount of GL and GA, and the activity of beta-glucuronidase, were monitored simultaneously. GA was separated and purified by macroporous resin, silica gel column chromatography followed by recrystalization with the final purity over 98%. Purified product was identified as GA by the infrared absorption spectrum, molecular weight, and nuclear magnetic resonance. This study provided a new and efficient approach of obtaining GA by microbial transformation.

Rhizobia form symbiotic nodules on host legumes and fix nitrogen for their hosts in exchange for nutrients. In order to establish this mutually beneficial relationship, rhizobia must compete with other soil bacteria in the host legume rhizosphere to colonize plant roots efficiently. A promoter-trap transposon screen in Mesorhizobium tianshanense, a Rhizobium that forms nodules on licorice (Glycyrrhiza uralensis) plants revealed that the expression of msiA, which encodes a putative exporter protein belonging to the LysE family of translocators, is activated by both legume exudates and MsiR, a LysR family transcriptional regulator. Chemical analysis suggests that the msiA-inducing signal in exudates is canavanine, an anti-metabolite present in the seeds and exudates of a variety of legume plants. We show that MsiA serves as a canavanine exporter that is indispensable for canavanine resistance in M. tianshanense. We also show that the expression of MsiA homologues in other rhizobial species is induced by canavanine and is critical for canavanine resistance. Furthermore, rhizobial canavanine resistance is important for root hair adherence as well as for survival in a canavanine-producing legume rhizosphere. Together, these data suggest that host legumes may exude specific antimetabolites into their surroundings to optimize the bacterial population in order to have successful symbiotic events with rhizobia.

Sinorhizobium fredii HH103 produces cyclic beta glucans (CG) composed of 18 to 24 glucose residues without or with 1-phosphoglycerol as the only substituent. The S. fredii HH103-Rifr cgs gene (formerly known as ndvB) was sequenced and mutated with the lacZ-gentamicin resistance cassette. Mutant SVQ562 did not produce CG, was immobile, and grew more slowly in the hypoosmotic GYM medium, but its survival in distilled water was equal to that of HH103-Rifr. Lipopolysaccharides and K-antigen polysaccharides produced by SVQ562 were not apparently altered. SVQ562 overproduced exopolysaccharides (EPS) and its exoA gene was transcribed at higher levels than in HH103-Rifr. In GYM medium, the EPS produced by SVQ562 was of higher molecular weight and carried higher levels of substituents than that produced by HH103-Rifr. The expression of the SVQ562 cgsColon, two colonslacZ fusion was influenced by the pH and the osmolarity of the growth medium. The S. fredii cgs mutants SVQ561 (carrying cgs::Omega) and SVQ562 only formed pseudonodules on Glycine max (determinate nodules) and on Glycyrrhiza uralensis (indeterminate nodules). Although nodulation factors were detected in SVQ561 cultures, none of the cgs mutants induced any macroscopic response in Vigna unguiculata roots. Thus, the nodulation process induced by S. fredii cgs mutants is aborted at earlier stages in V. unguiculata than in Glycine max.

LuxR/LuxI-type quorum-sensing systems have been shown to be important for symbiotic interactions between a number of rhizobium species and host legumes. In this study, we found that different isolates of Mesorhizobium tianshanense, a moderately-growing Rhizobium that forms nodules on a number of types of licorice plants, produces several different N-acyl homoserine lactone-like molecules. In M. tianshanense CCBAU060A, we performed a genetic screen and identified a network of regulatory components including a set of LuxI/LuxR-family regulators as well as a MarR-family regulator that is required for quorum-sensing regulation. Furthermore, compared with the wild-type strains, quorum-sensing deficient mutants showed a reduced growth rate and were defective in nodule formation on their host plant Glycyrrhiza uralensis. These data suggest that different M. tianshanense strains may use diverse quorum-sensing systems to regulate symbiotic process.

Mesorhizobium tianshanense is a nitrogen-fixing bacterium that can establish symbiotic associations with Glycyrrhiza uralensis in the form of root nodules. Nodule formation in rhizobia often requires various secreted carbohydrates. To investigate exopolysaccharide (EPS) production and function in M. tianshanense, we performed a genome-wide screen using transposon mutagenesis to identify genes involved in EPS production. We identified seven mutants that produced significantly lower amounts of EPS as well as a two-component sensor kinase/response regulator system that is involved in the activation of EPS synthesis. EPS mutants formed significantly less biofilm and displayed severely reduced nodulation capacity than wild type bacteria, suggesting that EPS synthesis can play important roles in the symbiosis process.

The endophytic strain Mesorhizobium sp. CCNWGX022 was isolated from licorice (Glycyrrhiza uralensis Fisch.) grown in the arid and semi-arid regions of Northwest China. The new stearic acid derived gamma-lactone 1, named rhizobialide (= (5S)-4,5-dihydro-5-(8-oxotetradecyl)furan-2(3H)-one), was isolated from the petroleum-ether extract of the fermentation broth of this strain. The structure of 1 was elucidated on the basis of spectroscopic and mass-spectrometric analysis. This is the first report of this type of compound from rhizobia.

Objective: To study the induction of hairy roots of Glycyrrhiza uralensis and effect of some physical as well as chemical factors on the growth of hairy roots., Method: Hypocotyls and cotyledons of G. uralensis were infected with Agrobacterium rhizogenes LBA9402 and A. rhizogenes R1601, respectively., Result: Hairy roots were induced by two strains of A. rhizogenes, and LBA9402 showed astronger infective ability. The frequency of transformation of hypocotyls was higher than that of cotyledons. Among the different media tested, WP medium yielded the fastest growth rate of hairy roots. Light inhibited the growth of hairy roots. The total amout of 5 flavonoids in hairy roots was 1.5 times as much as that in the callus, among them licochalcone content was 15.5 times as that of callus., Conclusion: The in vitro culture system of the hairy roots established laid a foundation for mass production of flavonoids by hairy root culture.

Objective: To study the dominant seed-borne fungi of Glycyrrihiza seeds which were from different producing area and compare the disinfection effect of several fungicides on seed-borne fungi of Glycyrrihiza seed., Method: Petri-dish testing was used to determine the external and internal seed-borne fungi and the disinfection effect of fungicides., Result and Conclusion: The result showed that the amount of spore on the surface of one Glycyrrihiza seed varied from 0.3% to 37.0% among samples. Penicillium spp. and Aspergillus spp. were the two major dominant fungi and there was few differences in the type of the fungi among producing areas but differences in the isolation frequency of the furgi; Penicillium spp. , Rhizopus spp. , Aspergillus spp. and Alternaria spp. were the internally dominant seed-borne fungi, including seed capsule and the internal tissue of seed and their fungi-carrying percentage was 8.0%-48.3% and 3.5%-42.0% respectively. There were differences in fungi percentage and dominant seed borne fungi among different producing areas. The disinfection effect of both mancozeb and thriam was up to 89.0%, so it is suggested to use them to disinfect Glycyrrihiza seed.

The ability of rhizobia to symbiotically fix nitrogen from the atmosphere when forming nodules on their plant hosts requires various signal transduction pathways. LuxR-LuxI-type quorum-sensing systems have been shown to be one of the players in a number of rhizobium species. In this study, we found that Mesorhizobium tianshanense, a moderate-growth Rhizobium that forms nodules on a number of licorice plants, produces multiple N-acyl homoserine lactone (AHL)-like molecules. A simple screen for AHL synthase genes using an M. tianshanense genomic expression library in Escherichia coli, coupled with a sensitive AHL detector, uncovered a LuxI-type synthase, MrtI, and a LuxR-type regulator, MrtR, in M. tianshanense. Deletions of the mrtI or mrtR locus completely abolished AHL production in M. tianshanense. Using lacZ transcriptional fusions, we found that expression of the quorum-sensing regulators is autoinduced, as mrtI gene expression requires MrtR and cognate AHLs and mrtR expression is dependent on AHLs. Compared with the wild-type strains, quorum-sensing-deficient mutants showed a marked reduction in the efficiency of root hair adherence and, more importantly, were defective in nodule formation on their host plant, Glycyrrhiza uralensis. These data provide strong evidence that quorum sensing plays a critical role in the M. tianshanense symbiotic process.