Microbial amelioration of salinity stress in endangered accessions of Iranian licorice (Glycyrrhiza glabra L.).

BACKGROUND: Glycyrrhiza glabra L. is a medicinal and industrial plant that has gone extinct due to different abiotic stress caused by climate change. To understand how the plant-associated microorganism can support this plant under salinity, we collected sixteen Iranian accessions of G. glabra L., inoculated their rhizomes with Azotobacter sp. (two levels, bacterial treatments, and no-bacterial treatments, and grown them under salinity stress (NaCl levels; 0, and 200 mM). RESULTS: Two accessions of Bardsir and Bajgah significantly showed higher resistant to salinity, for example by increasing crown diameter (11.05 and 11 cm, respectively) compared to an average diameter of 9.5 in other accessions. Azotobacter inoculation caused a significant increase in plant height and crown diameter. Among studied accessions, Kashmar (46.21%) and Ilam (44.95%) had the highest rate of membrane stability index (MSI). Evaluation of enzyme activity represented that bacterial application under salinity, increased polyphenol oxidase (PPO) (0.21 U mg-1 protein), peroxidase (POD) (3.09 U mg-1 protein U mg-1 protein), and phenylalanine ammonia-lyase (PAL) (17.85 U mg-1 protein) activity. Darab accession showed the highest increase (6.45%) in antioxidant potential compared with all studied accessions under Azotobacter inoculation. According to principal component analysis (PCA), it was found that the accession of Meshkinshahr showed a more remarkable ability to activate its enzymatic defense system under salt stress. Also, three accessions of Meshkinshahr, Eghlid, and Ilam were categorized in separated clusters than other accessions regarding various studied treatments. CONCLUSION: Analysis indicated that five accessions of Meshkinshahr, Rabt, Piranshahr, Bardsir, and Kermanshah from the perspective of induced systematic resistance are the accessions that showed a greater morphophysiological and biochemical outcome under salinity. This study suggested that, inoculation of with Azotobacter on selected accession can relieve salt stress and support industrial mass production under abiotic condition.

Succession of endophytic fungi and arbuscular mycorrhizal fungi associated with the growth of plant and their correlation with secondary metabolites in the roots of plants.

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.

A disposable gold foil paper-based aptasensor for detection of enteropathogenic Escherichia coli with SERS analysis and magnetic separation technology.

Rapid and sensitive detection of enteropathogenic Escherichia coli (EPEC) in fluids with complex background is an important task for safety quality control in the field of medicine, environment, and food. In this study, a gold foil paper-based aptasensor was developed for the detection of enteropathogenic EPEC O26:K60 with surface-enhanced Raman spectroscopy (SERS) and magnetic separation technology mediated by Fe3O4@Au composite. The gold foil paper was firstly modified with thiolated capture probe and SERS tag. The thiolated aptamer probe for EPEC was immobilized onto a Fe3O4@Au composite. In the presence of EPEC, highly specific recognition between the aptamer probe and EPEC made the Fe3O4@Au composite partially dissociated from the gold foil paper. This led to a decreased Raman intensity response, which showed an obvious negative linear correlation with increasing concentration of EPEC over a wide concentration range from 10 to 107 CFU/mL under an excitation wavelength of 633 nm. The detection limit was about 2.86 CFU/mL in a buffer solution and a licorice extractum and the detection time was only 2.5 h. The results demonstrate that the gold foil paper-based aptasensor can be an excellent biosensing platform that offers a reliable, rapid, and sensitive alternative for EPEC detection.

Plant performance of enhancing licorice with dual inoculating dark septate endophytes and Trichoderma viride mediated via effects on root development.

BACKGROUND: This study aimed to assess whether licorice (Glycyrrhiza uralensis) can benefit from dual inoculation by Trichoderma viride and dark septate endophytes (DSE) isolated from other medicinal plants. METHODS: First, we isolated and identified three DSE (Paraboeremia putaminum, Scytalidium lignicola, and Phoma herbarum) and Trichoderma viride from medicinal plants growing in farmland of China. Second, we investigated the influences of these three DSE on the performance of licorice at different T. viride densities (1 × 106, 1 × 107, and 1 × 108 CFU/mL) under sterilised condition in a growth chamber. RESULTS: Three DSE strains could colonize the roots of licorice, and they established a positive symbiosis with host plants depending on DSE species and T. viride densities. Inoculation of P. putaminum increased the root biomass, length, surface area, and root:shoot ratio. S. lignicola increased the root length, diameter and surface area and decreased the root:shoot ratio. P. herbarum increased the root biomass and surface area. T. viride increased the root biomass, length, and surface area. Structural equation model (SEM) analysis showed that DSE associated with T. viride augmented plant biomass and height, shoot branching, and root surface area. Variations in root morphology and biomass were attributed to differences in DSE species and T. viride density among treatments. P. putaminum or P. herbarum with low- or medium T. viride density and S. lignicola with low- or high T. viride density improved licorice root morphology and biomass. CONCLUSIONS: DSE isolated from other medicinal plants enhanced the root growth of licorice plants under different densities T. viride conditions and may also be used to promote the cultivation of medicinal plants.

Root-associated endophytic bacterial community composition and structure of three medicinal licorices and their changes with the growing year.

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.

Actinophytocola glycyrrhizae sp. nov. isolated from the rhizosphere of Glycyrrhiza inflata.

A Gram-stain-positive, aerobic actinomycete, designated strain BMP B8152T, was isolated from the rhizosphere of Glycyrrhiza inflata collected ashore, in Kashi, Xinjiang province, northwest PR China. A polyphasic approach was used to establish the taxonomic position of this strain. BMP B8152T was observed to form non-fragmented substrate mycelium, and relatively scanty aerial mycelium with rod-shaped spores. Cell-wall hydrolysates contained meso-diaminopimelic acid, galactose, arabinose, glucose and rhamnose (trace). Mycolic acids were not detected. The diagnostic phospholipids were identified as diphosphatidylglycerol, phosphatidylethanolamine, hydroxyphosphatidylethanolamine, ninhydrin-positive phosphoglycolipid and phosphatidylinositol. The predominant menaquinone and fatty acid were MK-9(H4) and iso-branched hexadecanoate (iso-C16 : 0), respectively. The phylogenetic analyses based on the 16S rRNA gene sequences indicated that BMP B8152T formed a distinct monophyletic clade clustered with Actinophytocola timorensisID05-A0653T (98.8 % 16S rRNA gene sequence similarity), Actinophytocola oryzaeGMKU 367T (98.6 %), Actinophytocola corallinaID06-A0464T (98.2 %) and Actinophytocola burenkhanensisMN08-A0203T (97.5 %). In addition, DNA-DNA hybridization values between BMP B8152T and A. timorensisID05-A0653T(44.2±3.6 %) and A. oryzaeGMKU 367T(36.7±2.3 %) were well below the 70 % limit for species identification. The combined phenotypic and genotypic data indicate that the isolate represents a novel species of the genus Actinophytocola, for which the name Actinophytocola glycyrrhizae sp. nov., is proposed, with the type strain BMP B8152T (=KCTC 49002T=CGMCC 4.7433T).

[Application of DNA-based electrochemical biosensor in rapid detection of Escherichia coli exist in licorice decoction].

A new method for detection of Escherichia coli exist in licorice decoction was developed by using DNA-based electrochemical biosensor. The thiolated capture probe was immobilized on a gold electrode at first. Then the aptamer for Escherichia coli was combined with the capture probe by hybridization. Due to the stronger interaction between the aptamer and the E. coli, the aptamer can dissociate from the capture probe in the presence of E. coli in licorice decoction. The biotinylated detection probe was hybridized with the single-strand capture probe. As a result, the electrochemical response to Escherichia coli can be measured by using differential pulse voltammetric in the presence of α-naphthyl phosphate. The plot of peak current vs. the logarithm of concentration in the range from 2.7×10² to 2.7×108 CFU·mL⁻¹ displayed a linear relationship with a detection limit of 50 CFU·mL⁻¹. The relative standard deviation of 3 successive scans was 2.5%，2.1%，4.6% for 2×10²ï¼Œ2×104，2×106：6 CFU·mL⁻¹ E. coli, respectively. The proposed procedure showed better specificity to E. coli in comparison to Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis. In the detection of the real extractum glycyrrhizae, the results between the proposed strategy and the GB assay showed high degree of agreement, demonstrating the designed biosensor could be utilized as a powerful tool for microbial examination for traditional Chinese medicine.

Discovery of anti-microbial and anti-tubercular molecules from Fusarium solani: an endophyte of Glycyrrhiza glabra.

AIMS: Glycyrrhiza glabra is a high-value medicinal plant thriving in biodiversity rich Kashmir Himalaya. The present study was designed to explore the fungal endophytes from G. glabra as a source of bioactive molecules. METHODS AND RESULTS: The extracts prepared from the isolated endophytes were evaluated for anti-microbial activities using broth micro-dilution assay. The endophytic strain coded as A2 exhibiting promising anti-bacterial as well as anti-tuberculosis activity was identified as Fusarium solani by ITS-5.8S ribosomal gene sequencing technique. This strain was subjected to large-scale fermentation followed by isolation of its bioactive compounds using column chromatography. From the results of spectral data analysis and comparison with literature, the molecules were identified as 3,6,9-trihydroxy-7-methoxy-4,4-dimethyl-3,4-dihydro-1H-benzo[g]isochromene-5,10-dione (1), fusarubin (2), 3-O-methylfusarubin (3) and javanicin (4). Compound 1 is reported for the first time from this strain. All the four compounds inhibited the growth of various tested bacterial strains with MIC values in the range of <1 to 256 µg ml-1 . Fusarubin showed good activity against Mycobacterium tuberculosis strain H37Rv with MIC value of 8 µg ml-1 , whereas compounds 1, 3 and 4 exhibited moderate activity with MIC values of 256, 64, 32 µg ml-1 , respectively. CONCLUSIONS: To the best of our knowledge, this is the first study that reports significant anti-tuberculosis potential of bioactive molecules from endophytic F. solani evaluated against the virulent strain of M. tuberculosis. This study sets background towards their synthetic intervention for activity enhancement experiments in anti-microbial drug discovery programme. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the chemoprofile variation of same endophyte with respect to source plant and ecoregions, further studies are required to explore endophytes of medicinal plants of all unusual biodiversity rich ecoregions for important and or novel bioactive molecules.

Isolation and antimicrobial activities of actinobacteria closely associated with liquorice plants Glycyrrhiza glabra L. and Glycyrrhiza inflate BAT. in Xinjiang, China.

A total of 218 actinobacteria strains were isolated from wild perennial liquorice plants Glycyrrhiza glabra L. and Glycyrrhiza. inflate BAT. Based on morphological characteristics, 45 and 32 strains from G. inflate and G. glabra, respectively, were selected for further analyses. According to 16S rRNA sequence analysis, most of the strains belonged to genus Streptomyces and a few strains represented the rare actinobacteria Micromonospora, Rhodococcus and Tsukamurella. A total of 39 strains from G. inflate and 27 strains from G. glabra showed antimicrobial activity against at least one indicator organism. The range of the antimicrobial activity of the strains isolated from G. glabra and G. inflate was similar. A total of 34 strains from G. inflate and 29 strains from G. glabra carried at least one of the genes encoding polyketide synthases, non-ribosomal peptide synthetase and FADH2-dependent halogenase. In the type II polyketide synthase KSα gene phylogenetic analysis, the strains were divided into two major clades: one included known spore pigment production-linked KSα sequences and other sequences were linked to the production of different types of aromatic polyketide antibiotics. Based on the antimicrobial range, the isolates that carried different KSα types were not separated from each other or from the isolates that did not carry KSα. The incongruent phylogenies of 16S rRNA and KSα genes indicated that the KSα genes were possibly horizontally transferred. In all, the liquorice plants were a rich source of biocontrol agents that may produce novel bioactive compounds.

Antimicrobial Potential of Thiodiketopiperazine Derivatives Produced by Phoma sp., an Endophyte of Glycyrrhiza glabra Linn.

During the screening of endophytes obtained from Glycyrrhiza glabra Linn., the extract from a fungal culture designated as GG1F1 showed significant antimicrobial activity. The fungus was identified as a species of the genus Phoma and was most closely related to Phoma cucurbitacearum. The chemical investigation of the GG1F1 extract led to the isolation and characterization of two thiodiketopiperazine derivatives. Both the compounds inhibited the growth of several bacterial pathogens especially that of Staphylococcus aureus and Streptococcus pyogenes, with IC50 values of less than 10 µM. The compounds strongly inhibited biofilm formation in both the pathogens. In vitro time kill kinetics showed efficient bactericidal activity of these compounds. The compounds were found to act synergistically with streptomycin while producing varying effects in combination with ciprofloxacin and ampicillin. The compounds inhibited bacterial transcription/translation in vitro, and also inhibited staphyloxanthin production in S. aureus. Although similar in structure, they differed significantly in some of their properties, particularly the effect on the expression of pathogenecity related genes in S. aureus at sub-lethal concentrations. Keeping in view the antimicrobial potential of these compounds, it would be needful to scale up the production of these compounds through fermentation technology and further explore their potential as antibiotics using in vivo models.

Alleviation of Salt Stress and Changes in Glycyrrhizic Acid Accumulation by Dark Septate Endophytes in Glycyrrhiza glabra Grown under Salt Stress.

This study aimed to investigate the mechanisms by which dark septate endophytes (DSE) regulate salt tolerance and the accumulation of bioactive constituents in licorice. First, the salt stress tolerance and resynthesis with the plant effect of isolated DSE from wild licorice were tested. Second, the performance of licorice inoculated with DSE, which had the best salt-tolerant and growth-promoting effects, was examined under salt stress. All isolated DSE showed salt tolerance and promoted plant growth, withCurvularia lunata D43 being the most effective. Under salt stress, C. lunata D43 could promote growth, increase antioxidant enzyme activities, enhance glycyrrhizic acid accumulation, improve key enzyme activities in the glycyrrhizic acid synthesis pathway, and induce the expression of the key enzyme gene and salt tolerance gene of licorice. The structural equation model demonstrated that DSE alleviate the negative effects of salt stress through direct and indirect pathways. Variations in key enzyme activities, gene expression, and bioactive constituent concentration can be attributed to the effects of DSE. These results contribute to revealing the value of DSE for cultivating medicinal plants in saline soils.

Isolation and characterization of three fungi with the potential of transforming glycyrrhizin.

Three fungi with different types of transformation of glycyrrhizin (GL) were isolated from the soil samples of glycyrrhiza glabra planting area in China. According to their morphologies and 18 S rDNA gene sequence analysis, the three fungi were identified and named as Penicillium purpurogenum Li-3, Aspergillus terreus Li-20 and Aspergillus ustus Li-62. Transforming products analysis by TLC and HPLC-MS indicated that P. purpurogenum Li-3, A. terreus Li-20 and A. ustus Li-62 could stably transform GL into GAMG, GAMG and GA, and GA, respectively. P. purpurogenum Li-3 was especially valuable to directly prepare GAMG for applications in the pharmaceutical industry.

[Endophytic bacterial diversity in Glycyrrhiza inflata Bat. from Xinjiang by culture-independent method].

OBJECTIVE: We investigated endophytic bacterial diversity in Glycyrrhiza inflata Bat. from Xinjiang. METHODS: We investigated endophytic bacterial diversity in root of Glycyrrhiza inflata Bat. from Xingjian by culture-independent method. Total DNA genome of Glycyrrhiza sample was extracted using CTAB (Hexadecyl trimethyl ammonium Bromide) procedure with some modifications. A pair of bacterial PCR primers were used for endophytic bacterial 16S rDNA gene amplification and a clone library was constructed for the Glycyrrhiza DNA samples. Clones screened from clone library on the basis of Hae III digestion patterns were sequenced and compared, flowed by constructing Neighbor-Joining tree. RESULTS: In total 150 clones were grouped to 32 operational taxonomic units, most of the clones showed high similarity to the known cultured bacteria. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, gamma subclasses of the Proteobacteria, Bacteroides, Firmicutes, Actinobacteria, and Uncultured bacteria; 74% of the clones were highly related to the known bacteria in the genus Sphingobium, Phyllobacterium, Hyphomonas, Agrobacterium etc (> 96% sequence similarity); whereas 26% of the clones showed lower affiliation with known genus (< 96% sequence similarity) and may represent novel taxa. CONCLUSION: There was abundant endophytic bacterial diversity in Glycyrrhiza inflata Bat. from Xinjiang as well as many unknown organisms.

Community structure, spatial distribution, diversity and functional characterization of culturable endophytic fungi associated with Glycyrrhiza glabra L.

A total of 266 endophytic fungal isolates were recovered from 1019 tissue segments of Glycyrrhiza glabra collected from four different locations in the North-Western Himalayas. The endophytes grouped into 21 genera and 38 different taxa. The host had strong affinity for the genus Phoma, followed by Fusarium. The species richness was highest at the sub-tropical location, followed by the sub-temperate location and the temperate locations, respectively. The tissue specificity of endophytes was also evident. Some endophytes showed potential antimicrobial activity against phyto-pathogens indicating that they may be helpful to the host in evading pathogens. All the endophytic taxa produced the plant growth promoting hormone, indole acetic acid (IAA), though in varying concentrations. None of these endophytes caused any symptoms of disease in co-cultivation with the tissue cultured plants. Further, all the endophytes had a positive influence on the phenolic and flavonoid content of the host. Three endophytes, Stagonosporopsis cucurbitacearum, Bionectria sp. and Aspergillus terreus also increased the host root (rhizome) and shoot growth visibly. Such endophytes are potential candidates for developing endophyte-based technologies for sustainable cultivation and enhanced productivity of G. glabra. This is the first report of community structure and biological properties of fungal endophytes associated with G. glabra.

Optimized QuEChERS Method Combined with UHPLC-MS/MS for the Simultaneous Determination of 15 Mycotoxins in Liquorice.

In our study, a reliable and rapid analytical method for the simultaneous determination of 15 mycotoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, alternariol, agroclavine, citrinin, diacetoxyscirpenol, deoxynivalenol, fumonisin B1, fumonisin B2, ochratoxin A, sterigmatocystin, T-2 toxin, and zearalenone) in liquorice using ultra-HPLC coupled to tandem MS was developed and validated. Due to the complex ingredients in liquorice, we chose a QuEChERS-based extraction procedure as the sample pretreatment. Meanwhile, for the first time, acetate buffer was used to replaced water, which can greatly reduce the concentration of formic acid in acetonitrile, which further reduces the extraction efficiency of impurities. The optimal combination of adsorbents is 150 mg primary secondary amine, 150 mg silica gel, 600 mg octadecylsilane, and 900 mg anhydrous magnesium sulfate. Electrospray ionization in both positive- and negative-ionization modes was applied to detect all the mycotoxins in a single run time of 15 min, with LOQs in the range of 0.125-2.5 µg/kg. The recoveries of determination obtained were in the range of 81.0-104.7%, whereas the analytes could be accurately quantified in the 0.25-625 µg/kg concentration range, with all coefficients being >0.992. Intra- and interday reproducibility were lower than 5.5 and 8.9%, respectively, for all analytical mycotoxins. The validated method was finally applied to screen mycotoxins in 31 batches of real samples collected from drugstores and hospitals in Shanghai, China. Our survey findings show that six mycotoxins were detected, including alternariol, citrinin, deoxynivalenol, fumonisin B1, ochratoxin, and zearalenone, and that the positive rate of mycotoxins was 54.8% in real samples, ranging from 3.37 to 520.6 µg/kg.

Actinobacteria associated with Glycyrrhiza inflata Bat. are diverse and have plant growth promoting and antimicrobial activity.

Many of the plant associated microbes may directly and indirectly contribute to plant growth and stress resistance. Our aim was to assess the plant growth-promoting and antimicrobial activities of actinobacteria isolated from Glycyrrhiza inflata Bat. plants to find strains that could be applied in agricultural industry, for example in reclaiming saline soils. We isolated 36 and 52 strains that showed morphological characteristics of actinobacteria from one year old and three year old G. inflata plants, respectively. Based on 16S rRNA gene sequence analysis, the strains represented ten actinobacterial genera. Most of the strains had plant growth promoting characteristics in vitro, tolerated 200 mM NaCl and inhibited the growth of at least one indicator organism. The eight selected Streptomyces strains increased the germination rate of G. inflata seeds under salt stress. In addition, the four best seed germination promoters promoted the growth of G. inflata in vivo. The best promoters of G. inflata growth, strains SCAU5283 and SCAU5215, inhibited a wide range of indicator organisms, and may thus be considered as promising candidates to be applied in inoculating G. inflata.

Microbial cooperation in the rhizosphere improves liquorice growth under salt stress.

Liquorice (Glycyrrhiza uralensis Fisch.) is one of the most widely used plants in food production, and it can also be used as an herbal medicine or for reclamation of salt-affected soils. Under salt stress, inhibition of plant growth, nutrient acquisition and symbiotic interactions between the medicinal legume liquorice and rhizobia have been observed. We recently evaluated the interactions between rhizobia and root-colonizing Pseudomonas in liquorice grown in potting soil and observed increased plant biomass, nodule numbers and nitrogen content after combined inoculation compared to plants inoculated with Mesorhizobium alone. Several beneficial effects of microbes on plants have been reported; studies examining the interactions between symbiotic bacteria and root-colonizing Pseudomonas strains under natural saline soil conditions are important, especially in areas where a hindrance of nutrients and niches in the rhizosphere are high. Here, we summarize our recent observations regarding the combined application of rhizobia and Pseudomonas on the growth and nutrient uptake of liquorice as well as the salt stress tolerance mechanisms of liquorice by a mutualistic interaction with microbes. Our observations indicate that microbes living in the rhizosphere of liquorice can form a mutualistic association and coordinate their involvement in plant adaptations to stress tolerance. These results support the development of combined inoculants for improving plant growth and the symbiotic performance of legumes under hostile conditions.

Evolution and taxonomy of native mesorhizobia nodulating medicinal Glycyrrhiza species in China.

Previously, 159 bacterial strains were isolated from the root nodules of wild perennial Glycyrrhiza legume species grown on 40 sites in central and north-western China, in which 57 strains were classified as "true symbionts" belonging to the genus Mesorhizobium based on amplified fragment length polymorphism (AFLP) genomic fingerprinting and partial sequences of the 16S rRNA gene [20]. In the present work, the phylogeny of Glycyrrhiza nodulating mesorhizobia was further examined by multilocus sequence analysis (MLSA). The concatenated gene tree of three housekeeping genes (16S rRNA, recA, and rpoB) of 59 strains including the 29 mesorhizobial test strains and 30 type mesorhizobial species, was constructed applying the maximum likelihood method and Bayesian inference. In the concatenated gene tree, the 29 test strains were distributed in seven separate clades. Seventeen test strains clustered with Mesorhizobium tianshanense, Mesorhizobium temperatum, Mesorhizobium muleiense, and Mesorhizobium alhagi with high bootstrap support (BS>85%). Eight test strains did not cluster with any of the described Mesorhizobium species. Based on the results, we proposed these eight test strains might belong to a putative new species of the genus Mesorhizobium. The sequences of three accessory genes (nodA, nodC, and nifH) of the test strains were also analyzed and were compared with those of representatives of the 30 described mesorhizobial species. The results showed that mesorhizobia involved in symbiosis with Glycyrrhiza plants probably have acquired some genetic material from other rhizobia in co-evolution with Glycyrrhiza and other legume species.

Comparative effect of irradiation and heating on the microbiological properties of licorice (Glycyrrhiza glabra L.) root powders.

PURPOSE: To determine the effect of gamma irradiation, steaming and heating on microbial load and chemical composition of licorice root powder. MATERIALS AND METHODS: Powders were exposed to hot air treatment at 60°C for 24 h, steaming at 1.1 bar and about 121°C for 15 min, and irradiation at 10 kGy using a (60)Co source. Microbial load, moisture, ash, and electrical conductivity (EC) values of licorice root powders were evaluated immediately after treatment, and after 12 months of storage. RESULTS: Total aerobic plate count (TAPC) of licorice roots powder (control sample), including coliform, Escherichia coli, and Klebsiella spp. were relatively high. Irradiation reduced the TAPC by 4 logs and total coliform to negative (less than 1 log(10) colony forming units (CFU) g(-1)) Klebsiella spp., E. coli, and Salmonella spp. were not detected (less than 1 log(10) CFU/g) in irradiated sample. Meanwhile, heating and steaming reduced the total count by 1 and 2 logs with slight effect on reducing the total coliforms counts. Also, irradiated samples were free of coliforms. No considerable changes in the moisture and ash were observed in irradiated powders, but the moisture increased due to steaming, and decreased due to dried heating. CONCLUSION: Gamma irradiation at 10 kGy was more effective than heating and steaming treatments in their microbial decontamination effect on the licorice root powders.

Ochratoxin A in liquorice products - a review.

Liquorice is a herbal medicine produced mainly in China and Iran. This plant is suspected to contain ochratoxin A (OTA), a secondary metabolite produced by fungi. Although liquorice is not included in the daily dietary of humans, the high levels of OTA reported in this product have concerned consumers. Registration of a standard method for measuring the amount of this mycotoxin in liquorice-derived products is an important challenge and requires the introduction of a reliable, simple, fast-performance and reproducible technique. This review examines studies carried out concerning the occurrence of OTA in liquorice products. Recent information regarding contaminated liquorice, the regulatory framework and methods to degrade OTA in liquorice are discussed.