Endophytic Bacillus pumilus G5 Interacting with Silicon to Improve Drought Stress Resilience in Glycyrrhiza uralensis Fisch. by Modulating Nitrogen Absorption, Assimilation, and Metabolism Pathways.

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.

Bacillus cereus G2 alleviate salt stress in Glycyrrhiza uralensis Fisch. by balancing the downstream branches of phenylpropanoids and activating flavonoid biosynthesis.

The salinity environment is one of the biggest threats to Glycyrrhiza uralensis Fisch. (G. uralensis) growth, resulting from the oxidative stress caused by excess reactive oxygen species (ROS). Flavonoids are the main pharmacodynamic composition and help maintain ROS homeostasis and mitigate oxidative damage in G. uralensis in the salinity environment. To investigate whether endophytic Bacillus cereus G2 can improve the salt-tolerance of G. uralensis through controlling flavonoid biosynthesis, the transcriptomic and physiological analysis of G. uralensis treated by G2 in the saline environment was conducted, focused on flavonoid biosynthesis-related pathways. Results uncovered that salinity inhibited flavonoids synthesis by decreasing the activities of phenylalanine ammonialyase (PAL) and 4-coumarate-CoA ligase (4CL) (42% and 39%, respectively) due to down-regulated gene Glyur000910s00020578 at substrate level, and then decreasing the activities of chalcone isomerase (CHI) and chalcone synthase (CHS) activities (50% and 42%, respectively) due to down-regulated genes Glyur006062s00044203 and Glyur000051s00003431, further decreasing isoliquiritigenin content by 53%. However, salt stress increased liquiritin content by 43%, which might be a protective mechanism of salt-treated G. uralensis seedlings. Interestingly, G2 enhanced PAL activity by 27% whereas reduced trans-cinnamate 4-monooxygenase (C4H) activity by 43% which could inhibit lignin biosynthesis but promote flavonoid biosynthesis of salt-treated G. uralensis at the substrate level. G2 decreased shikimate O-hydroxycinnamoyltransferase (HCT) activity by 35%, increased CHS activity by 54% through up-regulating the gene Glyur000051s00003431 encoding CHS, and increased CHI activity by 72%, thereby decreasing lignin (34%) and liquiritin (24%) content, but increasing isoliquiritigenin content (35%), which could mitigate oxidative damage and changed salt-tolerance mechanism of G. uralensis.

Bacillus cereus G2 Facilitates N Cycle in Soil, Further Improves N Uptake and Assimilation, and Accelerates Proline and Glycine Betaine Metabolisms of Glycyrrhiza uralensis Subjected to Salt Stress.

Soil salinity is a severe abiotic stress that reduces crop productivity. Recently, there has been growing interest in the application of microbes, mainly plant-growth-promoting bacteria (PGPB), as inoculants for saline land restoration and plant salinity tolerance. Herein, the effects of the plant endophyte G2 on regulating soil N cycle, plant N uptake and assimilate pathways, proline and glycine betaine biosynthesis, and catabolic pathways were investigated in Glycyrrhiza uralensis exposed to salinity. The results indicated that G2 improved the efficiency of N absorption and assimilation of plants by facilitating soil N cycling. Then, G2 promoted the synthesis substrates of proline and glycine betaine and accelerated its synthesis rate, which increased the relative water content and reduced the electrolyte leakage, eventually protecting the membrane system caused by salt stress in G. uralensis. These findings will provide a new idea from soil to plant systems in a salinity environment.

Plant-beneficial Streptomyces dioscori SF1 potential biocontrol and plant growth promotion in saline soil within the arid and semi-arid areas.

Environmental challenges like salinity, drought, fungal phytopathogens, and pesticides directly or/and indirectly influence the environment and agricultural yields. Certain beneficial endophytic Streptomyces sp. can ameliorate environmental stresses and be utilized as crop growth promoters under adverse conditions. Herein, Streptomyces dioscori SF1 (SF1) isolated from seeds of Glycyrrhiza uralensis tolerated fungal phytopathogens and abiotic stresses (drought, salt, and acid base). Strain SF1 showed multifarious plant growth promotion characteristics, including the production of indole acetic acid (IAA), ammonia, siderophores, ACC deaminase, extracellular enzymes, the ability of potassium solubilization, and nitrogen fixation. The dual plate assay showed that strain SF1 inhibited 63.21 ± 1.53%, 64.84 ± 1.35%, and 74.19 ± 2.88% of Rhizoctonia solani, Fusarium acuminatum, and Sclerotinia sclerotiorum, respectively. The detached root assays showed that strain SF1 significantly reduced the number of rotten sliced roots, and the biological control effect on sliced roots of Angelica sinensis, Astragalus membranaceus, and Codonopsis pilosula was 93.33%, 86.67%, and 73.33%, respectively. Furthermore, the strain SF1 significantly increased the growth parameters and biochemical indicators of adversity in G. uralensis seedlings under drought and/or salt conditions, including radicle length and diameter, hypocotyl length and diameter, dry weight, seedling vigor index, antioxidant enzyme activity, and non-enzymatic antioxidant content. In conclusion, the strain SF1 can be used to develop environmental protection biological control agents, improve the anti-disease activity of plants, and promote plant growth in salinity soil within arid and semi-arid regions.

Eco-friendly bio-encapsulation from sodium alginate-trehalose-kaolin and its performance evaluation in improving plant growth under salt or/and drought conditions.

Plant growth-promoting bacterium plays a significant role in improving plant tolerance to abiotic stresses. However, there are low survival and poor effect in field application, especially in unfavorable environments. Our previous study suggested that encapsulation of Bacillus pumilus G5 from polyvinyl alcohol­sodium alginate could improve plant growth and soil fertility under drought and salt soil conditions. However, in the G5 microbeads, the polyvinyl alcohol could not be degraded after entering the soil, and the loss of viable bacteria was severe during the drying process. Achieving a more eco-friendly and efficient formulation based on biodegradable polymers can have significant effects on increasing the quantity and quality of agricultural products. Herein, G5 has immobilized in the composite wall of sodium alginate-trehalose-kaolin microbeads and then evaluated the performance, and applied on the Pharbitis nil under salt or/and drought stress by pot experiment. A 2 % sodium alginate, 1 % trehalose, and 1 % kaolin formulation for the coating films resulted in optimal G5 microbeads embedding efficiency, viable bacteria, degradation rate, and sustained release. Also, the G5 microbeads exhibited longer storage life than that of the G5 suspension. Scanning electron microscopy revealed that the G5 microcapsules had a near-spherical structure with a particle size of around 1000 µm forming a continuous dense composite wall membrane with obvious protrusions and folds on the surface, which facilitated the release of the G5 strain. The interior of the G5 capsule was rough and suitable for bacterial attachment. Infrared spectroscopy showed that the G5 microcapsules are a simple physical mixture with no chemical reaction between the excipients, making the G5 microcapsules chemically stable. The inclusion of the G5 microcapsules considerably induced Pharbitis nil seedlings growth and biomass under drought and/or salt stress. In the rhizosphere soil of Pharbitis nil, the G5 microcapsules increased the total cultivable bacteria population, the activities of invertase, urease, phosphatase, and catalase, and the contents of available nitrogen and available phosphorus. We concluded that a suitable formulation by bio-encapsulation with eco-friendly excipients for alleviating drought and/or salt stress in plants will be advantageous in sustainable agriculture.

Comprehensive physiological, transcriptomic, and metabolomic analyses reveal the synergistic mechanism of Bacillus pumilus G5 combined with silicon alleviate oxidative stress in drought-stressed Glycyrrhiza uralensis Fisch.

Glycyrrhiza uralensis Fisch. is often cultivated in arid, semi-arid, and salt-affected regions that suffer from drought stress, which leads to the accumulation of reactive oxygen species (ROS), thus causing oxidative stress. Plant growth-promoting bacteria (PGPB) and silicon (Si) have been widely reported to be beneficial in improving the tolerance of plants to drought stress by maintaining plant ROS homeostasis. Herein, combining physiological, transcriptomic, and metabolomic analyses, we investigated the response of the antioxidant system of G. uralensis seedlings under drought stress to Bacillus pumilus (G5) and/or Si treatment. The results showed that drought stress caused the overproduction of ROS, accompanied by the low efficiency of antioxidants [i.e., superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), the ascorbate (AsA)-glutathione (GSH) pool, total carotenoids, and total flavonoids]. Inversely, supplementation with G5 and/or Si enhanced the antioxidant defense system in drought-stressed G. uralensis seedlings, and the complex regulation of the combination of G5 and Si differed from that of G5 or Si alone. The combination of G5 and Si enhanced the antioxidant enzyme system, accelerated the AsA-GSH cycle, and triggered the carotenoid and flavonoid metabolism, which acted in combination via different pathways to eliminate the excess ROS induced by drought stress, thereby alleviating oxidative stress. These findings provide new insights into the comparative and synergistic roles of PGPB and Si in the antioxidant system of plants exposed to drought and a guide for the application of PGPB combined with Si to modulate the tolerance of plants to stress.

Physio-biochemical and transcriptomic analysis reveals that the mechanism of Bacillus cereus G2 alleviated oxidative stress of salt-stressed Glycyrrhiza uralensis Fisch. seedlings.

Salt stress severely affects the growth and productivity of Glycyrrhiza uralensis. Our previous research found that the endophyte Bacillus cereus G2 alleviated the osmotic and oxidative stress in G. uralensis exposed to salinity. However, the mechanism is still unclear. Here, a pot experiment was conducted to analyse the change in parameters related to osmotic adjustment and antioxidant metabolism by G2 in salt-stressed G. uralensis at the physio-biochemistry and transcriptome levels. The results showed that G2 significantly increased proline content by 48 %, glycine betaine content by 75 % due to activated expression of BADH1, and soluble sugar content by 77 % due to upregulated expression of α-glucosidase and SS, which might help to decrease the cell osmotic potential, enable the cell to absorb water, and stabilize the cell's protein and membrane structure, thereby alleviating osmotic stress. Regarding antioxidant metabolism, G2 significantly decreased malondialdehyde (MDA) content by 27 %, which might be ascribed to the increase in superoxide dismutase (SOD) activity that facilitated the decrease in the superoxide radical (O2‾) production rate; it also increased the activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GPX), which helped stabilize the normal level of hydrogen peroxide (H2O2). G2 also increased glutathione (GSH) content by 65 % due to increased glutathione reductase (GR) activity and GSH/GSSG ratio, but G2 decreased oxidized glutathione (GSSG) content by 13 % due to decreased activity of dehydroascorbate reductase (DHAR), which could provide sufficient substrates for the ascorbate-glutathione (AsA-GSH) cycle to eliminate excess H2O2 that was not cleared in a timely manner by the antioxidant enzyme system. Taken together, G2 alleviated osmotic stress by increasing proline, soluble sugar, and glycine betaine contents and alleviated oxidative stress by the synergistic effect of antioxidant enzymes and the AsA-GSH cycle. Therefore, the results may be useful for explaining the mechanism by which endophyte inoculation regulates the salt tolerance of crops.

Bacillus cereus Enhanced Medicinal Ingredient Biosynthesis in Glycyrrhiza uralensis Fisch. Under Different Conditions Based on the Transcriptome and Polymerase Chain Reaction Analysis.

The aim of this study was to evaluate the effect of Bacillus cereus (B. cereus) on the seedling growth and accumulation of medicinal ingredients of Glycyrrhiza uralensis Fisch. (G. uralensis) under control and salt stress conditions. Our results revealed the different effects of B. cereus on the seedling growth and accumulation of medicinal ingredients particularly in different conditions based on the transcriptome and polymerase chain reaction (PCR) analysis. Under the control condition, B. cereus significantly increased the expression level of the ß-AS, SQS, CHS, LUS, UGAT, CYP72A154, and CYP88D6 genes and liquiritigenin content. Under salt stress, B. cereus significantly increased root length and lateral root number of G. uralensis seedlings, the expression level of HMGR, ß-AS, CHS, LUS, UGAT, CYP72A154, CYP88D6, and SE genes, and the contents of glycyrrhizic acid and glycyrrhetinic acid. Notably, the effect of B. cereus on the seedling growth and the medicinal ingredient biosynthesis was different under control and salt stress conditions. Specifically, the effect of B. cereus on the seedling growth under salt stress was greater than that under the control condition. Moreover, B. cereus increased liquiritigenin content under the control condition, which is closely related to flavone and flavonol biosynthesis, while it increased the contents of glycyrrhizic acid and glycyrrhetinic acid under salt stress, which is closely related to phenylpropanoid biosynthesis, and the MVA pathway is also involved. All in all, endophytes B. cereus could be used as a sustainable tool to develop effective bioinoculants to enhance the contents of medicinal ingredients in G. uralensis.

Encapsulation of Bacillus pumilus G5 from polyvinyl alcohol­sodium alginate (PVA-SA) and its implications in improving plant growth and soil fertility under drought and salt soil conditions.

Drought and salt stresses adversely affect the growth and yield of plants in agricultural production. Bacillus pumilus, an important plant growth-promoting bacterium, play a significant role in improving plant tolerance to abiotic stresses. In this study, B. pumilus G5 were immobilized in polyvinyl alcohol­sodium alginate (PVA-SA) microbeads and then applied on the Pharbitis nil under drought and salt stresses by pot experiment. Orthogonal array experiments showed that the optimal immobilization conditions of PVA-SA immobilized G5 microbeads were adsorbent 6.0%, PVA: SA 1:1 (3.0%), CaCl2 4.0%, and bacterium: embedding agent (PVA-SA) 3:4; And the G5 microbeads produced at the optimal condition exhibited better cultivable bacteria count, encapsulation rate, expansion rate and mechanical strength. Pot experiment showed that G5 microbeads significantly increased the length and diameter of root and stem, and dry weight of P. nil during experimental stage under drought and salt stress. G5 microbeads also increased the total cultivable bacteria population, the activities of invertase (INV), urease (URE), phosphatase (PHO) and catalase (CAT), and the contents of available nitrogen (AN) and available phosphorus (AP) in the rhizosphere soil of P. nil. Therefore, our study obtained the optimal process of G5 microbeads, and confirmed its effect on improved plant growth and soil chemical and biological properties of P. nil. Thus it can be used as sustainable tool for eco-friendly bio-inoculants at salinity soil within arid and semi-arid areas.

Bacillus pumilus improved drought tolerance in Glycyrrhiza uralensis G5 seedlings through enhancing primary and secondary metabolisms.

It has been reported that drought stress adversely affects the growth and yield of Glycyrrhiza uralensis, Chinese liquorice, in agricultural production. Bacillus pumilus, an important plant growth-promoting bacterium, play a significant role in improving plant tolerance to abiotic stress. However, the role of Bacillus pumilus G5 in resisting drought stress is largely unknown. In the present study, we found that drought stress significantly inhibited the growth and reduced the biomass of G. uralensis seedlings by restraining C- and N-metabolism, while this could be effectively reversed by B. pumilus G5 inoculation. Specifically, B. pumilus G5 significantly increased the content of primary metabolites such as soluble sugar, soluble protein, and free amino acids by regulating the C and N metabolic processes in G. uralensis seedlings. Moreover, B. pumilus G5 increased the content of glycyrrhizic acid, one of the important secondary metabolites, likely mediated through the increased content of primary metabolites and by recovering the expression of three key enzymes, HMGR, SQS, and ß-AS, in the biosynthesis of glycyrrhizic acid. Interestingly, the regulating effect of B. pumilus G5 inoculation on promoting the accumulation of glycyrrhizic acid and increasing the expression of synthesis-related genes is spatially selective. In summary, our findings suggest that B. pumilus G5 could alleviate adverse effects induced by drought stress on the growth of G. uralensis seedlings by regulating C- and N-metabolisms that further triggered the accumulation of secondary metabolites, and this finally improved the drought tolerance of cultivated G. uralensis seedlings.

Physiological Biochemistry-Combined Transcriptomic Analysis Reveals Mechanism of Bacillus cereus G2 Improved Salt-Stress Tolerance of Glycyrrhiza uralensis Fisch. Seedlings by Balancing Carbohydrate Metabolism.

Salt stress severely threatens the growth and productivity of Glycyrrhiza uralensis. Previous results found that Bacillus cereus G2 enhanced several carbohydrate contents in G. uralensis under salt stress. Here, we analyzed the changes in parameters related to growth, photosynthesis, carbohydrate transformation, and the glycolysis Embden-Meyerhof-Parnas (EMP) pathway-tricarboxylic acid (TCA) cycle by G2 in G. uralensis under salt stress. Results showed that G2 helped G. uralensis-accumulating photosynthetic pigments during photosynthesis, which could further increase starch, sucrose, and fructose contents during carbohydrate transformation. Specifically, increased soluble starch synthase (SSS) activity caused to higher starch content, which could induce α-amylase (AM) and ß-amylase (BM) activities; increased sucrose content due to the increase of sucrose synthase (SS) activity through upregulating the gene-encoding SS, which decreased cell osmotic potential, and consequently, induced invertase and gene-encoding α-glucosidase that decomposed sucrose to fructose, ultimately avoided further water loss; increased fructose content-required highly hexokinase (HK) activity to phosphorylate in G. uralensis, thereby providing sufficient substrate for EMP. However, G2 decreased phosphofructokinase (PFK) and pyruvate kinase (PK) activities during EMP. For inducing the TCA cycle to produce more energy, G2 increased PDH activity that enhanced CA content, which further increased isocitrate dehydrogenase (ICDH) activity and provided intermediate products for the G. uralensis TCA cycle under salt stress. In sum, G2 could improve photosynthetic efficiency and carbohydrate transformation to enhance carbohydrate products, thereby releasing more chemical energy stored in carbohydrates through the EMP pathway-TCA cycle, finally maintain normal life activities, and promote the growth of G. uralensis under salt stress.

The roles of methyl jasmonate to stress in plants.

Plants are constantly exposed to various stresses, which can degrade their health. The stresses can be alleviated by the application of methyl jasmonate (MeJA), which is a hormone involved in plant signalling. MeJA induces synthesis of defensive compounds and initiates the expression of pathogenesis-related genes involved in systemic acquired resistance and local resistance. Thus, MeJA may be used against pathogens, salt stress, drought stress, low temperature, heavy metal stress and toxicities of other elements. The application of MeJA improves growth, induces the accumulation of active compounds, and affects endogenous hormones levels, and other physiological and biochemical characteristics in stressed plants. Furthermore, MeJA antagonises the adverse effects of osmotic stress by regulating inorganic penetrating ions or organic penetrants to suppress the absorption of toxic ions. MeJA also mitigates oxidative stress by activating antioxidant systems to scavenge reactive oxygen species (ROS) in stressed plants. For these reasons, we reviewed the use of exogenous MeJA in alleviating biotic (pathogens and insects) and abiotic stresses in plants.

Silicon improves salt tolerance of Glycyrrhiza uralensis Fisch. by ameliorating osmotic and oxidative stresses and improving phytohormonal balance.

Si has a beneficial effect on improving plant tolerance to salt stress. Nevertheless, the mechanisms of Si in mediating the stress responses are still poorly understood. Glycyrrhiza uralensis Fisch. (G. uralensis), a well-known medicinal plant, possesses vast therapeutic potentials. In the present study, a pot experiment was conducted to investigate the long-term effects of Si on growth and physiobiochemical characteristics in 2-year-old G. uralensis subjected to different levels of salinity. Si markedly affected G. uralensis growth in a salt concentration-dependent manner and had no effect on G. uralensis growth under 6 g/kg NaCl. However, it partly reversed the reduction effect induced by 9 g/kg NaCl. In addition, Si significantly increased the contents of soluble sugar and protein but deceased proline content and thus increased water relations; Si markedly increased the activities of SOD, peroxidase, and CAT and further resulted in decreased MDA content and membrane permeability. Moreover, Si altered the levels of phytohormones and their balances. With correlation analysis and principal component analysis (PCA), root biomass had a significant negative correlation with MDA and membrane permeability while a positive correlation with indole-3-acetic acid and GA3. The PCA partitioned the total variance into three PCs contributing maximum (88.234%) to the total diversity among the salt stress with or without Si due to the study of various traits. In conclusion, Si exerts a beneficial property on salt-induced harmful effects in G. uralensis by relieving osmotic stress, improving water relations, and alleviating oxidative stress; thus, altering the levels and balance of phytohormones results in improved growth of salt-stressed G. uralensis.

Silicon alleviates salt and drought stress of Glycyrrhiza uralensis seedling by altering antioxidant metabolism and osmotic adjustment.

This study was conducted to determine effect and mechanism of exogenous silicon (Si) on salt and drought tolerance of Glycyrrhiza uralensis seedling by focusing on the pathways of antioxidant defense and osmotic adjustment. Seedling growth, lipid peroxidation, antioxidant metabolism, osmolytes concentration and Si content of G. uralensis seedlings were analyzed under control, salt and drought stress [100 mM NaCl with 0, 10 and 20% of PEG-6000 (Polyethylene glycol-6000)] with or without 1 mM Si. Si addition markedly affected the G. uralensis growth in a combined dose of NaCl and PEG dependent manner. In brief, Si addition improved germination rate, germination index, seedling vitality index and biomass under control and NaCl; Si also increased radicle length under control, NaCl and NaCl-10% PEG, decreased radicle length, seedling vitality index and germination parameters under NaCl-20% PEG. The salt and drought stress-induced-oxidative stress was modulated by Si application. Generally, Si application increased catalase (CAT) activity under control and NaCl-10% PEG, ascorbate peroxidase (APX) activity under all treatments and glutathione (GSH) content under salt combined drought stress as compared with non-Si treatments, which resisted to the increase of superoxide radicals and hydrogen peroxide caused by salt and drought stress and further decreased membrane permeability and malondialdehyde (MDA) concentration. Si application also increased proline concentration under NaCl and NaCl-20% PEG, but decreased it under NaCl-10% PEG, indicating proline play an important role in G. uralensis seedling response to osmotic stress. In conclusion, Si could ameliorate adverse effects of salt and drought stress on G. uralensis likely by reducing oxidative stress and osmotic stress, and the oxidative stress was regulated through enhancing of antioxidants (mainly CAT, APX and GSH) and osmotic stress was regulated by proline.

[Effect of Water Extracts from Rhizosphere Soil of Cultivated Astragalus membranaceus var. mongholicus on It's Seed Germination and Physiological Characteristics].

OBJECTIVE: To explore the relationship between continuous cropping obstacle and autotoxicity of Astragalus membranaceus var. mongholicus. METHODS: Distilled water(CK), water extracts of rhizosphere soil(50, 100, 200 and 400 mg/mL) were applied to test their effect on early growth and physiological characteristics of Astragalus membranaceus var. mongholicus. RESULTS: The water extracts from rhizospher soil of cultivated Astragalus membranaceus var. mongholicus significantly increased seedling emergence rate, root length and vigor index of Astragalus membranaceus var. mongholicus seedling when at the concentration of 100 mg/mL or below, however,there was no significant effect at 200 mg/mL or higher. The water extracts from rhizosphere soil of cultivated Astragalus membranaceus var. mongholicus significantly reduced the SOD activity in Astragalus membranaceus var. mongholicus seedling at 400 mg/mL and POD activity at 200 mg/mL and 400 mg/mL,while significantly increased the MDA content. CONCLUSION: Water extracts from Astragalus membranaceus var. mongholicus rhizosphere soil significantly affected Astragalus membranaceus var. mongholicus germination and seedling growth in a concentration-dependent manner, generally, low concentrations increased the SOD and POD activity which improved seed germination and seedling growth, while high concentrations caused cell membrane damage of the seedling.

[Effect of drought stress on growth and physiological-biochemical characteristics of Stellaria dichotoma].

A pot experiment was conducted to study effect of drought stress on leaf physiological characteristics and growth of one year old Stellaria dichotoma seedlings. The result showed that plant height and shoot dry weight significantly decreased with decrease in soil water content; however, root length and root dry weight increased at light drought stress and decreased at severe drought stress. The result also showed that with the decrease of soil water content, proline content in S. dichotoma leaves decreased then increase, while solube protein content decreased. Activities of SOD and POD in S. dichotoma leaves significantly decreased as soil water content decreased, while activity of CAT significantly decreased at severe drought stress. Membrane permeability in S. dichotoma leaves increased, while MDA content decreased then increased as soil water decreased. These results suggest that S. dichotoma had osmotic stress resistance ability and reactive oxygen scavenging capacity at light drought stress, which caused S. dichotoma growth was no inhibited at a certain extent drought stress.

[Autotoxicity of aqueous extracts from plant of cultivated Astragalus membranaceus var. mongholicus].

OBJECTIVE: To exploring the relationship between continuous cropping obstacle and autotoxicity of Astragalus membranaceus var. mongholicus, autotoxic effect of plant aqueous extract were determined. METHODS: Distilled water (CK), aqueous extract of plant, including root, stem and leaf (12.5, 25, 50 and 100 mg/mL respectively)were applied to testing their effect on early growth of Astragalus membranaceus var. mongholicus. Specifically, seed germination rate, germination index, emergence rate, elongation of radical and embryo, and seedling vigor index were determined. RESULTS: The aqueous extract of root, stem, and leaf at 25 mg/mL significantly inhibited the seed germination and seedling growth of Astragalus membranaceus var. mongholicus, and this inhibitory effect generally increased with the increase of the concentration of aqueous extracts. To the comprehensive allelopathic effect, the extracts from Astragalus membranaceus var. mongholicus stem were more inhibitory than those from leaf and root. The germination index and seedling vigor index were more sensitive to extract than other determined parameters. CONCLUSION: Aqueous extracts from Astragalus membranaceus var. mongholicus plant gave inhibitory effects on Astragalus. membranaceus var. mongholicus germination and seedling growth, and this inhibitory effect generally increased with the increases of aqueous extract concentration at a certain ranges. In conclusion, there is an autotoxicity in continuous cropping of Astragalus membranaceus var. mongholicus.

[Construction and immunogenicity analysis of the attenuated recombinant Salmonella typhimurium strains expressing Echinococcus granulosus Eg95 antigen].

OBJECTIVE: To study the feasibility of using attenuated Salmonella typhimurium as carrier for oral immunization of Eg95 antigen of Echinococcus granulosus. METHODS: The recombinant plasmid pYA3341-Eg95 was constructed by inserting the Eg95 gene into expression vector pYA3341, and identified by the methods of PCR and enzyme digestion. The recombinant plasmid pYA3341-Eg95 was electro-transformed into attenuated S. typhimurium strains X3730 and X4550 one by one to construct the recombinant strain St-Eg95. The expression of recombinant Eg95 protein in the recombinant strains St-Eg95 was analyzed by Western blotting. The strains of St-Eg95 were passaged 10 times in vitro and the recombinant plasmids were extracted at one generation interval. The genetic stability of recombinant plasmids was identified by PCR. BALB/c mice were randomly divided into six groups (five mice per group) and inoculated orally with St-Eg95, 100 µl/mouse, at dosage of 1 x 10(9), 1 x 10(10), 1 x 10(11), and 1 x 10(12) cfu/ml, wild-type S. typhimurium strain(l x l0(7)cfu/ml), and PBS, respectively. The survival rate was monitored daily for 30 days. Another 15 mice were divided into three groups and inoculated orally with St-Eg95(5 x 10(5) cfu/ml), X4550(pYA3341)(5 x 10(5) cfu/ml), and PBS, respectively, for 2 times, 0.5 ml/mouse/time, at biweekly intervals. On weeks 0, 2, 4, and 6 after the second immunization, sera were collected and tested for the presence of Eg95 antibody titers using commercially Eg antibody detection ELISA kit. The splenic lymphocyte proliferation was detected by MTT assay at 6 weeks after the second immunization. RESULTS: The constructed recombinant plasmid pYA3341-Eg95 was identified by enzyme digestion and PCR identification. The Eg95 protein (M, 18000) was expressed in the recombinant strains St-Eg95. After the recombinant strains St-Eg95 were passaged 10 times, the Eg95 gene (about 486 bp) was still amplified from St-Eg95. Safety results showed that mice inoculated orally with the St-Eg95 or PBS were all survival on the 30th day after immunization. However, all mice taking wild virulent S. typhimurium strain diedr within 4 days. The Eg95-specific antibodies examined by indirect ELISA were significantly higher in mice immunized with St-Eg95 than that of mice immunized with X4550 (pYA3341) or PBS at 2 weeks after the second immunization (P<0.05). The average Eg95-specific antibody titers reached up to the highest value of 1:1700 in mice immunized with St-Eg95 at 4 weeks after the second immunization. The lymphocyte proliferation test showed that the stimulation index value was significantly higher(P<0.05) in mice immunized with the St-Eg95(reached up to 1.94±0.15) than that in mice immunized with X4550 (pYA3341) or PBS at 6 weeks after the second immunization. CONCLUSION: The recombinant oral attenuated S. typhimurium St-Eg95 was successfully constructed, and has a good safety and immunogenicity profile in mouse.

[Effects of continuous cropping obstacle on growth of Angelica sinensis and its mechanism].

OBJECTIVE: To study the effects of continuous cropping obstacles on growth of Angelica sinensis. METHOD: The growth indexes, photosynthetic characteristic, activity of leaf protective enzymes, and the yield, essential oil content and extract content were determined. RESULT: Continuous cropping decreased the growth and the yield, essential oil content, extract content of A. sinensis. Photosynthetic pigment, which include Chla and Chlb, and photosynthetic rate of A. sinensis leaves decreased. Activity of leaf SOD, POD and CAT were also inhibited. The content of proline, soluble sugar and MDA increased. CONCLUSION: Through decreasing the activity of protective enzymes and their ability of cleaning free radical, continuous cropping made free radical remain in plant so that induced membrane lipid peroxidization, electrolytic leakage became heavier, content of proline and soluble sugar increased. The external manifestation of this influences were that plant growth was inhibited, content of photosynthetic pigments decreased, so did the intensity of photosynthesis and respiration, content of dry mass.