Increase in Cd Tolerance through Seed-Borne Endophytic Fungus Epichloë gansuensis Affected Root Exudates and Rhizosphere Bacterial Community of Achnatherum inebrians.

Soil cadmium (Cd) pollution is a serious environmental problem imperiling food safety and human health. The endophyte Epichloë gansuensis can improve the tolerance of Achnatherum inebrians to Cd stress. However, it is still unknown whether and how the endophyte helps host plants build up a specific bacterial community when challenged by CdCl2. In this study, the responses of the structure and function of bacterial community and root exudates of E+ (E. gansuensis infected) and E- (E. gansuensis uninfected) plants to Cd stress were investigated. Analysis of bacterial community structure indicated that the rhizosphere bacterial community predominated over the root endosphere bacterial community in enhancing the resistance of CdCl2 in a host mediated by E. gansuensis. E+ plant strengthened the interspecific cooperation of rhizosphere bacterial species. Moreover, the analysis of root exudates demonstrated E. gansuensis and increased the contents of organic acids and amino acids under Cd stress, and most root exudates were significantly correlated with rhizosphere bacteria. These results suggested that E. gansuensis employed a specific strategy to recruit distinct rhizosphere bacterial species and relevant functions by affecting root exudates to improve the tolerance of the host to Cd stress. This study provides a firm foundation for the potential application of symbionts in improving phytostabilization efficiency.

First Report of Ergot (Claviceps purpurea) on Drunken Horse Grass (Achnatherum inebrians) in China.

Drunken horse grass (Achnatherum inebrians) belongs to the family Poaceae: it is mainly distributed in the natural grasslands of northern and northwestern in China. Ergot is a disease that can not only affect the growth of the grass, but also cause livestock poisoning (Coufal-Majewski et al. 2016). In September 2018, ergot was observed in a large area (about 15 ha) in Xinghai county, Qinghai province, China (35° 47' N, 99° 53' E, Altitude 3559 m). Around 65% of the plants of Achnatherum inebrians were affected. Symptoms initially showed drop-like honeydew on the ears of drunken horse grass, and later brown to dark brown sclerotia were observed. These were straight to slightly curved, measured 6.7 to 13.5 × 1.5 to 2.1 mm, which was approximately 1 to 4 times the size of healthy seeds. Sixteen spikes with typical symptoms were collected from eight different fields. Sclerotia were disinfested by immersion in 75% ethanol for 30 s and 1% NaClO for 90 s, rinsed three times in sterilized water, plated on potato dextrose agar (PDA) medium, incubated at 24°C in the dark and isolates purified by culturing from single spores. Finally, 16 single-spore cultures with similar phenotypes were obtained from these sclerotia. Colonies produced on PDA for 15 days at 24 ℃ were grayish white with fluffy aerial mycelium, about 60 mm in diameter. Conidia were hyaline, ovoid to cylindrical, 5.42 to 7.69 × 2.85 to 3.75 µm (avg. 5.67 × 3.2; n = 50). These morphological characteristics were consistent with the descriptions of Claviceps species in general (PíchovÁ et al. 2018). To further identify the Claviceps spp., isolate NSZJ (=MHLZU-AI20201012) was selected as a representative for molecular characterization. Two nuclear protein-coding genes TUB2 and MCM7 were amplified by T2/T12 (O'Donnell and Cigelnik 1997) and CARCA-F/M456-5R (Rehner and Buckley 2005), respectively, and sequenced. Sequences were deposited in GenBank (accession nos. MW115640 for TUB2 and MW115641 for MCM7). A BLAST analysis of these two segments showed >99% identity with those sequences of isolate W3 of C. purpurea (Pazoutová et al. 2014). To confirm the pathogenicity on drunken horse grass, 20 healthy plants (2-year-old) grown in an experimental field at the College of Pasture Agriculture Science and Technology, Yuzhong Campus of Lanzhou University in China (104° 39' E, 35° 89' N, altitude1653m) were spray-inoculated with conidial suspension (1 × 106 conidia/ml) during the flowering period. Another 20 plants in the field were sprayed with sterilized distilled water as controls. All plants were individually covered with transparent polyethylene bags for 24 h to maintain high relative humidity. After 7 to 10 days, small yellowish-white drops of honeydew were observed in some florets. At 22 days post inoculation, all the inoculated panicles developed three to six sclerotia per head, ranging in size from 6.7 to 13.5 mm, while control plants remained healthy. The same pathogen was consistently re-isolated from inoculated spikes and confirmed by morphological and molecular characterization as described above. Claviceps purpurea was reported to be associated with ergot in A. lemmonii in Idaho and A. robustum in Montana (Alderman et al. 2004). To our knowledge, this is the first report of C. purpurea causing ergot in A. inebrians in China.

Effects of feeding drunken horse grass infected with Epichloë gansuensis endophyte on animal performance, clinical symptoms and physiological parameters in sheep.

BACKGROUND: Many reports showed that grass-endophyte symbiosis induced livestock poisoned. Yet, there is no study evaluating clinical symptoms and physiological parameters in sheep fed Epichloë gansuensis endophyte-infected grass. The objective of the present study was to investigate these indexes by feeding sheep with endophyte-infected A. inebrians (E+ Group) or endophyte-free A. inebrians (E- Group) drunken horse grass or alfalfa hay (Control Group). RESULTS: The Epichloë endophyte caused obvious toxicity symptoms in the sheep fed E+ A. inebrians, with 1 of the 5 sheep having died by the 35th day. The feed intake and body weight gain of the E+ Group were significantly less than the E- and control groups (P < 0.05). Serum concentrations of alanine aminotransferase (ALT, 45.5 mmol/L) and aspartate aminotransferase for the E+ group (AST, 139.3 mmol/L) were significantly (P < 0.05) greater than for the E- (ALT, 31.2 mmol/L; AST, 78.6 mmol/L) and control (ALT, 32.6 mmol/L; AST, 56.6 mmol/L) groups at the fifth week; serum concentration of creatinine for the E+ group (63.8 mmol/L) was also significantly (P < 0.05) greater than for E- (56.6 mmol/L) and control groups (58.5 mmol/L). Meanwhile, urine biochemical indices for the E+ group indicated that ketone and occult blood were significantly (P < 0.05) elevated compared to the other groups while urine pH values were significantly (P < 0.05) acidic. The relative weight of heart, brain, liver, lung and kidney for Group E+ were almost two fold more than the other groups, but uterus weight was about half that found for Group E- or Control. CONCLUSIONS: We conclude that the Epichloë endophyte infection is the cause of A. inebrians toxicity to sheep. Interestingly, none of the measured parameters differed significantly between E- and the control groups, which implied that drunken horse grass could be utilized efficiently by sheep when not infected by the Epichloë endophyte.

Interactions between Epichloë endophyte and the plant microbiome impact nitrogen responses in host Achnatherum inebrians plants.

The clavicipitaceous fungus Epichloë gansuensis forms symbiotic associations with drunken horse grass (Achnatherum inebrians), providing biotic and abiotic stress protection to its host. However, it is unclear how E. gansuensis affects the assembly of host plant-associated bacterial communities after ammonium nitrogen (NH4+-N) treatment. We examined the shoot- and root-associated bacterial microbiota and root metabolites of A. inebrians when infected (I) or uninfected (F) with E. gansuensis endophyte. The results showed more pronounced NH4+-N-induced microbial and metabolic changes in the endophyte-infected plants compared to the endophyte-free plants. E. gansuensis significantly altered bacterial community composition and ß-diversity in shoots and roots and increased bacterial α-diversity under NH4+-N treatment. The relative abundance of 117 and 157 root metabolites significantly changed with E. gansuensis infection under water and NH4+-N treatment compared to endophyte-free plants. Root bacterial community composition was significantly related to the abundance of the top 30 metabolites [variable importance in the projection (VIP) > 2 and VIP > 3] contributing to differences between I and F plants, especially alkaloids. The correlation network between root microbiome and metabolites was complex. Microorganisms in the Proteobacteria and Firmicutes phyla were significantly associated with the R00693 metabolic reaction of cysteine and methionine metabolism. Co-metabolism network analysis revealed common metabolites between host plants and microorganisms.IMPORTANCEOur results suggest that the effect of endophyte infection is sensitive to nitrogen availability. Endophyte symbiosis altered the composition of shoot and root bacterial communities, increasing bacterial diversity. There was also a change in the class and relative abundance of metabolites. We found a complex co-occurrence network between root microorganisms and metabolites, with some metabolites shared between the host plant and its microbiome. The precise ecological function of the metabolites produced in response to endophyte infection remains unknown. However, some of these compounds may facilitate plant-microbe symbiosis by increasing the uptake of beneficial soil bacteria into plant tissues. Overall, these findings advance our understanding of the interactions between the microbiome, metabolome, and endophyte symbiosis in grasses. The results provide critical insight into the mechanisms by which the plant microbiome responds to nutrient stress in the presence of fungal endophytes.

Effect of Epichloë gansuensis Endophyte on Seed-Borne Microbes and Seed Metabolites in Achnatherum inebrians.

The seed-borne microbiota and seed metabolites of the grass Achnatherum inebrians, either host to Epichloë gansuensis (endophyte infected [EI]) or endophyte free (EF), were investigated. This study determined the microbial communities both within the seed (endophytic) and on the seed surface (epiphytic) and of the protective glumes by using Illumina sequencing technology. Epichloë gansuensis decreased the richness of the seed-borne microbiota except for the epiphytic fungi of glumes and also decreased the diversity of seed-borne microbiota. In addition, metabolites of seeds and glumes were detected using liquid chromatography-mass spectrometry (LC-MS). Unlike with the seeds of EF plants, the presence of E. gansuensis resulted in significant changes in the content of 108 seed and 31 glume metabolites. A total of 319 significant correlations occurred between seed-borne microbiota and seed metabolites; these correlations comprised 163 (147 bacterial and 16 fungal) positive correlations and 156 (136 bacterial and 20 fungal) negative correlations. Meanwhile, there were 42 significant correlations between glume microbiota and metabolites; these correlations comprised 28 positive (10 bacterial and 18 fungal) and 14 negative (9 bacterial and 5 fungal) correlations. The presence of E. gansuensis endophyte altered the communities and diversities of seed-borne microbes and altered the composition and content of seed metabolites, and there were many close and complex relationships between microbes and metabolites. IMPORTANCE The present study was to investigate seed-borne microbiota and seed metabolites in Achnatherum inebrians using high-throughput sequencing and LC-MS technology. Epichloë gansuensis decreased the richness of the seed-borne microbiota except for the epiphytic fungi of glumes and also decreased the diversity of seed-borne microbiota. Compared with endophyte-free plants, the content of 108 seed and 31 glume metabolites of endophyte-infected plants was significantly changed. There were 319 significant correlations between seed-borne microbiota and seed metabolites and 42 significant correlations between glume microbiota and metabolites.

Responses of community traits and soil characteristics of Achnatherum inebrians-type degraded grassland to grazing systems in alpine meadows on the Qinghai-Tibet Plateau.

Introduction: Scientific grazing management is of great significance for the ecological health and sustainable use of alpine meadows. Methods: To explore appropriate management methods of alpine grasslands of the Qinghai-Tibet Plateau degraded by Achnatherum inebrians (Hance) Keng ex Tzvele presence, we studied the effects of different grazing systems on the A. inebrians population, grassland vegetation community traits, soil characteristics and soil microbial community structure for cold- season grazing plus supplementary feeding pasture (CSF) and four-season open public pasture (FOP) in Tianzhu County, Gansu Province. Results: Compared with FOP, the CSF site showed significantly inhibited reproduction of A. inebrians, especially the crown width, seed yield and number of reproductive branches per plant were as high as 50%, significantly increased the aboveground biomass of edible forage and soil water content by 57% and 43-55%, better soil nutrients, and significantly reduced soil bulk density by 10- 29%. Different grazing systems affected the composition and diversity of soil microbial communities, with a greater effect on fungi than on bacterial flora. The most abundant phyla of bacteria and fungi were Proteobacteria and Ascomycota for CSF (by 30-38% and 24-28%) and for FOP (by 67-70% and 68-73%), and the relative abundance and species of bacterial and fungal genera were greater for CSF than FOP. The α-diversity indexes of fungi were improved, and the ß-diversity of fungi was significant difference between CSF and FOP. However, the grazing utilization time was prolonged in FOP, which reduced the diversity and abundance of soil bacteria and increased soil spatial heterogeneity. The use of A. inebrians-type degraded grassland in the cold season, and as a winter supplementary feeding and resting ground, could effectively inhibit expansion of A. inebrians, promote edible forage growth, enhance grassland productivity and community stability, and improve soil structure. Discussion: The results guide healthy and sustainable utilization of A. inebrians-type degraded grassland in the Qinghai-Tibet Plateau.

Directional screening and identification of potential cytotoxic components from Achnatherum inebrians by a combination of surface palsmon resonance and chromatography.

Objective: To establish a method for directional screening of the cytotoxic components from the medicinal herb of Achnatherum inebrians by a combination of surface plasmon resonance (SPR) biosensor and chromatographic isolation technology. Methods: Under the guidance of bioactive assessment based on binding abilities between objects and the α-Mannosidase (α-Man) target, the active components from different solvents extracts, different polar extraction parts and fractions were screened orderly and directionally using SPR. Components with a high binding ability to α-Man can be precisely oriented in a narrower fractions range and are easy to isolate. Three human cancer cells were used to evaluate the cytotoxic activity of component with the highest affinity to α-Man. Results: Eight compounds were isolated and identificated from A. inebrians for the first time. Deoxyvasicinone possessed the highest affinity to α-Man among them. Moreover, deoxyvasicinone showed good effects on inhibited proliferation of human hepatoma cells HepG2 (IC50 = 5.7 µmol/L), human breast cancer cells MCF7 (IC50 = 7.21 µmol/L) and human lung cancer cells HCC827 (IC50 = 0.75 µmol/L), respectively. In particular, its inhibitory effect on HCC827 was stronger than the positive drug gefitinib (IC50 = 1.65 µmol/L). Conclusion: A comprehensive strategy of directional screening potential cytotoxic components from herb based on biomolecular interaction and chromatography was established. Deoxyvasicinone as an effective anti-cancer component was initially isolated from A. inebrians. It is expected that this screening strategy could provide new perspectives for rapid screening and identification of active components from natural plants with the complex matrix.

Vertically Transmitted Epichloë Systemic Endophyte Enhances Drought Tolerance of Achnatherum inebrians Host Plants through Promoting Photosynthesis and Biomass Accumulation.

Achnatherum inebrians (drunken horse grass, DHG) plants, a dominant grass species in the arid and semi-arid regions of northwest China, symbiotic with an Epichloë fungal endophyte, is well adapted to drought. However, little is known about how the presence of the foliar Epichloë endophyte enhances the tolerance of DHG to drought at the molecular level. This study explored the positive effects of the presence of the Epichloë endophyte on plant growth, biomass, and photosynthetic efficiency and processes of DHG under non-drought and two drought (moderate and severe) treatments, using RNA sequencing to compare transcriptomes. The transcriptome results showed that 32 selected unigenes involved in the photosynthesis processes within Epichloë symbiotic plants were differently expressed (DEGs) versus non-symbiotic plants. The majority of these selected DEGs were upregulated in Epichloë symbiotic plants versus non-symbiotic plants, such as upregulated unigenes (c51525.graph_c1, c47798.graph_c0 & c64087.graph_c0) under drought conditions. In line with the transcriptomes data, the presence of the Epichloë endophyte promoted the photosynthetic rate and biomass accumulation of DHG plants, and the relationship between the photosynthetic rate and biomass is linear and significant. The presence of the endophyte only increased the biomass per tiller of DHG plants under drought. This study provides further insights into the molecular mechanisms that underlie the enhanced plant growth and drought tolerance of Epichloë-symbiotic DHG plants.

Transcriptomic Analyses Reveals Molecular Regulation of Photosynthesis by Epichloë endophyte in Achnatherum inebrians under Blumeria graminis Infection.

Photosynthesis is essential for the growth of all green plants, and the presence of an Epichloë endophyte enhances the photosynthesis of Achnatherum inebrians (drunken horse grass, DHG), including when it is under attack by fungal pathogens. However, few studies have examined the mechanism of the increased photosynthetic activity at the molecular level of A. inebrians when it is under pathogen stress. The present study investigated the effects of the presence of the Epichloë endophyte on the net photosynthetic rate, intercellular CO2 concentration, stomatal conductance, and transpiration rate of DHG plants under a Blumeria graminis infection condition, and we compared the transcriptomes using RNA sequencing. The results showed that the photosynthetic rate of Epichloë endophyte-infected (E+) plants was higher under the B. graminis infection condition, and also without this pathogen, when it was compared with Epichloë endophyte-free (E-) plants. The E+ plants uninfected with B. graminis had 15 up-regulated unigenes that are involved in photosynthesis which were compared to the E- plants that were uninfected with this pathogen. This suggests that the presence of an Epichloë endophyte up-regulates the genes that are involved in the process of photosynthesis.

Cuticular Wax Modification by Epichloë Endophyte in Achnatherum inebrians under Different Soil Moisture Availability.

The cuticular wax serves as the outermost hydrophobic barrier of plants against nonstomatal water loss and various environmental stresses. An objective of this study was to investigate the contribution of the mutualistic fungal endophyte Epichloë gansuensis to leaf cuticular wax of Achnatherum inebrians under different soil moisture availability. Through a pot experiment and gas chromatography-mass spectrometry (GC-MS) analysis, our results indicated that the hydrocarbons were the dominant components of leaf cuticular wax, and the proportion of alcohols, aldehydes, amines, and ethers varied with the presence or absence of E. gansuensis and different soil moisture availability. Amines and ethers are unique in endophyte-free (EF) A. inebrians plants and endophyte-infected (EI) A. inebrians plants, respectively. By transcriptome analysis, we found a total of 13 differentially expressed genes (DEGs) related to cuticular biosynthesis, including FabG, desB, SSI2, fadD, BiP, KCS, KAR, FAR, and ABCB1. A model is proposed which provides insights for understanding cuticular wax biosynthesis in the association of A. inebrians plants with E. gansuensis. These results may help guide the functional analyses of candidate genes important for improving the protective layer of cuticular wax of endophyte-symbiotic plants.

Response of sheep rumen fermentation and microbial communities to feed infected with the endophyte Epichloë gansuensis as evaluated with rumen-simulating technology.

Achnatherum inebrians, a perennial grass, is widely distributed in China. When infected by the endophyte Epichloë gansuensis, A. inebrians produces an abundance of alkaloids that enhance plant survival but are toxic to animals. Here we used in vitro fermentation to study the impact of endophyte- infected A. inebrians (E+) addition on rumen fermentation characteristics and on microbial community and diversity as assessed with amplicon sequencing technology. We examined E+ addition at five levels, E0, E25, E50, E75, and E100, corresponding to 0%, 25%, 50%, 75%, and 100% of the fermentation substrate, respectively. Both the fermentation characteristics and rumen microbial community structure differed significantly among treatments. E100 resulted in the highest values for pH, the Shannon index, Kiritimatiellaeota, and Lentisphaerae levels relative to the other treatments. In contrast, E25 was associated with higher levels of ammonia nitrogen, total volatile fatty acid, propionate, butyrate, isobutyrate, valerate, of the phyla Bacteroidetes and Firmicutes, and of the genus Prevotella_1, Succiniclasticum, Family_XIII_AD3011_group, Rikenellaceae_RC9_gut_group, Prevotellaceae_UCG-001, and Pyramidobacter as compared with other treatments. E50 resulted in the greatest values for the abundance-based coverage estimator (ACE) and the Chao1 index as compared with other treatments. E0 resulted in the greatest values for digestibility of dry matter, gas production, acetate, and Ruminobacter as compared with other treatments. This approach avoided animal toxicity experiments and confirmed that rumen fermentation characteristics and rumen microbiota were affected by E+ toxin. Therefore, E25 showed higher abundance in Prevotella_1, Prevotellaceae_ UCG-001, and Lachnospiraceae_XPB1014_group that implied they should play significant roles in E+ alkaloids degradation. And then, we can infer that rumen microorganisms should function as an antidote with respect to this poisoning reaction at moderate dietary percentages of E+.

Complete chloroplast genomes of Achnatherum inebrians and comparative analyses with related species from Poaceae.

This article reports the complete chloroplast genome of Achnatherum inebrians, a poisonous herb that is widely distributed in the rangelands of Northern China. The genome is 137 714 bp in total and consists of a large single-copy (81 758 bp) region and small single-copy (12 682 bp) region separated by a pair of inverted repeats (21 637 bp). The genome contains 130 genes, including 84 protein-coding genes, 38 tRNA genes and 8 ribosomal RNA genes, and the guanine + cytosine content is 36.17%. We subsequently performed comparative analysis of complete genomes from A. inebrians and other Poaceae-related species from GenBank. Thirty-eight simple sequence repeats were identified, further demonstrating rapid evolution in Poaceae. Finally, the phylogenetic trees of 37 species of Poaceae and 2 species of Amaranthaceae were constructed by using maximum likelihood and Bayesian inference methods, based on the genes of the complete chloroplast genome. We identified hotspots that can be used as molecular markers and barcodes for phylogenetic analysis, as well as for species identification. Phylogenetic analysis indicated that A. inebrians is a member of the genus Stipa rather than Achnatherum.

Effects of Aqueous Extracts of Endophyte-Infected Grass Achnatherum inebrians on Growth and Development of Pea Aphid Acyrthosiphon pisum.

The pea aphid Acyrthosiphon pisum has a worldwide distribution and causes serious losses for agricultural production. Drunken horse grass Achnatherum inebrians is a widely distributed perennial poisonous grass on the grasslands of Northern and Northwestern China. The present study focused on contact toxicity activity of aqueous extracts of endophyte-infected (E+) and endophyte-free (E-) A. inebrians in different growth periods of pea aphids, and the growth and development of two color morphs of F1 generation nymphs. Both of the color morphs had development durations in E+ treatments that tended to be longer at 1st, 2nd, 3rd, and 4th instars than E- and control (CK). The E+ treated aphids also showed decreased weights at maturity with over all lower mean relative growth rates (MRGR). Aphid survival of E+ treated aphids was lower than that of E- and CK at all growth periods. Seeding stage E+ extracts showed a greater propensity for negatively affecting aphids than did E+ extract at maturity and the yellowing stage. These results show that extracts from endophyte-containing plants may contain compounds that may be used to control insects.

Transcriptome Analysis Revealed Plant Hormone Biosynthesis and Response Pathway Modification by Epichloëgansuensis in Achnatheruminebrians under Different Soil Moisture Availability.

The present study was designed to explore the effects of the endophyte Epichloë gansuensis on gene expression related to plant hormone biosynthesis and response pathways and the content of salicylic acid (SA) and jasmonic acid (JA) hormones of Achnatherum inebrians, under different moisture conditions. Through a pot experiment and transcriptome analysis, we found a total of 51 differentially expressed genes (DEGs) related to hormone biosynthesis and response pathways, including 12 auxin related genes, 8 cytokinin (CTK) related genes, 3 gibberellin (GA) related genes, 7 abscisic acid (ABA) related genes, 7 ethylene (ET) related genes, 12 JA related genes and 4 SA related genes. Furthermore, key genes of JA and SA biosynthesis and response pathways, such as LOX2S, AOS, OPR, ACX, JMT, JAZ, PAL, NPR1, TGA and PR-1, showed different degrees of upregulation or downregulation. Under 60% soil moisture content, the JA content of endophyte-free (EF) A. inebrians was significantly (p < 0.05) higher than that of endophyte-infected (EI) A. inebrians. Under 30% and 60% soil moisture content, the SA content of EF A. inebrians was significantly (p < 0.05) higher than that of EI A. inebrians. SA content of EI A. inebrians under 30% and 60% soil moisture content was significantly (p < 0.05) higher than that under 15% soil moisture content. With both EI and EF plants, the SA and JA levels, respectively, are very similar at 15% soil moisture content. This study has revealed that E. gansuensis differentially activated plant hormone synthesis and signal transduction pathways of A. inebrians plants under different soil moisture availability.

The fungal endophyte Epichloë gansuensis increases NaCl-tolerance in Achnatherum inebrians through enhancing the activity of plasma membrane H+-ATPase and glucose-6-phosphate dehydrogenase.

Salt stress negatively affects plant growth, and the fungal endophyte Epichloëgansuensis increases the tolerance of its host grass species, Achnatherum inebrians, to abiotic stresses. In this work, we first evaluated the effects of E. gansuensis on glucose-6-phosphate dehydrogenase (G6PDH) and plasma membrane (PM) H+-ATPase activity of Achnatherum inebrians plants under varying NaCl concentrations. Our results showed that the presence of E. gansuensis increased G6PDH, PM H+-ATPase, superoxide dismutase and catalase activity to decrease O2•-, H2O2 and Na+ contents in A. inebrians under NaCl stress, resulting in enhanced salt tolerance. In addition, the PM NADPH oxidase activity and NADPH/NADP+ ratios were all lower in A. inebrians with E. ganusensis plants than A. inebrians plants without this endophyte under NaCl stress. In conclusion, E. gansuensis has a positive role in improving host grass yield under NaCl stress by enhancing the activity of G6PDH and PM H+-ATPase to decrease ROS content. This provides a new way for the selection of stress-resistant and high-quality forage varieties by the use of systemic fungal endophytes.

Effects of Epichloë gansuensis Endophyte on the Root and Rhizosphere Soil Bacteria of Achnatherum inebrians Under Different Moisture Conditions.

This study was conducted to explore effects of the systemic fungal endophyte Epichloë gansuensis on root and rhizosphere soil bacterial diversity of Achnatherum inebrians host plants growing under different moisture conditions. Soil properties of different treatments were compared using standard techniques. A total of 4371379 16S rRNA gene sequences were obtained and assigned to 5025 operational taxonomic units (OTUs). These OTUs in roots and rhizosphere soil were divided into 13 and 17 phyla, respectively, and the Actinobacteria and Proteobacteria were the most abundant phyla both in roots and rhizosphere soil. Shannon diversity and Chao1 richness index of bacteria in rhizosphere soil was significantly higher than in roots. E. gansuensis decreased the Shannon diversity of the root-associated bacterial community, and increased Shannon diversity and Chao1 richness index of the rhizosphere soil bacterial community of A. inebrians. Meanwhile, Chao1 richness of the rhizosphere soil bacterial community of A. inebrians significantly increased with the increase of the soil moisture level. Structural equation modeling also emphasized that E. gansuensis decreased the diversity of the root-associated bacterial community and increased the diversity of the rhizosphere soil bacterial community through decreasing soil available N. Additionally, soil moisture increased the diversity of the rhizosphere soil bacterial community through increased soil pH, C/N, and NN, and decreased soil AP. The E. gansuensis endophyte and soil moisture effects on root and rhizosphere soil bacterial diversity were likely to be from responses to modifications of the rhizosphere soil properties.

Effect of Epichloë gansuensis Endophyte on the Nitrogen Metabolism, Nitrogen Use Efficiency, and Stoichiometry of Achnatherum inebrians under Nitrogen Limitation.

The systemic fungal endophyte of the grass Achnatherum inebrians, Epichloë gansuensis, has important roles in enhancing resistance to biotic and abiotic stresses. In this work, we first evaluated the effects of E. gansuensis on nitrogen metabolism, nitrogen use efficiency, and stoichiometry of A. inebrians under varying nitrogen concentrations. The results demonstrated that E. gansuensis significantly improved the growth of A. inebrians under low nitrogen conditions. The fresh and dry weights, nitrogen reductase, nitrite reductase, and glutamine synthetase activity, NO3-, NH4+, N, and P content, and also the total N accumulation, N utilization efficiency, and N uptake efficiency were all higher in leaves of A. inebrians with E. ganusensis (E+) plants than A. inebrians plants without this endophyte (E-) under low nitrogen availability. In conclusion, E. gansuensis has positive effects on improving the growth of A. inebrians under low-nitrogen conditions by modulating the enzymes of nitrogen metabolism and enhancing nitrogen use efficiency.

Directional screening and identification of potential cytotoxic components from Achnatherum inebrians by a combination of surface palsmon resonance and chromatography / 中草药·英文版

OBJECTIVE@#To establish a method for directional screening of the cytotoxic components from the medicinal herb of Achnatherum inebrians by a combination of surface plasmon resonance (SPR) biosensor and chromatographic isolation technology.@*METHODS@#Under the guidance of bioactive assessment based on binding abilities between objects and the α-Mannosidase (α-Man) target, the active components from different solvents extracts, different polar extraction parts and fractions were screened orderly and directionally using SPR. Components with a high binding ability to α-Man can be precisely oriented in a narrower fractions range and are easy to isolate. Three human cancer cells were used to evaluate the cytotoxic activity of component with the highest affinity to α-Man.@*RESULTS@#Eight compounds were isolated and identificated from A. inebrians for the first time. Deoxyvasicinone possessed the highest affinity to α-Man among them. Moreover, deoxyvasicinone showed good effects on inhibited proliferation of human hepatoma cells HepG2 (IC50 = 5.7 μmol/L), human breast cancer cells MCF7 (IC50 = 7.21 μmol/L) and human lung cancer cells HCC827 (IC50 = 0.75 μmol/L), respectively. In particular, its inhibitory effect on HCC827 was stronger than the positive drug gefitinib (IC50 = 1.65 μmol/L).@*CONCLUSION@#A comprehensive strategy of directional screening potential cytotoxic components from herb based on biomolecular interaction and chromatography was established. Deoxyvasicinone as an effective anti-cancer component was initially isolated from A. inebrians. It is expected that this screening strategy could provide new perspectives for rapid screening and identification of active components from natural plants with the complex matrix.

The Effect of Seed-Borne Fungi and Epichloë Endophyte on Seed Germination and Biomass of Elymus sibiricus.

The interactive effects of asexual Epichloë (formerly known as Neotyphodium) endophytes isolated from Hordeum brevisubulatum, Elymus tangutorum and Achnatherum inebrians, and seed-borne fungi on Elymus sibiricus seeds, were determined by an in vitro study using supernatants from liquid cultures of the endophyte strains. In an 8 week greenhouse study, the effects on the seedlings growth was measured. The in vitro study was carried out with the seed-borne fungi Alternaria alternata, Bipolaris sorokiniana, Fusarium avenaceum, and a Fusarium sp. isolated from E. sibiricus. Different concentrations and combinations of the liquid cultures of endophytic fungi enhanced the interim germination, germination rate, length of coleoptile and radicle, and seedling dry weight of E. sibiricus under stress from seed-borne fungi. In the greenhouse study, different concentrations of the supernatant of the endophytes from H. brevisubulatum and E. tangutorum but not A. inebrians, signficantly (P < 0.05) enhanced E. sibiricus seed germination. There was no significant (P > 0.05) increase of the tiller numbers after 2 weeks. However, later on, there were significant (P < 0.05) increases in tiller number (4-8 weeks), seedling height (2-8 weeks) and dry weight (2-8 weeks). The application of Epichloë endophyte culture supernatants was an effective strategy to improve seed germination and growth under greenhouse conditions.

Establishment of antioxidative activity determination for Achnatherum inebrians by graphene nanoenzyme / 中草药

Objective: To develop a new method for the determination of anti-oxidant activity of drugs by using the peroxidase activity of graphene nanoenzyme, and apply it for the determination of the anti-oxidant activity of the national medicine Achnatherum inebrians. Methods: The anti-oxidant activity of 18 batches of samples was measured after optimizing H2O2 concentration and reaction time. At the same time, the anti-oxidative activity was measured again using the DPPH method. The Bland-Altman and Passing-Bablok regression methods were used to compare the detection data of the two methods. Results: Using the nanoenzyme method, the inhibition rates of 18 batches of A. inebrians were between 37.28% and 71.58%, with an average of 50.87% and a median of 47.09%. Free radical scavenging rates of 18 batches of A. inebrians measured by the DPPH method were between 36.06% and 83.11%, with an average of 54.89% and a median of 50.83%. The statistical results showed that the measured values of nanoenzyme method and DPPH method were similar; There was a linear relationship between the two methods, and different methods can be transformed by Passing-Bablok regression method. Conclusion: The graphene nanoenzyme was successfully applied to the detection of anti-oxidant activity, and the anti-oxidant activity of A. inebrians was also found. The nanoenzyme method can avoid the problem of light sensitivity of the reagent, shorten the reaction time, reduce the amount of medicinal solution, and the results are consistent with the DPPH method, which is suitable for the determination of antioxidant activity.