[Objectives]To systematically investigate the microbial community composition of rhizosphere soil and endophytes associated with Pogostemon cablin,and to explore the relationships between endophytes and rhizosphere mi...[Objectives]To systematically investigate the microbial community composition of rhizosphere soil and endophytes associated with Pogostemon cablin,and to explore the relationships between endophytes and rhizosphere microorganisms as well as their potential applications.[Methods]Microbial isolates were obtained from rhizosphere soil,root tissues,and stem tissues using the serial dilution and spread plate method.These isolates were identified through morphological characterization,physiological and biochemical assays,and molecular biological techniques.[Results]A total of 18 microbial strains were isolated,including 7 bacterial and 11 fungal strains.Among the bacterial isolates,Pseudomonas spp.and Bacillus spp.were predominant,while the fungal isolates were mainly represented by Aspergillus spp.Certain bacterial strains,notably Pseudomonas spp.,exhibited potential abilities for indole-3-acetic acid(IAA)production,nitrogen fixation,and antagonistic activity against pathogenic microorganisms,suggesting their potential utility as biocontrol agents and promoters of plant growth.[Conclusions]This study establishes a foundational understanding of the microbial community characteristics in the rhizosphere and tissues of P.cablin,as well as their roles in plant growth and development.展开更多
[ Objective] The aim of this study was to isolate the endophyte of three solanaceae fruits and vegetables such as tomato, pepper and eggplant, to screen and identify the bacterial wilt antagonistic bacteria. [ Methodl...[ Objective] The aim of this study was to isolate the endophyte of three solanaceae fruits and vegetables such as tomato, pepper and eggplant, to screen and identify the bacterial wilt antagonistic bacteria. [ Methodl According to the lapping liquid culture method, the endophyte of three plants was isolated by the selective medium and purified by the plate streaking method, so the purified enclophyte was screened by the hyphal pieces confront culture method. Furthermore, the screened antagonistic and endophyteic bacteria was identified and classified through culture characteristics of isolates and morphological features of thallus, Gram stain as well as physiological and biochemical reactions. [ Result] Fifty-three endophytic bacteria, fifty-three endophytic fungi and forty-four endophytic actinomycetes were separated from the endophyte of three plants. The screened fourteen endophytic bacteria with strong antagonistic effect on the bacterial wilt were classified to Bacillus, Escherichia, Klebsiella, Agromonas, Erwinia and Curto Bacterium respectively. Especially, Bacillus was the dominant species, which had the strongest antagonistic effect on the bacterial wilt. [ Conclusion] This study provides an effective way for biological control of the bacterial wilt in solanaceae.展开更多
After insufflating extended endophytes phomopsis B3 ferment diluent on seedling bed of rice, the effect of microbial inoculum on rice growth was studied. The investigation result of seedlings before transplanting seed...After insufflating extended endophytes phomopsis B3 ferment diluent on seedling bed of rice, the effect of microbial inoculum on rice growth was studied. The investigation result of seedlings before transplanting seedling showed that leaf age,the number of green leaves,the number of tiller of individual plant and the weight of per 100 fresh plants in microbial inoculum treatment group increased 0.11,0.12 ,0. 03 and 2.6 g respectively compared with those of control group. 20 days after transplanting, increased tiller number in microbial inoculum treatment group was 55 500 hm^2 more than that in control group. 30 days after transplanting, tiller number in microbial inoculum treatment group was 42 000 hm^2 more than that in control group. In harvest season, the yield ,weight of I 0130 grain and seed-setting rate increased 300 kg/hm2, 0. 7 g and 0. 23% respectively compared with these in control group. The result of preliminary study demonstrated that microbial inoculum confected by endophytes phomopsis B3 could increase the number of strong seedling and yield.展开更多
Mangrove endophytic fungus 1893 was isolated from Kandelia candel from an estuarine mangrove on the South China Sea Coast Two new lactones 1893A and 1893B, together with other known compounds, have been isolated from ...Mangrove endophytic fungus 1893 was isolated from Kandelia candel from an estuarine mangrove on the South China Sea Coast Two new lactones 1893A and 1893B, together with other known compounds, have been isolated from its fermentation broth. To classify the endophyte correctly for further industrial application, a combination of morphological and molecular techniques was used to approach its identity. The endophyte was compared with similar species having trichogynes or trichogyne-like hyphae which apparently fused with an- theridium-like hyphae, and perithecia initials developing from an ascogonial coil surrounded by enveloping hyphae in early developmental stages on pure culture. Further morphological characteristics on host and non-host were used for comparison with similar species when the endophyte was cultivated on leaves ofKandelia candel and Mangifera indica, respectively, which resulted in classifying the endophyte as a Phomopsis specics. The ITS sequence of rDNA was used to infer its phylogenetic relationships with Phomopsis species that resembled the strain in morphology or ecology. Finally, the endophyte was identified as Diaporthe phaseolorum var. sojae based on morphological and molecular evidence. Our study is a first report ofDiaporthephaseolorum var. sojae isolated from mangrove Kandelia candel.展开更多
This study identifies the potential role in heat-stress mitigation of phytohormones and other secondary metabolites produced by the endophytic fungus Paecilomyces formosus LWL1 in japonica rice cultivar Dongjin. The j...This study identifies the potential role in heat-stress mitigation of phytohormones and other secondary metabolites produced by the endophytic fungus Paecilomyces formosus LWL1 in japonica rice cultivar Dongjin. The japonica rice was grown in controlled chamber conditions with and without P. formosus LWL1 under no stress （NS） and prolonged heat stress （HS） conditions. Endophytic association under NS and HS conditions significantly improved plant growth attributes, such as plant height, fresh weight, dry weight, and chlorophyll content. Furthermore, P. for- mosus LWL1 protected the rice plants from HS compared with controls, indicated by the lower endogenous level of stress-signaling compounds such as abscisic acid （25.71%） and jasmonic acid （34.57%） and the increase in total protein content （18.76%-33.22%）. Such fungal endophytes may be helpful for sustainable crop production under high environmental temperatures.展开更多
Sweet cherry is one of the most popular fresh fruits in the world.Previously,we isolated a soilborne dark septate endophyte(DSE)strain,S16,which promoted the growth of Gisela 5 sweet cherry rootstock.However,little is...Sweet cherry is one of the most popular fresh fruits in the world.Previously,we isolated a soilborne dark septate endophyte(DSE)strain,S16,which promoted the growth of Gisela 5 sweet cherry rootstock.However,little is known about the molecular mechanism of the effect of S16 on the growth of sweet cherry.In this study,the physiological parameters and transcript profiles of sweet cherry roots were analyzed under S16 treatment compared with a control to elucidate the molecular mechanisms of the effect of this strain on sweet cherry growth.After inoculation with S16,sweet cherry seedlings exhibited more vigorous growth.Moreover,we identified 4249 differentially expressed genes(DEGs)between S16-treated plants and the control.Many of the DEGs are involved in pathways related to plant growth,such as cellular metabolic and plant hormone pathways.Additionally,some genes involved in nitrate regulation were also enriched;and these genes may be involved in the regulation of nitrate uptake in plants.Physiological index detection demonstrated that S16 could improve the nitrate assimilation of sweet cherry via NO3–transporters.This RNA-seq dataset provides comprehensive insight into the transcriptomic landscape to reveal the molecular mechanisms whereby the DSE influences the growth of sweet cherry.展开更多
Medicinal plants have been used by marginal communities to treat various ailments. However, the potential of endophytes within these bio-prospective medicinal plants remains unknown. The present study elucidates the e...Medicinal plants have been used by marginal communities to treat various ailments. However, the potential of endophytes within these bio-prospective medicinal plants remains unknown. The present study elucidates the endophytic diversity of medicinal plants（Caralluma acutangula, Rhazya stricta, and Moringa peregrina） and the endophyte role in seed growth and oxidative stress. Various organs of medicinal plants yielded ten endophytes, which were identified as Phoma sp.（6 isolates）, Alternaria sp.（2）, Bipolaris sp.（1）, and Cladosporium sp.（1） based on 18 S rD NA sequencing and phylogenetic analysis. The culture filtrates（CFs; 25%, 50%, and 100% concentrations） from these endophytes were tested against the growth of normal and dwarf mutant rice lines. Endophytic CF exhibited dose-dependent growth stimulation and suppression effects. CF（100%） of Phoma sp. significantly increased rice seed germination and growth compared to controls and other endophytes. This growth-promoting effect was due to the presence of indole acetic acid in endophytic CF. The gas chromatography/mass spectrometry（GC/MS） analysis showed the highest indole acetic acid content（（54.31±0.21） μmol/L） in Bipolaris sp. In addition, the isolate of Bipolaris sp. exhibited significantly higher radical scavenging and anti-lipid peroxidation activity than the other isolates. Bipolaris sp. and Phoma sp. also exhibited significantly higher flavonoid and phenolic contents. The medicinal plants exhibited the presence of bio-prospective endophytic strains, which could be used for the improvement of crop growth and the mitigation of oxidative stresses.展开更多
An endophyte is a fungus or bacterium that lives within a plant in a symbiotic relationship.Extensive colonization of the plant tissue by endophytes creates a barrier effect,where they outcompete and prevent pathogen...An endophyte is a fungus or bacterium that lives within a plant in a symbiotic relationship.Extensive colonization of the plant tissue by endophytes creates a barrier effect,where they outcompete and prevent pathogenic organisms from taking hold.This happens by producing secondary metabolites that inhibit the growth of the competitors or pathogens.In this way they play a very important role in the plant defence mechanisms.The metabolites produced by these endophytes fall within a wide range of classes of compounds that include peptides which are the focus of this review.Peptides are increasingly being selected for drug development because they are specific for their targets and have a higher degree of interactions.There have been quite a number of endophytic peptides reported in the recent past indicating that endophytes can be used for the production of peptide based drugs.Molecular screening for NRPS,which shows peptide producing capability,has also shown that endophytes are potential producers of peptides.The presence of NRPS also offers the possibility of genetic modifications which may generate peptides with high pharmacological activities.This review,therefore,aims to show the current status of peptides isolated from endophytic bacteria and fungi in the recent decade.Endophytes as potential sources of peptides according to NRPS studies will also be discussed.展开更多
Endophytes are hypothesized to be transferred across the soil-plant continuum, suggesting both the transfers of endophytes from environment to plant and from plant to soil. To verify this hypothesis and to assess the ...Endophytes are hypothesized to be transferred across the soil-plant continuum, suggesting both the transfers of endophytes from environment to plant and from plant to soil. To verify this hypothesis and to assess the role of locality, we evaluated the similarity of microbial communities commonly found both in soils and endophytic communities in three arid regions, i.e. the Jornada LTER （Long Term Ecological Research） site in New Mexico, USA, and the research station of Jordan University of Science and Technology （JUST） and Khanasd research station in Badia region of Jordan. Rhizosphere and non-rhizosphere soils, leaves and seeds of Atriplex spp. were sampled. Diversity and distribution of bacteria and fungi across the soil-plant continuums were assessed by tag-encoded FLX amplicon pyrosequencing and sequence alignment. Of the total bacterial OTUs （operational taxonomic units）, 0.17% in Khanasri research station, 0.16% in research station of JUST, and 0.42% in Jornada LTER site were commonly found across all the plant and soil compartments. The same was true for fungi in two regions, i.e. 1.56% in research station of JUST and 0.86% in Jornada LTER site. However, in Khanasri research station, 12.08% of total fungi OTUs were found in at least one soil compartment and one plant compartment. Putative Arthrobacter, Sporosarcina, Cladosporium and members of Proteobacteria and Actinobacteria were found across all the soil-plant continuums. Ascomycota, mainly including Didymellaceae, Pleosporaceae and Davidiellaceae were present across all the soil-plant continuums. Microbial communities in two regions of Jordan were similar to each other, but both of them were different from the Jornada LTER site of USA. SIMPER （similarity percentage） analysis of bacterial and fungal taxa for both soil and endophyte communities revealed that dissimilarities of two bacterial genera （Arthrobacter and Sporosarcina） and two fungal genera （Cladosporium and Alternaria） are very high, so they play key roles in the soil-plant continuums. A weighed Pearson correlation analysis for the specific bacterial OTUs in the soil-plant continuums only showed high similarity between the two regions of Jordan. However, fungal groups showed higher similarities among all regions. This research supports the hypothesis of continuity of certain bacterial and fungal communities across the soil-plant continuums, and also explores the influences of plant species and geographic specificity on diversity and distribution of bacteria and fungi.展开更多
The present study was designed to isolate and purify chemical constituents from solid culture of endophyte Aspergillus terreus LQ, using silica gel column chromatography, gel filtration with Sephadex LH-20, and HPLC. ...The present study was designed to isolate and purify chemical constituents from solid culture of endophyte Aspergillus terreus LQ, using silica gel column chromatography, gel filtration with Sephadex LH-20, and HPLC. Fumigaclavine I(1), a new alkaloid, was obtained, along with seven known compounds, including fumigaclavine C(2), rhizoctonic acid(3), monomethylsulochrin(4), chaetominine(5), spirotryprostatin A(6), asperfumoid(7), and lumichrome(8). The structure of compound 1 was elucidated by various spectroscopic analyses(UV, MS, 1D and 2D NMR). The in vitro cytotoxicity of compound 1 was determined by MTT assay in human hepatocarcinoma cell line SMMC-7721, showing weaker cytotoxicity, compared with cisplatin, a clinically used cancer chemotherapeutic agent.展开更多
Leishmaniases and chronic inflammatory diseases are the cause of millions of deaths in the world each year.The treatment of leishmaniasis is facing serious drawbacks particularly due to the limited number of effective...Leishmaniases and chronic inflammatory diseases are the cause of millions of deaths in the world each year.The treatment of leishmaniasis is facing serious drawbacks particularly due to the limited number of effective medicines,the resistance,and the toxicity of available drugs.On the other hand,many drugs are used for the management of inflammatory disorders.However,the most commonly prescribed although efficient is highly toxic with multiples side effects.New leads compounds for the development of new anti-leishmanial and anti-inflammatory drugs are needed.Over the past decade,several studies on the potential of endophytes to produce bioactive metabolites have been reported.We are presenting in the present review the status of research from 2000 to 2019 on the anti-leishmanial and anti-inflammatory metabolites isolated from endophytes from diverse habitats.An emphasis was put on existing gaps in the literature to inspire and guide future investigations.We hope that this review will help accelerate the drug discovery against leishmaniases and inflammation-associated disorders.展开更多
A survey of 35 tree species （belonging to 28 genera in 19 families） in Aliyar, South India was carried out to ascertain their arbuscular mycorrhizal （AM） and dark septate endophyte （DSE） fungal status. All the t...A survey of 35 tree species （belonging to 28 genera in 19 families） in Aliyar, South India was carried out to ascertain their arbuscular mycorrhizal （AM） and dark septate endophyte （DSE） fungal status. All the tree species examined had AM association. AM and DSE coloni- zation is reported for the first time in 20 and 14 species respectively. Co- occurrence of AM and DSE was observed in 14 （40%） tree species. The extent of DSE colonization was inversely related to the extent of AM fungal colonization. Six tree species had Arum-type, 18 had intermediate- type and 11 had typical Paris-type AM morphology. AM fungal spore morphotypes belonging to 11 species in two genera were isolated from the rhizosphere soil. AM fungal spore numbers were not related to the ex- tent of AM colonization and Glomus dominated spore diversity. AM association individually and along with DSE were found respectively in the 63% and 44% of the economically important tree species. The occur- rence of AM and DSE fungal association in economically important indigenous tree species indicates the possibility of exploiting this asso- ciation in future conservation programmes of these species.展开更多
Bacterial endophytes have been shown to improve abiotic and biotic stress responses in plants. Plants growing under harsh conditions along the Oregon Coast could contain bacterial endophytes that improve persistence a...Bacterial endophytes have been shown to improve abiotic and biotic stress responses in plants. Plants growing under harsh conditions along the Oregon Coast could contain bacterial endophytes that improve persistence and growth of grasses in this environment. Thirty-four plants consisting of eight different species were collected along the Oregon coast from four different sites. Bacterial endophytes were isolated from root crown, stem and leaf tissues. A portion of the 16S rRNA ITS regions of each isolate was amplified, sequenced, and used to perform a BLAST search against the nucleotide database collection at National Center for Biotechnology Information. One-hundred and thirty-three different bacterial isolates, ninety-four of which were unique, representing thirty-six different taxonomic groups were found. Over 50% of the total bacteria isolates were in just five taxonomic groups. Pseudomonads were the most predominant bacteria isolated, making up 20.3% of the total isolates, followed by Curtobacterium and Microbacterium, each at 8.2%, Bacillus at 7.5% and Xanthomomas at 6%. Forty-seven percent (17 of 36) of the taxonomic groups contained only a single isolate. Fourteen bacterial isolates from five taxonomic groups, nine of which were from the genus Pseudomonas, were found to have 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, an enzyme associated with improving plant growth under stress. These newly discovered bacterial endophytes will be a valuable biological resource to develop approaches to increase the yield and adaptability of grasses and other crops grown in diverse environments and to meet the challenges associated with an unpredictable climate.展开更多
5 totally different endophytic fungal species were isolated from bark and leaf parts of Calophyllum inophyllum. Leaf part yielded Trichoderma harzianum and Alternaria species, whereas bark showed the presence of Fusar...5 totally different endophytic fungal species were isolated from bark and leaf parts of Calophyllum inophyllum. Leaf part yielded Trichoderma harzianum and Alternaria species, whereas bark showed the presence of Fusarium species, Aspergillus species and unidentified fungi. Two solvents (hexane and methanol) were used for endophytic fungal extraction and the Alternaria species had shown the presence of coumarin whereas Trichoderma harzianum in methanol extract and Fusarium species in hexane extract had shown the coumarin(s) in all the four methods tested. The total coumarin yield was more in microwave assistance method, the methanol Alternaria species (3.941 ± 0.082) stood first, followed by hexane extract of Alternaria species (3.254 ± 0.082), Fusarium species (2.532 ± 0.082) and Trichoderma harzianum (2.294 ± 0.082), the plant extract showed 4.149 + 0.053. The methanol extract of Alternaria species inhibited the activity of HIV-Reverse Transcriptase (RT) (82.81 ± 1.0), integrase (98%) and protease (78) in maximum level followed by hexane extract of Alternaria species (71.12 ± 0.9, 89, 68), Fusarium species (63.92 ± 1.8, 67, 66) and Trichoderma harzianum (56.69 ± 0.9, 71, 63). The endophytic fungi Alternaria species inhibited all the three viral enzymes at maximum level and it was more than standard drug. However, in order to know possible anti-HIV, it is necessary to isolate active coumarin from the Alternaria species and the mechanism of action will be studied in future studies.展开更多
Objective: To determine ex vivo antimalarial activity and cytotoxicity of endophytic Streptomyces SUK 08 as well as the main core structure fractionated from its crude extract.Methods: The activities of SUK 08 crude e...Objective: To determine ex vivo antimalarial activity and cytotoxicity of endophytic Streptomyces SUK 08 as well as the main core structure fractionated from its crude extract.Methods: The activities of SUK 08 crude extract were evaluated by using the Plasmodium lactate dehydrogenase assay and synchronization test against rodent malaria parasite Plasmodium berghei, instead of human malarial parasite Plasmodium falciparum. The cytotoxicity of the crude extract was determined by MTT assay. The crude extract was analyzed by thin-layer chromatography and gas chromatography–mass spectrophotometry.Results: The ethyl acetate crude extract showed very promising antimalarial activity with IC50 of 1.25 mg/m L. The synchronization tests showed that ethyl acetate extraction could inhibit all stages of the Plasmodium life cycle, but it was most effective at the Plasmodium ring stage. On the basis of a MTT assay on Chang Liver cells, ethyl acetate and ethanol demonstrated IC50 values of >1.0 mg/m L. The IC50 of parasitemia at 5% and30% for this extract was lower than chloroquine. Thin-layer chromatography, with 1: 9 ratio of ethyl acetate: hexane, was used to isolate several distinct compounds. Based on gas chromatography–mass spectrophotometry analysis, three core structures were identified as cyclohexane, butyl propyl ester, and 2,3-heptanedione. Structurally, these compounds were similar to currently available antimalarial drugs.Conclusions: The results suggest that compounds isolated from Streptomyces SUK 08 are viable antimalarial drug candidates that require further investigations.展开更多
Drought can limit the growth and reduce the yield of crops,but the safe and effective bio-approach to improve the drought resistance of crops is very little.We conducted an experiment in which we monitored the effects...Drought can limit the growth and reduce the yield of crops,but the safe and effective bio-approach to improve the drought resistance of crops is very little.We conducted an experiment in which we monitored the effects of polysaccharide from the endophyte Bionectria sp.Fat6 on the growth of Tartary buckwheat(Fagopyrum tataricum(L.)Gaertn)seedlings under control and drought-stressed conditions by determining gas exchange,photosynthesis parameters,photosynthetic pigment contents,and metabolite accumulation.Results indicated that the polysaccharide from endophyte stimulated plant growth and increased the aboveground biomass,root mass,and root/shoot ratio of Tartary buckwheat.Application of the polysaccharide to drought-stressed plants resulted in a significant increase in the net photosynthetic rate,stomatal conductance,and transpiration rate of Tartary buckwheat and decreased the intercellular CO_(2) concentration.The contents of chlorophyll a,chlorophyll b,chlorophyll a+b,and carotenoids in leaves were higher in polysaccharide-treated seedlings than that in control.Polysaccharide notably increased the soluble protein and proline content and decreased the malondialdehyde content in Tartary buckwheat leaves.The endophytic polysaccharide may protect Tartary buckwheat against drought by improving leaf gas exchange and photosynthetic capacity,and altering concentrations of protective metabolites.Together,these changes may compensate for the negative impacts of drought stress on the growth of Tartary buckwheat.Thus,the polysaccharide from the endophyte Bionectria sp.Fat6 may be an effective biotic elicitor and a promising bio-approach to improve Tartary buckwheat production worldwide.展开更多
The aim of this study is to investigate in vitro antioxidant activities and the phytochemical screening endophytes. Seven different endophytic fungi were isolated from different parts of the plant and their extracts s...The aim of this study is to investigate in vitro antioxidant activities and the phytochemical screening endophytes. Seven different endophytic fungi were isolated from different parts of the plant and their extracts subjected to know antioxidant properties and phytochemical screening. Phytochemical analysis revealed the presence of tannins, flavonoids, steroids, alkaloids, phenols and proteins from different solvents extracts of different endophytes. The antioxidant activity was evaluated by six separated methods: scavenging of free radical DPPH, FRAP, TBA, superoxide radical, FTC and iron methods. All seven different endophytes yielded almost all phytochemicals in methanol extracts which were tested. The endophytes A. niger, Penicillium sp. and Trichoderma sp. have shown potential in vitro antioxidant activities. Further work is needful to isolate the exact compound which is responsible for antioxidant activity and biophysical characterization will be done in the future.展开更多
Fungal endophytes have been shown to improve abiotic and biotic stress response in plants. Grasses growing along the Oregon coast are exposed to harsh conditions and may harbor endophytes that enable them to survive a...Fungal endophytes have been shown to improve abiotic and biotic stress response in plants. Grasses growing along the Oregon coast are exposed to harsh conditions and may harbor endophytes that enable them to survive and grow under these conditions. Endophytic fungi were isolated from thirty-four grass plants representing eight different grass species at four different locations along the Oregon coast. The ITS-1, 5.8S, and ITS-2 regions of each isolate were amplified, sequenced, and used to perform a BLAST search against the nucleotide database collection at National Center for Biotechnology Information. One-hundred-eleven different fungal isolates were classified into thirtynine genera with two isolates that did not show a match greater than 95%. These endophytes will be investigated to determine their potential for improving the adaptability of grasses and other crop plants to grow in diverse environments where they are subjected to multiple biotic and abiotic stresses.展开更多
Eclipta prostrata has been extensively studied for its promoting effect on human health. Previous study proved that E. prostrata possessed anti-osteoporosis function in SAMP6 mice via gut microbiota (GM). Endophytes a...Eclipta prostrata has been extensively studied for its promoting effect on human health. Previous study proved that E. prostrata possessed anti-osteoporosis function in SAMP6 mice via gut microbiota (GM). Endophytes are widespread in plants, especially in Chinese herbal medicines. However, little is known regarding the endophytes of E. prostrata. In this study, we focus on screening and identifying the endophytes from plant E. prostrata and exploring their roles in modulating GM. According to biochemical, physiological tests and 16S rDNA sequence analysis, ten endophytes were characterized in different locations of plant E. prostrata belonging to Lactococcus, Bacillus, Enterococcus, Exiguobacterium and Pantoea. The antimicrobial activity of endophytes EP01-10 was investigated via the oxford cup method. Furthermore, the acid and bile salt resistant ability of EP01-10 was detected to explore their survival ability in gastrointestinal tract. Results indicated that strains of Lactococcus, Bacillus and Exiguobacterium (EP01, 03 and 05) showed strong antimicrobial activity against Escherichia coli and Staphylococcus aureus and strains of Lactococcus, Bacillus and Enterococcus (EP01, 02, 04, 08 and 10) have the ability to survive in the gastrointestinal tract. 16S rRNA sequencing of GM in C57BL/6J mice was performed for diversity and structure analysis responded to the administration of E. prostrata extract. E. prostrata extract acted on improving the microflora diversity, particularly in increasing the abundance of Lactococcus significantly. Thus, as an endophyte, Lactococcus plays an important role in E. prostrata modulating intestinal tract. Our study suggested that endophytes of Chinese herbal medicines might be a novel target for the treatment of diseases by regulating the GM.展开更多
基金Supported by Rural Science and Technology Commissioner Project of Guangdong Province(KTP20240806).
文摘[Objectives]To systematically investigate the microbial community composition of rhizosphere soil and endophytes associated with Pogostemon cablin,and to explore the relationships between endophytes and rhizosphere microorganisms as well as their potential applications.[Methods]Microbial isolates were obtained from rhizosphere soil,root tissues,and stem tissues using the serial dilution and spread plate method.These isolates were identified through morphological characterization,physiological and biochemical assays,and molecular biological techniques.[Results]A total of 18 microbial strains were isolated,including 7 bacterial and 11 fungal strains.Among the bacterial isolates,Pseudomonas spp.and Bacillus spp.were predominant,while the fungal isolates were mainly represented by Aspergillus spp.Certain bacterial strains,notably Pseudomonas spp.,exhibited potential abilities for indole-3-acetic acid(IAA)production,nitrogen fixation,and antagonistic activity against pathogenic microorganisms,suggesting their potential utility as biocontrol agents and promoters of plant growth.[Conclusions]This study establishes a foundational understanding of the microbial community characteristics in the rhizosphere and tissues of P.cablin,as well as their roles in plant growth and development.
文摘[ Objective] The aim of this study was to isolate the endophyte of three solanaceae fruits and vegetables such as tomato, pepper and eggplant, to screen and identify the bacterial wilt antagonistic bacteria. [ Methodl According to the lapping liquid culture method, the endophyte of three plants was isolated by the selective medium and purified by the plate streaking method, so the purified enclophyte was screened by the hyphal pieces confront culture method. Furthermore, the screened antagonistic and endophyteic bacteria was identified and classified through culture characteristics of isolates and morphological features of thallus, Gram stain as well as physiological and biochemical reactions. [ Result] Fifty-three endophytic bacteria, fifty-three endophytic fungi and forty-four endophytic actinomycetes were separated from the endophyte of three plants. The screened fourteen endophytic bacteria with strong antagonistic effect on the bacterial wilt were classified to Bacillus, Escherichia, Klebsiella, Agromonas, Erwinia and Curto Bacterium respectively. Especially, Bacillus was the dominant species, which had the strongest antagonistic effect on the bacterial wilt. [ Conclusion] This study provides an effective way for biological control of the bacterial wilt in solanaceae.
基金Supported by the National Natural Science Foundation of China(30770073,30500066)~~
文摘After insufflating extended endophytes phomopsis B3 ferment diluent on seedling bed of rice, the effect of microbial inoculum on rice growth was studied. The investigation result of seedlings before transplanting seedling showed that leaf age,the number of green leaves,the number of tiller of individual plant and the weight of per 100 fresh plants in microbial inoculum treatment group increased 0.11,0.12 ,0. 03 and 2.6 g respectively compared with those of control group. 20 days after transplanting, increased tiller number in microbial inoculum treatment group was 55 500 hm^2 more than that in control group. 30 days after transplanting, tiller number in microbial inoculum treatment group was 42 000 hm^2 more than that in control group. In harvest season, the yield ,weight of I 0130 grain and seed-setting rate increased 300 kg/hm2, 0. 7 g and 0. 23% respectively compared with these in control group. The result of preliminary study demonstrated that microbial inoculum confected by endophytes phomopsis B3 could increase the number of strong seedling and yield.
基金supported partly by the Guangzhou Natural Science Foundation (Grant No.2007Z3-EO581)the Guangdong Provincial Natural Science Foundation (Grant No.2007A0200300001-7)+1 种基金the Chinese High-Tech 863 Project (Grant No.2006AA09Z422)the National Natural Science Foundation of China(Grant No. 20572136).
文摘Mangrove endophytic fungus 1893 was isolated from Kandelia candel from an estuarine mangrove on the South China Sea Coast Two new lactones 1893A and 1893B, together with other known compounds, have been isolated from its fermentation broth. To classify the endophyte correctly for further industrial application, a combination of morphological and molecular techniques was used to approach its identity. The endophyte was compared with similar species having trichogynes or trichogyne-like hyphae which apparently fused with an- theridium-like hyphae, and perithecia initials developing from an ascogonial coil surrounded by enveloping hyphae in early developmental stages on pure culture. Further morphological characteristics on host and non-host were used for comparison with similar species when the endophyte was cultivated on leaves ofKandelia candel and Mangifera indica, respectively, which resulted in classifying the endophyte as a Phomopsis specics. The ITS sequence of rDNA was used to infer its phylogenetic relationships with Phomopsis species that resembled the strain in morphology or ecology. Finally, the endophyte was identified as Diaporthe phaseolorum var. sojae based on morphological and molecular evidence. Our study is a first report ofDiaporthephaseolorum var. sojae isolated from mangrove Kandelia candel.
基金supported by the National Research Foundation of Korea(NRF)Ministry of Science,ICT and Future-Planning through Basic-Science Research Program(No.2014R1A1A2A10058022)
文摘This study identifies the potential role in heat-stress mitigation of phytohormones and other secondary metabolites produced by the endophytic fungus Paecilomyces formosus LWL1 in japonica rice cultivar Dongjin. The japonica rice was grown in controlled chamber conditions with and without P. formosus LWL1 under no stress （NS） and prolonged heat stress （HS） conditions. Endophytic association under NS and HS conditions significantly improved plant growth attributes, such as plant height, fresh weight, dry weight, and chlorophyll content. Furthermore, P. for- mosus LWL1 protected the rice plants from HS compared with controls, indicated by the lower endogenous level of stress-signaling compounds such as abscisic acid （25.71%） and jasmonic acid （34.57%） and the increase in total protein content （18.76%-33.22%）. Such fungal endophytes may be helpful for sustainable crop production under high environmental temperatures.
基金supported by the National Natural Science Foundation of China(31570649)the Key Research and Development Program of Yantai City,Shandong Province,China(2019XDHZ089)+1 种基金the Natural Science Foundation of Shandong Province,China(ZR2019PC052 and 2017C03)the Key Research and Development Program of Shandong Province,China(2019GSF107091,2019GSF109114,2018YYSP004 and 2018GNC110028)。
文摘Sweet cherry is one of the most popular fresh fruits in the world.Previously,we isolated a soilborne dark septate endophyte(DSE)strain,S16,which promoted the growth of Gisela 5 sweet cherry rootstock.However,little is known about the molecular mechanism of the effect of S16 on the growth of sweet cherry.In this study,the physiological parameters and transcript profiles of sweet cherry roots were analyzed under S16 treatment compared with a control to elucidate the molecular mechanisms of the effect of this strain on sweet cherry growth.After inoculation with S16,sweet cherry seedlings exhibited more vigorous growth.Moreover,we identified 4249 differentially expressed genes(DEGs)between S16-treated plants and the control.Many of the DEGs are involved in pathways related to plant growth,such as cellular metabolic and plant hormone pathways.Additionally,some genes involved in nitrate regulation were also enriched;and these genes may be involved in the regulation of nitrate uptake in plants.Physiological index detection demonstrated that S16 could improve the nitrate assimilation of sweet cherry via NO3–transporters.This RNA-seq dataset provides comprehensive insight into the transcriptomic landscape to reveal the molecular mechanisms whereby the DSE influences the growth of sweet cherry.
基金Project supported by the Oman Research Council(FURAP Program)the Korea Institute of Planning and Evaluation for Technology in Food,Agriculture,Forestry and Fisheries(IPET) through Agriculture,Food and Rural Affairs Research Center Support Program,funded by Ministry of Agriculture,Food and Rural Affairs(MAFRA)(716001-7)
文摘Medicinal plants have been used by marginal communities to treat various ailments. However, the potential of endophytes within these bio-prospective medicinal plants remains unknown. The present study elucidates the endophytic diversity of medicinal plants（Caralluma acutangula, Rhazya stricta, and Moringa peregrina） and the endophyte role in seed growth and oxidative stress. Various organs of medicinal plants yielded ten endophytes, which were identified as Phoma sp.（6 isolates）, Alternaria sp.（2）, Bipolaris sp.（1）, and Cladosporium sp.（1） based on 18 S rD NA sequencing and phylogenetic analysis. The culture filtrates（CFs; 25%, 50%, and 100% concentrations） from these endophytes were tested against the growth of normal and dwarf mutant rice lines. Endophytic CF exhibited dose-dependent growth stimulation and suppression effects. CF（100%） of Phoma sp. significantly increased rice seed germination and growth compared to controls and other endophytes. This growth-promoting effect was due to the presence of indole acetic acid in endophytic CF. The gas chromatography/mass spectrometry（GC/MS） analysis showed the highest indole acetic acid content（（54.31±0.21） μmol/L） in Bipolaris sp. In addition, the isolate of Bipolaris sp. exhibited significantly higher radical scavenging and anti-lipid peroxidation activity than the other isolates. Bipolaris sp. and Phoma sp. also exhibited significantly higher flavonoid and phenolic contents. The medicinal plants exhibited the presence of bio-prospective endophytic strains, which could be used for the improvement of crop growth and the mitigation of oxidative stresses.
文摘An endophyte is a fungus or bacterium that lives within a plant in a symbiotic relationship.Extensive colonization of the plant tissue by endophytes creates a barrier effect,where they outcompete and prevent pathogenic organisms from taking hold.This happens by producing secondary metabolites that inhibit the growth of the competitors or pathogens.In this way they play a very important role in the plant defence mechanisms.The metabolites produced by these endophytes fall within a wide range of classes of compounds that include peptides which are the focus of this review.Peptides are increasingly being selected for drug development because they are specific for their targets and have a higher degree of interactions.There have been quite a number of endophytic peptides reported in the recent past indicating that endophytes can be used for the production of peptide based drugs.Molecular screening for NRPS,which shows peptide producing capability,has also shown that endophytes are potential producers of peptides.The presence of NRPS also offers the possibility of genetic modifications which may generate peptides with high pharmacological activities.This review,therefore,aims to show the current status of peptides isolated from endophytic bacteria and fungi in the recent decade.Endophytes as potential sources of peptides according to NRPS studies will also be discussed.
文摘Endophytes are hypothesized to be transferred across the soil-plant continuum, suggesting both the transfers of endophytes from environment to plant and from plant to soil. To verify this hypothesis and to assess the role of locality, we evaluated the similarity of microbial communities commonly found both in soils and endophytic communities in three arid regions, i.e. the Jornada LTER （Long Term Ecological Research） site in New Mexico, USA, and the research station of Jordan University of Science and Technology （JUST） and Khanasd research station in Badia region of Jordan. Rhizosphere and non-rhizosphere soils, leaves and seeds of Atriplex spp. were sampled. Diversity and distribution of bacteria and fungi across the soil-plant continuums were assessed by tag-encoded FLX amplicon pyrosequencing and sequence alignment. Of the total bacterial OTUs （operational taxonomic units）, 0.17% in Khanasri research station, 0.16% in research station of JUST, and 0.42% in Jornada LTER site were commonly found across all the plant and soil compartments. The same was true for fungi in two regions, i.e. 1.56% in research station of JUST and 0.86% in Jornada LTER site. However, in Khanasri research station, 12.08% of total fungi OTUs were found in at least one soil compartment and one plant compartment. Putative Arthrobacter, Sporosarcina, Cladosporium and members of Proteobacteria and Actinobacteria were found across all the soil-plant continuums. Ascomycota, mainly including Didymellaceae, Pleosporaceae and Davidiellaceae were present across all the soil-plant continuums. Microbial communities in two regions of Jordan were similar to each other, but both of them were different from the Jornada LTER site of USA. SIMPER （similarity percentage） analysis of bacterial and fungal taxa for both soil and endophyte communities revealed that dissimilarities of two bacterial genera （Arthrobacter and Sporosarcina） and two fungal genera （Cladosporium and Alternaria） are very high, so they play key roles in the soil-plant continuums. A weighed Pearson correlation analysis for the specific bacterial OTUs in the soil-plant continuums only showed high similarity between the two regions of Jordan. However, fungal groups showed higher similarities among all regions. This research supports the hypothesis of continuity of certain bacterial and fungal communities across the soil-plant continuums, and also explores the influences of plant species and geographic specificity on diversity and distribution of bacteria and fungi.
基金financially supported by National Natural Science Foundation of China(Nos.21372191 and 31370079)Jiangsu Province Natural Science Foundation(No.BK20130437)
文摘The present study was designed to isolate and purify chemical constituents from solid culture of endophyte Aspergillus terreus LQ, using silica gel column chromatography, gel filtration with Sephadex LH-20, and HPLC. Fumigaclavine I(1), a new alkaloid, was obtained, along with seven known compounds, including fumigaclavine C(2), rhizoctonic acid(3), monomethylsulochrin(4), chaetominine(5), spirotryprostatin A(6), asperfumoid(7), and lumichrome(8). The structure of compound 1 was elucidated by various spectroscopic analyses(UV, MS, 1D and 2D NMR). The in vitro cytotoxicity of compound 1 was determined by MTT assay in human hepatocarcinoma cell line SMMC-7721, showing weaker cytotoxicity, compared with cisplatin, a clinically used cancer chemotherapeutic agent.
文摘Leishmaniases and chronic inflammatory diseases are the cause of millions of deaths in the world each year.The treatment of leishmaniasis is facing serious drawbacks particularly due to the limited number of effective medicines,the resistance,and the toxicity of available drugs.On the other hand,many drugs are used for the management of inflammatory disorders.However,the most commonly prescribed although efficient is highly toxic with multiples side effects.New leads compounds for the development of new anti-leishmanial and anti-inflammatory drugs are needed.Over the past decade,several studies on the potential of endophytes to produce bioactive metabolites have been reported.We are presenting in the present review the status of research from 2000 to 2019 on the anti-leishmanial and anti-inflammatory metabolites isolated from endophytes from diverse habitats.An emphasis was put on existing gaps in the literature to inspire and guide future investigations.We hope that this review will help accelerate the drug discovery against leishmaniases and inflammation-associated disorders.
文摘A survey of 35 tree species （belonging to 28 genera in 19 families） in Aliyar, South India was carried out to ascertain their arbuscular mycorrhizal （AM） and dark septate endophyte （DSE） fungal status. All the tree species examined had AM association. AM and DSE coloni- zation is reported for the first time in 20 and 14 species respectively. Co- occurrence of AM and DSE was observed in 14 （40%） tree species. The extent of DSE colonization was inversely related to the extent of AM fungal colonization. Six tree species had Arum-type, 18 had intermediate- type and 11 had typical Paris-type AM morphology. AM fungal spore morphotypes belonging to 11 species in two genera were isolated from the rhizosphere soil. AM fungal spore numbers were not related to the ex- tent of AM colonization and Glomus dominated spore diversity. AM association individually and along with DSE were found respectively in the 63% and 44% of the economically important tree species. The occur- rence of AM and DSE fungal association in economically important indigenous tree species indicates the possibility of exploiting this asso- ciation in future conservation programmes of these species.
文摘Bacterial endophytes have been shown to improve abiotic and biotic stress responses in plants. Plants growing under harsh conditions along the Oregon Coast could contain bacterial endophytes that improve persistence and growth of grasses in this environment. Thirty-four plants consisting of eight different species were collected along the Oregon coast from four different sites. Bacterial endophytes were isolated from root crown, stem and leaf tissues. A portion of the 16S rRNA ITS regions of each isolate was amplified, sequenced, and used to perform a BLAST search against the nucleotide database collection at National Center for Biotechnology Information. One-hundred and thirty-three different bacterial isolates, ninety-four of which were unique, representing thirty-six different taxonomic groups were found. Over 50% of the total bacteria isolates were in just five taxonomic groups. Pseudomonads were the most predominant bacteria isolated, making up 20.3% of the total isolates, followed by Curtobacterium and Microbacterium, each at 8.2%, Bacillus at 7.5% and Xanthomomas at 6%. Forty-seven percent (17 of 36) of the taxonomic groups contained only a single isolate. Fourteen bacterial isolates from five taxonomic groups, nine of which were from the genus Pseudomonas, were found to have 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, an enzyme associated with improving plant growth under stress. These newly discovered bacterial endophytes will be a valuable biological resource to develop approaches to increase the yield and adaptability of grasses and other crops grown in diverse environments and to meet the challenges associated with an unpredictable climate.
文摘5 totally different endophytic fungal species were isolated from bark and leaf parts of Calophyllum inophyllum. Leaf part yielded Trichoderma harzianum and Alternaria species, whereas bark showed the presence of Fusarium species, Aspergillus species and unidentified fungi. Two solvents (hexane and methanol) were used for endophytic fungal extraction and the Alternaria species had shown the presence of coumarin whereas Trichoderma harzianum in methanol extract and Fusarium species in hexane extract had shown the coumarin(s) in all the four methods tested. The total coumarin yield was more in microwave assistance method, the methanol Alternaria species (3.941 ± 0.082) stood first, followed by hexane extract of Alternaria species (3.254 ± 0.082), Fusarium species (2.532 ± 0.082) and Trichoderma harzianum (2.294 ± 0.082), the plant extract showed 4.149 + 0.053. The methanol extract of Alternaria species inhibited the activity of HIV-Reverse Transcriptase (RT) (82.81 ± 1.0), integrase (98%) and protease (78) in maximum level followed by hexane extract of Alternaria species (71.12 ± 0.9, 89, 68), Fusarium species (63.92 ± 1.8, 67, 66) and Trichoderma harzianum (56.69 ± 0.9, 71, 63). The endophytic fungi Alternaria species inhibited all the three viral enzymes at maximum level and it was more than standard drug. However, in order to know possible anti-HIV, it is necessary to isolate active coumarin from the Alternaria species and the mechanism of action will be studied in future studies.
基金financial assistance provided by Malaysia's Ministry of Higher Education (Grant number: FRGS/2/2010/SG/ UKM/01/9)the Universiti Kebangsaan Malaysia (Grant number: UKM_GUP-TKP-08-22-074) for making this study possible
文摘Objective: To determine ex vivo antimalarial activity and cytotoxicity of endophytic Streptomyces SUK 08 as well as the main core structure fractionated from its crude extract.Methods: The activities of SUK 08 crude extract were evaluated by using the Plasmodium lactate dehydrogenase assay and synchronization test against rodent malaria parasite Plasmodium berghei, instead of human malarial parasite Plasmodium falciparum. The cytotoxicity of the crude extract was determined by MTT assay. The crude extract was analyzed by thin-layer chromatography and gas chromatography–mass spectrophotometry.Results: The ethyl acetate crude extract showed very promising antimalarial activity with IC50 of 1.25 mg/m L. The synchronization tests showed that ethyl acetate extraction could inhibit all stages of the Plasmodium life cycle, but it was most effective at the Plasmodium ring stage. On the basis of a MTT assay on Chang Liver cells, ethyl acetate and ethanol demonstrated IC50 values of >1.0 mg/m L. The IC50 of parasitemia at 5% and30% for this extract was lower than chloroquine. Thin-layer chromatography, with 1: 9 ratio of ethyl acetate: hexane, was used to isolate several distinct compounds. Based on gas chromatography–mass spectrophotometry analysis, three core structures were identified as cyclohexane, butyl propyl ester, and 2,3-heptanedione. Structurally, these compounds were similar to currently available antimalarial drugs.Conclusions: The results suggest that compounds isolated from Streptomyces SUK 08 are viable antimalarial drug candidates that require further investigations.
基金This work was funded by National Key R&D Program of China(Grant No.2019YFD1001302/2019YFD1001300)Supported by National Natural Science Foundation of China(31771716+2 种基金3160126031701358)the earmarked fund for China Agriculture Research System(CARS-07-02A).
文摘Drought can limit the growth and reduce the yield of crops,but the safe and effective bio-approach to improve the drought resistance of crops is very little.We conducted an experiment in which we monitored the effects of polysaccharide from the endophyte Bionectria sp.Fat6 on the growth of Tartary buckwheat(Fagopyrum tataricum(L.)Gaertn)seedlings under control and drought-stressed conditions by determining gas exchange,photosynthesis parameters,photosynthetic pigment contents,and metabolite accumulation.Results indicated that the polysaccharide from endophyte stimulated plant growth and increased the aboveground biomass,root mass,and root/shoot ratio of Tartary buckwheat.Application of the polysaccharide to drought-stressed plants resulted in a significant increase in the net photosynthetic rate,stomatal conductance,and transpiration rate of Tartary buckwheat and decreased the intercellular CO_(2) concentration.The contents of chlorophyll a,chlorophyll b,chlorophyll a+b,and carotenoids in leaves were higher in polysaccharide-treated seedlings than that in control.Polysaccharide notably increased the soluble protein and proline content and decreased the malondialdehyde content in Tartary buckwheat leaves.The endophytic polysaccharide may protect Tartary buckwheat against drought by improving leaf gas exchange and photosynthetic capacity,and altering concentrations of protective metabolites.Together,these changes may compensate for the negative impacts of drought stress on the growth of Tartary buckwheat.Thus,the polysaccharide from the endophyte Bionectria sp.Fat6 may be an effective biotic elicitor and a promising bio-approach to improve Tartary buckwheat production worldwide.
文摘The aim of this study is to investigate in vitro antioxidant activities and the phytochemical screening endophytes. Seven different endophytic fungi were isolated from different parts of the plant and their extracts subjected to know antioxidant properties and phytochemical screening. Phytochemical analysis revealed the presence of tannins, flavonoids, steroids, alkaloids, phenols and proteins from different solvents extracts of different endophytes. The antioxidant activity was evaluated by six separated methods: scavenging of free radical DPPH, FRAP, TBA, superoxide radical, FTC and iron methods. All seven different endophytes yielded almost all phytochemicals in methanol extracts which were tested. The endophytes A. niger, Penicillium sp. and Trichoderma sp. have shown potential in vitro antioxidant activities. Further work is needful to isolate the exact compound which is responsible for antioxidant activity and biophysical characterization will be done in the future.
文摘Fungal endophytes have been shown to improve abiotic and biotic stress response in plants. Grasses growing along the Oregon coast are exposed to harsh conditions and may harbor endophytes that enable them to survive and grow under these conditions. Endophytic fungi were isolated from thirty-four grass plants representing eight different grass species at four different locations along the Oregon coast. The ITS-1, 5.8S, and ITS-2 regions of each isolate were amplified, sequenced, and used to perform a BLAST search against the nucleotide database collection at National Center for Biotechnology Information. One-hundred-eleven different fungal isolates were classified into thirtynine genera with two isolates that did not show a match greater than 95%. These endophytes will be investigated to determine their potential for improving the adaptability of grasses and other crop plants to grow in diverse environments where they are subjected to multiple biotic and abiotic stresses.
文摘Eclipta prostrata has been extensively studied for its promoting effect on human health. Previous study proved that E. prostrata possessed anti-osteoporosis function in SAMP6 mice via gut microbiota (GM). Endophytes are widespread in plants, especially in Chinese herbal medicines. However, little is known regarding the endophytes of E. prostrata. In this study, we focus on screening and identifying the endophytes from plant E. prostrata and exploring their roles in modulating GM. According to biochemical, physiological tests and 16S rDNA sequence analysis, ten endophytes were characterized in different locations of plant E. prostrata belonging to Lactococcus, Bacillus, Enterococcus, Exiguobacterium and Pantoea. The antimicrobial activity of endophytes EP01-10 was investigated via the oxford cup method. Furthermore, the acid and bile salt resistant ability of EP01-10 was detected to explore their survival ability in gastrointestinal tract. Results indicated that strains of Lactococcus, Bacillus and Exiguobacterium (EP01, 03 and 05) showed strong antimicrobial activity against Escherichia coli and Staphylococcus aureus and strains of Lactococcus, Bacillus and Enterococcus (EP01, 02, 04, 08 and 10) have the ability to survive in the gastrointestinal tract. 16S rRNA sequencing of GM in C57BL/6J mice was performed for diversity and structure analysis responded to the administration of E. prostrata extract. E. prostrata extract acted on improving the microflora diversity, particularly in increasing the abundance of Lactococcus significantly. Thus, as an endophyte, Lactococcus plays an important role in E. prostrata modulating intestinal tract. Our study suggested that endophytes of Chinese herbal medicines might be a novel target for the treatment of diseases by regulating the GM.
