Melatonin and dopamine alleviate waterlogging stress in apples by recruiting beneficial endophytes to enhance physiological resilience

Melatonin and dopamine can potentially prevent waterlogging stress in apples.The current study investigated the mechanism by which melatonin and dopamine alleviate apple waterlogging stress.This study demonstrated that melatonin and dopamine alleviated waterlogging by removing reactive oxygen species(ROS),and that the nitric oxide(NO)content and nitrate reductase(NR)activity were significantly correlated.Melatonin and dopamine were also found to recruit different candidate beneficial endophytes(melatonin:Novosphingobium,Propionivibrio,and Cellvibrio;dopamine:Hydrogenophaga,Simplicispira,Methyloversatilis,Candidatus_Kaiserbacteria,and Humicola),and these endophytes were significantly and positively correlated with plant growth.Network analyses showed that melatonin and dopamine significantly affected the endophytic bacterial and fungal communities under waterlogging stress.The metabolomic results showed that melatonin and dopamine led to waterlogging resistance by upregulating the abundance of beneficial substances such as amino acids,flavonoids,coumarins,and organic acids.In addition,melatonin and dopamine regulated the physicochemical properties of the soil,which altered the endophyte community and affected plant growth.The co-occurrence network demonstrated close and complex relationships among endophytes,metabolites,soil,and the plants.Our results demonstrate that melatonin and dopamine alleviate waterlogging stress in apples by recruiting beneficial endophytes to enhance physiological resilience.This study provides new insights into how melatonin and dopamine alleviate stress and a theoretical basis for synergistic beneficial microbial resistance to waterlogging stress.

Root Endophyte Shift and Key Genera Discovery in Rice under Barnyardgrass Stress

Despite increasing knowledge of barnyardgrass(Echinochloa crus-galli) interference with rice, relatively little is known how endophytes improve the ability of rice against barnyardgrass stress. Here, we provided a detailed temporal characterization of rice root-associated microbiomes during co-cultivation with barnyardgrass and a comparison with the microbiomes of weed-free rice plants. Alpha diversity analysis indicated that barnyardgrass had the opposite effects on endophytic bacteria and fungi in rice roots, in terms of the community diversity, richness and coverage at the rice seedling stage. Principal coordinate analysis showed that barnyardgrass had only a minor effect on the community composition of endophytes in rice roots at the rice seedling stage, but showed a significant and maximum interference at the heading stage. Rice recruited many endophytes to resist biotic stress from barnyardgrass, especially for fungi. PICRUSt(phylogenetic investigation of communities by reconstruction of unobserved states) predictive analysis indicated that 23 metabolic pathways of bacteria were overrepresented in rice. In addition, the main trophic mode of fungi was pathotroph according to FUNGuild analysis. A positive correlation between bacteria and fungi in rice roots was found via network analysis. Anaeromyxobacter, Azospira and Pseudolabrys were the vital bacteria, Phaeosphaeria and Funneliformis were the dominant fungi in maintaining the stability of the ecological network. These results provided data and a theoretical basis for the in-depth understanding of what role endophytes play in rice resistance to barnyardgrass stress and will have implications on improving the resistance of rice against biotic stress using root microbiota.

Isolation and Identification of Bacterial Endophytes from Grasses along the Oregon Coast

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.

Testing of Bacterial Endophytes from Non-Host Sources as Potential Antagonistic Agents against Tomato Wilt Pathogen <i>Ralstonia solanacearum</i>

The study was taken up with the objective of testing whether the endophytic organisms isolated from crops that are normally non-hosts to the bacterial wilt pathogen Ralstonia solanacearum possessed pathogen-antagonistic activity and to evaluate the selected isolates for the alleviation of wilt disease in the target tomato crop through horizontal movement of promising organisms. Sixteen endophytic bacteria (EB) isolated from the micropropagated cultures of grape, watermelon and papaya were tested for potential antagonistic effects against R. solanacearum tomato isolate “NH-01” through agar-well diffusion assay. Enterobacter cloacae from papaya (EB-11) displayed the maximum antagonistic effect followed by Bacillus subtilis (EB-06) and B. flexus (EB-07) from watermelon and B. pumilus (EB-02) from grape. Testing the above organisms for crop protection through seed fortification of susceptible tomato cv. Arka Vikas at sowing in R. solanacearum inoculated (Ral+) organic cocopeat showed EB-02 and EB-11 promising (33% and 32% survival, respectively, four weeks after sowing against 15% in Ral+ control). A second trial showed 37%, 28%, 21% and 55% seedling survival 6 weeks after sowing for EB-02, EB-06, EB-07 and EB-11 respectively, compared to 2.5% in non-treated control. Assessing the four endophytes for crop protection in Ral+ sick-soil through seedling fortification at transplanting indicated less disease incidence in treated sets (40%, 40%, 20% and 20% survival, respectively, six weeks after transplanting) over non-fortified control (5%). Endophytic fortification of seedlings through hypocotyl inoculation showed some systemic resistance induction upon seedling transplanting to sick soil but not with petiole fortification. Seedling growth was enhanced by the isolates EB-06 and EB-07. The study thus identifies four endophytic organisms from crops unrelated to tomato possessing potential antagonistic activity against the wilt pathogen and prospects for exploitation as biocontrol agents coupled with seedling growth promotion effects.

<i>In Vitro</i>Anti-HIV Activity of Partially Purified Coumarin(s) Isolated from Fungal Endophyte, <i>Alternaria</i>Species of <i>Calophyllum inophyllum</i>

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.

Characterization of Endophytes Isolated from <i>Eclipta prostrate</i>and Roles in Regulating the Gut Microbiota of C57BL/6J Mice

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.

一株产紫杉醇的曼地亚红豆杉内生真菌的分离及鉴定(摘要)(英文)

[目的]分离并鉴定1株产紫杉醇的曼地亚红豆杉内的内生真菌。[方法]从曼地亚红豆杉树皮内表皮中分离得到32株内生真菌,并通过高效液相色谱法检测其发酵产物。[结果]筛选获得1株可以产紫杉醇的内生真菌M57,其紫杉醇产量为45～50μg/L,并通过对M57菌落的形态学观察以及18S rDNA序列分析初步将其鉴定为根霉属(Rhizopus)真菌。[结论]该菌株的发现为微生物发酵法生产紫杉醇提供了具有潜在应用价值新的菌种。

Diversity Analysis of Endophytes in Wheat Infected by Powdery Mildew

Common wheat (Triticum aestivum L.) is one of the most important food crops. Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most serious diseases on wheat. In this study, the changes of endogenous bacteria in root, stem and leaf tissues of wheat infected and uninfected with powdery mildew were measured based on 16S rDNA. Integration, OTU cluster analysis, taxonomic analysis, diversity index, Shannon-Index curve, Rank-Abundance curve and PCoA analysis were carried out for each sample, and the roots, stems and leaves of different tissue parts were classified and summarized. The results showed that the infection of wheat powdery mildew had a certain effect on endophytic bacteria in stem tissue. There are also differences in the control and treatment of leaf tissue and root tissue. This indicated that endophytic bacteria were distributed differently in different parts of wheat.

Isolation and Identification of Fungal Endophytes from Grasses along the Oregon Coast

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.

Antimicrobial Activity of Ulva reticulata and Its Endophytes

Seaweeds are known to exhibit various antimicrobial properties,since it harbours an enormous range of indigenous bioactive compounds.The emergence of drug resistant strains has directed to the identification of prospective metabolites from seaweed and its endophytes,thereby exploiting the properties in resisting bacterial diseases.The current study was aimed to assess the antimicrobial activity of extracts obtained from Ulva reticulate,for which metabolites of Ulva reticulata and its endophytes were extracted and assessed against human pathogens like Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa,Salmonella typhi,and Bacillus subtilis.It was observed that the hexane extract of isolate VITDSJ2 was effective against all the tested pathogens but a significant inhibition was observed for Staphylococcus aureus and Escherichia coli.Further,Gas chromatography coupled with Mass spectroscopy(GC-MS) revealed the existence of phenol,3,5-bis(1,1-dimethylethyl) in the crude hexane extract which is well-known to possess antibacterial activity.The effective isolate VITDSJ2 was identified to be the closest neighbour of Pseudomonas stutzeri by phenotypic and genotypic methods.The crude extracts of the seaweed Ulva reticulata was also screened for antibacterial activity and the hexane extract was effective in showing inhibition against all the tested pathogens.The compound in the crude extract of Ulva reticulata was identified as hentriacontane using GC-MS.The extracts obtained from dichloromethane did not show significant activity in comparison with the hexane extracts.Hence the metabolites of Ulva reticulata and the bacterial secondary metabolites of the endophytes could be used in the treatment of bacterial infections.

Bioactive compounds fractionated from endophyte Streptomyces SUK 08 with promising ex-vivo antimalarial activity

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.

Antioxidant Activity and Phytochemical Screening of Crude Endophytes Extracts of <i>Tabebuia argentea</i>Bur. &K. Sch.

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.

Diversity of endophytes across the soil-plant continuum for Atriplex spp. in arid environments

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.

Transcriptome analysis for understanding the mechanism of dark septate endophyte S16 in promoting the growth and nitrate uptake of sweet cherry

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.

Endophytes as Producers of Peptides:An Overview About the Recently Discovered Peptides from Endophytic Microbes

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.

Analysis on Composition and Diversity of Endophytes in Moringa oleifera

[Objectives] To explore the diversity of endophytes in Moringa oleifera,and provide a scientific basis for better protection,development and use of Moringa oleifera resources. [Methods] With Moringa oleifera stems and leaves as materials,the Illumina Mi Seq highthroughput sequencing technology was used to study the endophyte diversity. [Results] A total of 199 OTUs were measured in the samples,belonging to 12 phyla such as Proteobacteria,Actinobacteria,Acidobacteria,Gemmatimonadetes,Bacteroidetes,Chloroflexi,DeinococcusThermus,Verrucomicrobia,Candidatus Saccharibacteria and Cyanobacteria. [Conclusions]Gemmatimonas is the dominant population of endophyte in Moringa oleifera.

植物内生真菌次生代谢产物的多样性及潜在应用价值

近些年来,植物内生真菌由于能够产生丰富多样的具有多种生物活性的次生代谢产物,在自然界中具有重要的生态学作用,受到了全世界的广泛关注并取得了极大进展。本文主要从生物活性角度综述了植物内生真菌次生代谢产物的主要类型,包括植物生长调节(赤霉素等)、杀虫(黑麦草碱)、抑菌(环已烷类等)、抗肿瘤活性物质(紫杉醇等),侧重介绍了一些产活性物质的植物(如红豆杉、长春花、雷公藤、苦皮藤等)内生真菌,并就植物内生真菌次生代谢产物生成的机理"内生共生理论"进行了分析。最后,还对他们在新医药和新农药等药物开发应用中的潜在价值作了探讨。

药用植物大戟的快速繁殖研究

以野生大戟为材料,探讨了大戟茎尖扦插繁殖和组织培养等快速繁殖技术的条件。结果表明:大戟茎 尖扦插繁殖,其成活率可以达到88.6%,用含有腋芽的茎在MS培养基上培养,发芽比例可以达到55%;用愈 伤组织诱导生芽,最高可以达到12%。嫩芽在不含激素的1/2MS培养基中培养,生根率达到47.1%。幼苗 接种本源的或同科外源植物5株内生真菌,比较其生长表明,接种大戟来源的两株内生真菌全苗重分别达到 对照的1.51和2.08倍,根重达到对照的2.09和3.68倍,最有利于宿主生长。

Polysaccharide Elicitor from the Endophyte Bionectria sp.Fat6 Improves Growth of Tartary Buckwheat under Drought Stress

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.