Efficient gene editing in the slow-growing, non-sporulating, melanized, endophytic fungus Berkleasmium sp. Dzf12 using a CRISPR/Cas9 system.

The endophytic fungus Berkleasmium sp. Dzf12 that was isolated from Dioscorea zingiberensis, is a proficient producer of palmarumycins, which are intriguing polyketides of the spirobisnaphthalene class. These compounds displayed a wide range of bioactivities, including antibacterial, antifungal, and cytotoxic activities. However, conventional genetic manipulation of Berkleasmium sp. Dzf12 is difficult and inefficient, partially due to the slow-growing, non-sporulating, and highly pigmented behavior of this fungus. Herein, we developed a CRISPR/Cas9 system suitable for gene editing in Berkleasmium sp. Dzf12. The protoplast preparation was optimized, and the expression of Cas9 in Berkleasmium sp. Dzf12 was validated. To assess the gene disruption efficiency, a putative 1, 3, 6, 8-tetrahydroxynaphthalene synthase encoding gene, bdpks, involved in 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, was selected as the target for gene disruption. Various endogenous sgRNA promoters were tested, and different strategies to express sgRNA were compared, resulting in the construction of an optimal system using the U6 snRNA-1 promoter as the sgRNA promoter. Successful disruption of bdpks led to a complete abolishment of the production of spirobisnaphthalenes and melanin. This work establishes a useful gene targeting disruption system for exploration of gene functions in Berkleasmium sp. Dzf12, and also provides an example for developing an efficient CRISPR/Cas9 system to the fungi that are difficult to manipulate using conventional genetic tools.

Endophytic fungi of Panax sokpayensis produce bioactive ginsenoside Compound K in flask fermentation.

Endophytes of Panax have the potential to produce their host plant secondary metabolites, ginsenosides. Panax sokpayensis, an endemic traditional medicinal plant of the Sikkim Himalayas was explored for the isolation of endophytic fungi. In the present study, we have isolated 35 endophytic fungal cultures from the rhizome of P. sokpayensis and screened for ginsenosides production by HPLC by comparing the peak retention time with that of standard ginsenosides. The HPLC analysis revealed that out of 35 isolates, the mycelial extracts of four fungal endophytes (PSRF52, PSRF53, PSRF49 and PSRF58) exhibited peaks with a similar retention time of the standard ginsenoside, Compound K (CK). LC-ESI-MS/MS analysis led to the confirmation of ginsenoside CK production by the four fungal endophytes which showed a compound with m/z 639.6278, similar to that of standard ginsenoside CK with yield in potato dextrose broth flask fermentation ranging from 0.0019 to 0.0386 mg/g of mycelial mass in dry weight basis. The four prospective fungal endophyte isolates were identified as Thermothielavioides terrestris PSRF52, Aspergillus sp. PSRF49, Rutstroemiaceae sp. strain PSRF53, and Phaeosphaeriaceae sp. strain PSRF58 based on ITS sequencing. The present finding highlights the need for further study on growth optimization and other culture parameters to exploit the endophytes as an alternative source for ginsenoside CK production.

Investigation the biological activities and the metabolite profiles of endophytic fungi isolated from Gundelia tournefortii L.

Endophytic fungi are microorganisms that are considered as a potential source of natural compounds, and can be applied in various industries. The aims of this research were molecular identification of endophytic fungi isolated from the Gundelia tournefortii stems, and investigation their biological activities as well as phenolic and fatty acid profile. Surface sterilized stems of G. tournefortii were placed on potato dextrose agar (PDA) to isolate the fungal endophytes. Genomic DNA was extracted by CTAB method, and PCR amplification was performed by ITS 1 and ITS 4 as primers. The enzyme production of endophytic fungi was determined based on the formation of a clear zone that appeared around the colonies of fungus. The anti-oxidant activity was evaluated by measuring the amount of free radicals DPPH. Also, the total phenol and flavonoid contents were measured obtained by Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. Moreover, the separation and identification of phenolic acids and fatty acids were done by HPLC and GC, respectively. Phylogenetic analysis was done based on the Internal Transcribed Spacer (ITS) region, and five isolates were identified as following: Aspergillus niger, Penicillium glabrum, Alternaria alternata, A. tenuissima, and Mucor circinelloides. Evaluation of the enzymatic properties showed that P. gabrum (31 ± 1.9 mm), and A. niger (23 ± 1.7) had more ability for producing pectinase and cellulase. The anti-oxidant activity of isolates showed that A. alternata extract (IC50 = 471 ± 29 µg/mL) had the highest anti-oxidant properties, followed by A. tenuissima extract (IC50 = 512 ± 19 µg/mL). Also, the extract of A. alternata had the greatest amount of total phenols and flavonoids contents (8.2 ± 0.4 mg GAL/g and 2.3 ± 0.3 mg QE/g, respectively). The quantification analysis of phenolic acid showed that rosmarinic acid, para-coumaric acid, and meta-coumaric acid (42.02 ± 1.31, 7.53 ± 0.19, 5.41 ± 0.21 mg/g, respectively) were the main phenolic acids in the studied fungi. The analysis of fatty acids confirmed that, in all fungi, the main fatty acids were stearic acid (27.9-35.2%), oleic acid (11.3-17.3%), palmitic acid (16.9-23.2%), linoleic acid (5.8-11.6%), and caprylic acid (6.3-10.9%). Our finding showed that endophytic fungi are a source of bioactive compounds, which could be used in various industries. This is the first report of endophytic fungi associated with G. tournefortii, which provides knowledge on their future use on biotechnological processes.

Foliar phenols and flavonoids level in pteridophytes: an insight to culturable fungal endophyte colonisation.

There are many available reports of secondary metabolites as bioactive molecules from culturable endophytes, nevertheless, there are scarce research pertaining to the levels of metabolites in plants with respect to the incidence and colonisation of fungal endophytes in the same foliar tissues. Therefore, the study was focussed to examine whether fungal endophyte colonisation and the accumulation of secondary metabolites, such as flavonoids and phenols, in the plants are related in any way. For this reason, the study aims to analyse phenols and flavonoids from the fronds of eleven pteridophytes along with the culture-dependent isolation of fungal endophytes from the host plants subsequently assigning them to morphological category and their quantitative analysis and further resolving its identities through molecular affiliation. The results revealed that nine morpho-categories of fungal endophytes were allotted based on culture attributes, hyphal patterns and reproductive structural characters. Highest numbers of species were isolated from Adiantum capillus-veneris and least was recorded from Pteris vittata and Dicranopteris linearis. Maximum phenol content was analysed from the fronds of P. vittata and lowest was recorded in A. capillus-veneris. Highest flavonoid content was measured in D. linearis and lowest was detected in Christella dentata. Significant negative correlation was observed between phenol content of ferns and species richness of fungi. Moreover, significant positive correlation was observed with the relative abundance of Chaetomium globosum and flavonoid content of ferns and negative significant relation was found between relative abundance of Pseudopestalotiopsis chinensis and phenol content of pteridophytes. The occurrence and the quantitative aspects of endophytes in ferns and their secondary metabolites are discussed.

Isolation of endophytes from Dioscorea nipponica Makino for stimulating diosgenin production and plant growth.

KEY MESSAGE: Both bacterial and fungal endophytes exhibited one or more plant growth-promoting (PGP) traits. Among these strains, the Paenibacillus peoriae SYbr421 strain demonstrated the greatest activity in the direct biotransformation of tuber powder from D. nipponica into diosgenin. Endophytes play crucial roles in shaping active metabolites within plants, significantly influencing both the quality and yield of host plants. Dioscorea nipponica Makino accumulates abundant steroidal saponins, which can be hydrolyzed to produce diosgenin. However, our understanding of the associated endophytes and their contributions to plant growth and diosgenin production is limited. The present study aimed to assess the PGP ability and potential of diosgenin biotransformation by endophytes isolates associated with D. nipponica for the efficient improvement of plant growth and development of a clean and effective approach for producing the valuable drug diosgenin. Eighteen bacterial endophytes were classified into six genera through sequencing and phylogenetic analysis of the 16S rDNA gene. Similarly, 12 fungal endophytes were categorized into 5 genera based on sequencing and phylogenetic analysis of the ITS rDNA gene. Pure culture experiments revealed that 30 isolated endophytic strains exhibited one or more PGP traits, such as nitrogen fixation, phosphate solubilization, siderophore synthesis, and IAA production. One strain of endophytic bacteria, P. peoriae SYbr421, effectively directly biotransformed the saponin components in D. nipponica. Moreover, a high yield of diosgenin (3.50%) was obtained at an inoculum size of 4% after 6 days of fermentation. Thus, SYbr421 could be used for a cleaner and more eco-friendly diosgenin production process. In addition, based on the assessment of growth-promoting isolates and seed germination results, the strains SYbr421, SYfr1321, and SYfl221 were selected for greenhouse experiments. The results revealed that the inoculation of these promising isolates significantly increased the plant height and fresh weight of the leaves and roots compared to the control plants. These findings underscore the importance of preparing PGP bioinoculants from selected isolates as an additional option for sustainable diosgenin production.

Production and bioprocessing of Taxol from Aspergillus niger, an endophyte of Encephalartos whitelockii, with a plausible biosynthetic stability: antiproliferative activity and cell cycle analysis.

The biosynthetic potency of Taxol by fungi raises their prospective to be a platform for commercial production of Taxol, nevertheless, the attenuation of its productivity with the fungal storage, is the challenge. Thus, screening for a novel fungal isolate inhabiting ethnopharmacological plants, with a plausible metabolic stability for Taxol production could be one of the most affordable approaches. Aspergillus niger OR414905.1, an endophyte of Encephalartos whitelockii, had the highest Taxol productivity (173.9 µg/L). The chemical identity of the purified Taxol was confirmed by HPLC, FTIR, and LC-MS/MS analyses, exhibiting the same molecular mass (854.5 m/z) and molecular fragmentation pattern of the authentic Taxol. The purified Taxol exhibited a potent antiproliferative activity against HepG-2, MCF-7 and Caco-2, with IC50 values 0.011, 0.016, and 0.067 µM, respectively, in addition to a significant activity against A. flavus, as a model of human fungal pathogen. The purified Taxol displayed a significant effect against the cellular migration of HepG-2 and MCF-7 cells, by ~ 52-59% after 72 h, compared to the control, confirming its interference with the cellular matrix formation. Furthermore, the purified Taxol exhibited a significant ability to prompt apoptosis in MCF-7 cells, by about 11-fold compared to control cells, suppressing their division at G2/M phase. Taxol productivity by A. niger has been optimized by the response surface methodology with Plackett-Burman Design and Central Composite Design, resulting in a remarkable ~ 1.6-fold increase (279.8 µg/L), over the control. The biological half-life time of Taxol productivity by A. niger was ~ 6 months of preservation at 4 â„ƒ, however, the Taxol yield by A. niger was partially restored in response to ethyl acetate extracts of E. whitelockii, ensuring the presence of plant-derived signals that triggers the cryptic Taxol encoding genes.

Unlocking the potential of bacterial endophytes from medicinal plants for drug discovery.

Among the plant-associated microorganisms, the so-called endophytes continue to attract much attention because of their ability not only to protect host plants from biotic and abiotic stress factors, but also the potential to produce bioactive secondary metabolites. The latter property can elicit growth-promoting effects on plants, as well as boost the production of plant-specific secondary metabolites with valuable pharmacological properties. In addition, endophyte-derived secondary metabolites may be a rich source for the discovery of drugs to treat various diseases, including infections and cancer. However, the full potential of endophytes to produce bioactive secondary metabolites is often not revealed upon conventional cultivation in the laboratory. New advances in genomics and metabolic engineering offer exciting opportunities for the exploration and exploitation of endophytes' biosynthetic potential. This review focuses on bacterial endophytes of medicinal plants, some of their secondary metabolites and recent advances in deciphering their biosynthesis. The latter may assist in genetic engineering efforts aimed at the discovery of novel bioactive compounds with the potential to be developed into drugs.

Multilevel analysis between Physcomitrium patens and Mortierellaceae endophytes explores potential long-standing interaction among land plants and fungi.

The model moss species Physcomitrium patens has long been used for studying divergence of land plants spanning from bryophytes to angiosperms. In addition to its phylogenetic relationships, the limited number of differential tissues, and comparable morphology to the earliest embryophytes provide a system to represent basic plant architecture. Based on plant-fungal interactions today, it is hypothesized these kingdoms have a long-standing relationship, predating plant terrestrialization. Mortierellaceae have origins diverging from other land fungi paralleling bryophyte divergence, are related to arbuscular mycorrhizal fungi but are free-living, observed to interact with plants, and can be found in moss microbiomes globally. Due to their parallel origins, we assess here how two Mortierellaceae species, Linnemannia elongata and Benniella erionia, interact with P. patens in coculture. We also assess how Mollicute-related or Burkholderia-related endobacterial symbionts (MRE or BRE) of these fungi impact plant response. Coculture interactions are investigated through high-throughput phenomics, microscopy, RNA-sequencing, differential expression profiling, gene ontology enrichment, and comparisons among 99 other P. patens transcriptomic studies. Here we present new high-throughput approaches for measuring P. patens growth, identify novel expression of over 800 genes that are not expressed on traditional agar media, identify subtle interactions between P. patens and Mortierellaceae, and observe changes to plant-fungal interactions dependent on whether MRE or BRE are present. Our study provides insights into how plants and fungal partners may have interacted based on their communications observed today as well as identifying L. elongata and B. erionia as modern fungal endophytes with P. patens.

Multiomics Reveals Mechanisms of Alternaria oxytropis Inhibiting Pathogenic Fungi in Oxytropis ochrocephala.

Endophytic fungi can benefit the host plant and increase the plant resistance. Now, there is no in-depth study of how Alternaria oxytropis (A. oxytropis) is enhancing the ability of inhibiting pathogenic fungi in Oxytropis ochrocephala (O. ochrocephala). In this study, the fungal community and metabolites associated with endophyte-infected (EI) and endophyte-free (EF) O. ochrocephala were compared by multiomics. The fungal community indicated that there was more A. oxytropis, less phylum Ascomycota, and less genera Leptosphaeria, Colletotrichum, and Comoclathris in the EI group. As metabolic biomarkers, the levels of swainsonine and apigenin-7-O-glucoside-4-O-rutinoside were significantly increased in the EI group. Through in vitro validation experiments, swainsonine and apigenin-7-O-glucoside-4-O-rutinoside can dramatically suppress the growth of pathogenic fungi Leptosphaeria sclerotioides and Colletotrichum americae-borealis by increasing the level of oxidative stress. This work suggested that O. ochrocephala containing A. oxytropis could increase the resistance to fungal diseases by markedly enhancing the content of metabolites inhibiting pathogenic fungi.

Antimicrobial metabolites produced by endophytic fungi associated with the leaves of Vochysia divergens.

Investigation of the endophytic fungi Nigrospora sphaerica, Nigrospora oryzae, and Pseudofusicoccum stromaticum MeOH fractions isolated from the leaves of Vochysia divergens, a medicinal species from the Brazilian Pantanal, led to the identification of five compounds, namely a new compound (1E,8Z)-10,11-dihydroxy-5,5,8-trimethyl-4-oxocycloundeca-1,8-diene-1-carbaldehyde (1) and four known compounds: 5-methylmellein (2), sclerone (3), daldinone A (4), and lasiodiplodin (5). All compounds were identified using spectroscopic methods, and 1 was corroborated with mass spectrometry, while the known compounds were compared with data in the literature. The relative configuration of compound 1 was determined based on theoretical conformational studies as well as the J experimental values between the hydroxymethyne hydrogens. The antimicrobial activity of the compounds was evaluated. Promising results were obtained for compounds 2, 4, and 5 since they inhibited the bacterium Pseudomonas aeruginosa, an opportunistic pathogen, suggesting the potential of these microorganisms as a source of new antibacterial agents.

Enhanced Cd activation by Coprinus comatus endophyte Bacillus thuringiensis and the molecular mechanism.

Fungal endophytes not only tolerate and activate Cd in soil but also promote host growth, yet its Cd activation capacity and mechanism remain unrevealed. Our previous study isolated a robust endophyte Bacillus thuringiensis L1 from Coprinus comatus fruiting body with splendid Cd resistance and activation abilities under laboratory conditions. In this study, those peculiarities were investigated in the actual soil environment. L1 could significantly increase the soil bioavailable Cd content and effectively compensate for alkali-hydro nitrogen losses and microbial inhibition caused by Cd. Furthermore, L1 inoculation improved the soil's bacterial community structure and increased the relative abundance of Cd-resistant bacteria, such as Actinobacteria, Chloroflexi, Acidobacter, and Firmicutes, closely associated with the soil enzyme activity shift. The genome sequencing analysis revealed the presence of genes related to growth promotion, resistance to Cd stress, and Cd activation, which were significantly up-regulated under Cd stress. Notably, L1 mainly activates Cd in soil by secreting citric acid, succinic acid, siderophore, and soluble phosphorus substances to chelate with Cd or dissolve bounded Cd. Meanwhile, the metal-responsive transcription repressor (CadC) and the Cd-translocating protein P-type ATPase (CadA) can help the L1 to suppress the toxicity of Cd. Those results help to unveil the possible mechanism of L1 in Cd-contaminated soil remediation, providing a clear strategy for Cd bio-extraction from soil.

Insight of endophytic fungi promoting the growth and development of woody plants.

Microorganisms play an important role in plant growth and development. In particular, endophytic fungi is one of the important kinds of microorganisms and has a mutually beneficial symbiotic relationship with host plants. Endophytic fungi have many substantial benefits to host plants, especially for woody plants, such as accelerating plant growth, enhancing stress resistance, promoting nutrient absorption, resisting pathogens and etc. However, the effects of endophytic fungi on the growth and development of woody plants have not been systematically summarized. In this review, the functions of endophytic fungi for the growth and development of woody plants have been mainly reviewed, including regulating plant growth (e.g., flowering, root elongation, etc.) by producing nutrients and plant hormones, and improving plant disease, insect resistance and heavy metal resistance by producing secondary metabolites. In addition, the diversity of endophytic fungi could improve the ability of woody plants to adapt to adverse environment. The components produced by endophytic fungi have excellent potential for the growth and development of woody plants. This review has systematically discussed the potential regulation mechanism of endophytic fungi regulating the growth and development of woody plants, it would be of great significance for the development and utilization of endophytic fungi resource from woody plants for the protection of forest resources.

Antifeedant, Antifungal Cryptic Polyketides with Six Structural Frameworks from Tea Endophyte Daldinia eschscholtzii Propelled by the Antagonistic Coculture with Phytopathogen Colletotrichum pseudomajus and Different Culture Methods.

The antagonistic coculture with tea phytopathogen Colletotrichum pseudomajus induces antifungal cryptic metabolites from isogenesis endophyte Daldinia eschscholtzii against tea phytopathogens. Sixteen new polyketides with six structural frameworks including ten cryptic ones, named coldaldols A-C (1-3), collediol (5), and daldinrins A-L (10-20 and 23), were found from the coculture of C. pseudomajus and D. eschscholtzii by different culture methods. The unique framework of compounds 11 and 12 featured a benzopyran-C7 polyketone hybrid, and compounds 13-16 were characterized by the novel benzopyran dimer. The structures were determined mainly by spectroscopic methods, including extensive one-dimensional (1D), two-dimensional (2D) NMR, high resolution electrospray ionisation mass spectroscopy (HRESIMS), ECD calculation, and single-crystal X-ray diffraction. The configuration of acyclic compounds 5 and 18 were determined by application of the universal NMR database. Most compounds showed significant antifungal activities against the tea pathogens C. pseudomajus and Alternaria sp. with MICs of 1-8 µg/mL. Compound 12 had stronger antifungal activity than that of positive drug nystatin. The ether bond at C-4 of the benzopyran derivative increased the antifungal activity. Compounds 4-9 and 11-23 showed antifeedant activities against silkworms with feeding deterrence indices of 15-100% at the concentration of 50 µg/cm2.

Epichloë fungal endophyte interactions in perennial ryegrass (Lolium perenne L.) modified to accumulate foliar lipids for increased energy density.

BACKGROUND: Commercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions. RESULTS: Expected relative increases in total fatty acids of 17-58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity. CONCLUSIONS: These results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species.

Bio Prospecting of Endophytes and PGPRs in Artemisinin Production for the Socio-economic Advancement.

The growing demand for Artemisia annua plants in healthcare, food, and pharmaceutical industries has led to increased cultivation efforts to extract a vital compound, Artemisinin. The efficacy of Artemisinin as a potent drug against malaria disease is well established but its limited natural abundance. However, the common practice of using chemical fertilizers for maximum yield has adverse effects on plant growth, development, and the quality of phytochemicals. To address these issues, the review discusses the alternative approach of harnessing beneficial rhizosphere microbiota, particularly plant growth-promoting rhizobacteria (PGPR). Microbes hold substantial biotechnological potential for augmenting medicinal plant production, offering an environmentally friendly and cost-effective means to enhance medicinal plant production. This review article aims to identify a suitable endophytic population capable of enabling Artemisia sp. to thrive amidst abiotic stress while simultaneously enhancing Artemisinin production, thereby broadening its availability to a larger population. Furthermore, by subjecting endophytes to diverse combinations of harsh conditions, this review sheds light on the modulation of essential artemisinin biosynthesis pathway genes, both up regulated and down regulated. The collective findings suggest that through the in vitro engineering of endophytic communities and their in vivo application to Artemisia plants cultivated in tribal population fields, artemisinin production can be significantly augmented. The overall aim of this review to explore the potential of harnessing microbial communities, their functions, and services to enhance the cultivation of medicinal plants. It outlines a promising path toward bolstering artemisinin production, which holds immense promise in the fight against malaria.

Plants and endophytes interaction: a "secret wedlock" for sustainable biosynthesis of pharmaceutically important secondary metabolites.

Many plants possess immense pharmacological properties because of the presence of various therapeutic bioactive secondary metabolites that are of great importance in many pharmaceutical industries. Therefore, to strike a balance between meeting industry demands and conserving natural habitats, medicinal plants are being cultivated on a large scale. However, to enhance the yield and simultaneously manage the various pest infestations, agrochemicals are being routinely used that have a detrimental impact on the whole ecosystem, ranging from biodiversity loss to water pollution, soil degradation, nutrient imbalance and enormous health hazards to both consumers and agricultural workers. To address the challenges, biological eco-friendly alternatives are being looked upon with high hopes where endophytes pitch in as key players due to their tight association with the host plants. The intricate interplay between plants and endophytic microorganisms has emerged as a captivating subject of scientific investigation, with profound implications for the sustainable biosynthesis of pharmaceutically important secondary metabolites. This review delves into the hidden world of the "secret wedlock" between plants and endophytes, elucidating their multifaceted interactions that underpin the synthesis of bioactive compounds with medicinal significance in their plant hosts. Here, we briefly review endophytic diversity association with medicinal plants and highlight the potential role of core endomicrobiome. We also propose that successful implementation of in situ microbiome manipulation through high-end techniques can pave the way towards a more sustainable and pharmaceutically enriched future.

The Root-Colonizing Endophyte Piriformospora indica Supports Nitrogen-Starved Arabidopsis thaliana Seedlings with Nitrogen Metabolites.

The root-colonizing endophytic fungus Piriformospora indica promotes the root and shoot growth of its host plants. We show that the growth promotion of Arabidopsis thaliana leaves is abolished when the seedlings are grown on media with nitrogen (N) limitation. The fungus neither stimulated the total N content nor did it promote 15NO3- uptake from agar plates to the leaves of the host under N-sufficient or N-limiting conditions. However, when the roots were co-cultivated with 15N-labelled P. indica, more labels were detected in the leaves of N-starved host plants but not in plants supplied with sufficient N. Amino acid and primary metabolite profiles, as well as the expression analyses of N metabolite transporter genes suggest that the fungus alleviates the adaptation of its host from the N limitation condition. P. indica alters the expression of transporter genes, which participate in the relocation of NO3-, NH4+ and N metabolites from the roots to the leaves under N limitation. We propose that P. indica participates in the plant's metabolomic adaptation against N limitation by delivering reduced N metabolites to the host, thus alleviating metabolic N starvation responses and reprogramming the expression of N metabolism-related genes.

Asteriquinones from Aspergillus sp. GZWMJZ-258 and Their Derivatives.

A new asteriquinone, ochrindole F (1), and five previously reported analogues (2-6) were isolated from the culture of the fungus Aspergillus sp. GZWMJZ-258, an endophyte of Garcinia multiflora. The structure of compound 1 was determined by a spectroscopic analysis. Furthermore, eight new derivatives (7-14) were synthesized from major metabolites 2 and 3. These compounds showed selective antiproliferative activity against the human acute myeloid leukemia (AML) cell line MV4-11, among which compound 12 showed the strongest activity with an IC50 value of 0.14 µM and the highest selectivity with a selectivity index greater than 710. An initial probe of the mechanism of action showed that compounds 12 and 14 could inhibit the expression of FLT-3 in the MV4-11 cell line.

Growth of alfalfa in the presence of metabolites from a dark septate endophyte strain Alternaria sp. 17463 cultured with a nonionic surfactant and emulsifier.

AIM: Dark septate endophytes (DSE) were widely used in the agriculture and ecological restoration. The objective of this work was to assess the effect of culture media nonionic surfactant and emulsifier on the biomass and metabolites of DSE strain Alternaria sp. 17463. METHODS AND RESULTS: Changes in the composition of DSE metabolites following the addition of Tween 80 during liquid culture of a DSE fungus were analyzed and used in growth tests of alfalfa.Shaking flask fermentation was carried out and the surfactant was fed to the fungus during the fermentation. The residual sugar content and pH declined significantly in the medium and the biomass of DSE increased by 7.27% over controls with no surfactant. Metabolomic analysis showed that adding the surfactant significantly increased the content of 63 metabolites (P < 0.05). These include lipids and lipid-like molecules, organooxygen compounds, amino acids and organic acids, and flavonoids. Enrichment analysis of metabolic pathways indicates that surfactant addition promoted carbohydrate metabolism and amino acid synthesis. A plant hydroponic experiment indicated that these changes in metabolites altered the root structure of alfalfa seedlings. They also promoted significant increases in root length and root surface area, and increased alfalfa total biomass by 50.2%. CONCLUSIONS: The addition of the surfactant promoted sugar utilization by the DSE fungus and increased the synthesis of lipids and amino acids, resulting in the ability of the fungal metabolites to change root structure and promote plant growth.

Diversity, Antimicrobial, Antioxidant, and Anticancer Activity of Culturable Fungal Endophyte Communities in Cordia dichotoma.

Endophytic fungi are a significant source of secondary metabolites, which are chemical compounds with biological activities. The present study emphasizes the first-time isolation and identification of such fungi and their pharmacological activities from the medicinal plant Cordia dichotoma, which is native to Jammu, India. The Shannon Wiener diversity index revealed a wide range of fungal endophytes in root (1.992), stem (1.645), and leaf (1.46) tissues. A total of 19 endophytic fungi belonging to nine different genera were isolated from this plant and the majority belonged to the Ascomycota phylum. ITS rRNA gene sequencing was used to identify the fungal strains and they were submitted in NCBI GenBank. The most potent fungal isolate Cladosporium cladosporioides OP870014 had strong antimicrobial, antioxidant, and anticancer activity against MCF-7, HCT-116, and PC-3 cancer cell lines. The LC-MS and GC-MS analyses of the ethyl acetate extract of C. cladosporioides were examined to identify the bioactive metabolites. The major compounds of the crude extract derived from C. cladosporioides OP870014, according to GC-MS, are spiculisporic acid; dibutyl phthalate; phenylethyl alcohol; cyclohexanone, 2,3,3-trimethyl-2-3-methylbutyl; pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-3-(phenylmethyl);2,5-piperazinedione,3,6-bis(2-methylpropyl); and heneicosane which possessed antimicrobial, anticancerous, and antioxidant activities. The findings revealed that C. dichotoma has the capacity to host a wide variety of fungal endophytes and that secondary metabolites from the endophytic fungus may be a source of alternative naturally occurring antimicrobial, antioxidant, and cytotoxic compounds.