Role of grass endophytic fungi as a natural resource of bioactive metabolites.

Grass endophytic fungi have garnered increasing attention as a prolific source of bioactive metabolites with potential application across various fields, including pharmaceticals agriculture and industry. This review paper aims to synthesize knowledge on the diversity, isolation, and bioactivity of metabolites produced by grass endophytic fungi. Additionally, this approach aids in the conservation of rare and endangered plant species. Advanced analytical techniques such as high-performance liquid chromatography, liquid chromatograpy-mass spectrometry and gas chromatography are discussed as critical tools for metabolite identification and characterization. The review also highlights significant bioactive metabolites discovered to date, emphasizing their antimicrobial, antioxidant, and insecticidal activities and plant growth regulation properties. Besides address the challenges and future prospects in harnessing grass endophytic fungi for sustainable biotenological applications. By consolidating recent advancements and identifying agaps in the current research, this paper provides a comprehensive overview of the potential grass endophytic fungi as a valuable resource for novel bioactive compounds.

Myco-nanotechnological approach to synthesize gold nanoparticles using a fungal endophyte, Penicillium oxalicum and unravelling their antibacterial activity and anti-breast cancer role via metabolic reprogramming.

The present study has been designed to fabricate fungal endophyte assisted gold nanoparticles and elucidate their anti-breast cancer potential. The aqueous extract of fungal endophyte, Penicillium oxalicum, associated with the medicinal plant Amoora rohituka has been used for the fabrication of gold nanoparticles (POAuNPs). The physicochemical characterization using UV-Vis spectroscopy, FTIR, XRD, DLS, Zeta potential, TEM and FESEM analysis revealed stable, uniform distribution, spherical-shape and crystalline nature of POAuNPs with size range of 3-46 nm. Further, POAuNPs potentially inhibited the growth of pathogenic bacterial strains, E. coli and S. aureus. The synthesized POAuNPs has shown potential antioxidant effects against DPPH, superoxide and nitric oxide radical scavenging assay with an EC50 value of 8.875±0.082, 52.593±2.506 and 43.717±1.449 µg/mL, respectively. Moreover, the value of EC50 for total antioxidant capacity of POAuNPs was found to be 23.667±1.361 µg/mL. The cell viability of human breast cancer cells, MDA-MB-231 and MCF-7 was found to be reduced after treatment with POAuNPs and IC50 values were found to be 19.753±0.640 and 35.035±0.439 µg/mL respectively. Further, in vitro biochemical assays revealed POAuNPs induced metabolic reprogramming in terms of reduced glucose uptake and increased LDH release and, disruption of oxidative balance through depletion of GSH level, increased nitric oxide level and lipid peroxidation as a possible pathway to suppress the human breast cancer cell proliferation. Apoptosis-specific nuclear modulations induced by POAuNPs in human breast cancer cells were validated through DAPI nuclear staining. The present investigation thus attempted to show first ever fabrication of gold nanoparticles using aqueous extract of P. oxalicum associated with A. rohituka. The results revealed unique physico-chemical characteristics of myogenic gold nanoparticles and screening their effect against breast cancer via metabolic reprogramming and induction of apoptosis thus adds great significance against cancer therapeutics, suggesting further exploration to develop nanotherapeutic drugs.

Bioprospecting of exopolysaccharides from the endophytic fungi Epicoccum sorghinum AMFS4, for its structure, composition, bioactivities and application in seed priming.

The endophytic fungi, Epicoccum sorghinum AMFS4 was investigated for its metabolic components and composition of bioactive exopolysaccharides (EPS). Metabolic analysis of the ethyl acetate extract majorly detected sugars derivatives such as, 4-Cholesten-3-one semicarbazone (20.9%), d-Fructose (18.96%), and α-d-Galactopyranosiduronicacid (1.71%). The growth curve and EPS yield were determined as 12.22 ± 1.02 g/L and 7.41 ± 0.32 g/L (dry weight) respectively on day 8. The deproteined EPS has been characterised with pyranose ring linked by α-glycosidic bonds, composing fructose, galactose and glucose monosaccharides validated by HPLC. Total sugar content was found to be 93.18 ± 0.81% with detection of proteins and uronate. The viscous EPS appeared filamentous under SEM observation and behaves as emulsifier with notable antioxidant properties. Priming of EPS on tomato seeds showed early induction of secondary rooting than in the control seedlings. Thus, E. sorghinum AMFS4 synthesises bioactive EPS with simple carbohydrate structure, good water absorption and significant metabolic influence on seed germination.

Secondary metabolites from Penicillium sp. HS-11, a fungal endophyte of Huperzia serrata.

Three new sorbicillinoids sorbicatechols E-G (1-3), along with seven known compounds 4-10, were obtained from the ethanol extract of Penicillium sp. HS-11, a fungal endophyte of the medicinal plant Huperzia serrata. The structures of 1-3 were established by detailed interpretation of the spectroscopic data and their absolute configurations were established by comparative analyses of the ECD spectra. Sorbicatechol G (3) represented the first hybrid sorbicillinoid bearing a tetralone skeleton. In the in-vitro bioassay, trichodimerol (5) exhibited moderate inhibitory activity against the Escherichia coli ß-glucuronidase (EcGUS) with an IC50 value of 92.0 ± 9.4 µM.

Weather in two climatic regions shapes the diversity and drives the structure of fungal endophytic community of bilberry (Vaccinium myrtillus L.) fruit.

BACKGROUND: Bilberry (Vaccinium myrtillus L.) is one of the most important economic and natural resources in Northern Europe. Despite its importance, the endophytic fungal community of the fruits has rarely been investigated. Biogeographic patterns and determinants of the fungal diversity in the bilberry fruit are poorly understood, albeit fungal endophytes can have a close relationship with the host plants. Here, we investigated the effect of climatic regions, and their weather conditions within growth season and soil properties on fungal endophytic communities of bilberry fruits collected from northern and southern regions of Finland using high-throughput sequencing technology targeting the internal transcribed spacer 2 ribosomal DNA region for fungi. RESULTS: Species richness and beta diversity (variation in community structure) were higher in the southern compared to the studied northern region. The weather condition of the growth season drove both fungal richness and community structure. Furthermore, abundance of the genera Venturia, Cladosporium, and Podosphaera was influenced by the weather, being different between the south and north regions. CONCLUSIONS: We conclude that diversity and assembly structure of the fungal endophytes in bilberry fruits follow similar patterns as for foliar fungal endophytes, being shaped by various environmental factors, such as the climate and surrounding vegetation.

Harnessing fungal endophytes for natural management: a biocontrol perspective.

In the ever-evolving realm of agriculture, the convoluted interaction between plants and microorganisms have assumed paramount significance. Fungal endophytes, once perceived as mere bystanders within plant tissues, have now emerged as dynamic defenders of plant health. This comprehensive review delves into the captivating world of fungal endophytes and their multifaceted biocontrol mechanisms. Exploring their unique ability to coexist with their plant hosts, fungal endophytes have unlocked a treasure trove of biological weaponry to fend off pathogens and enhance plant resilience. From the synthesis of bioactive secondary metabolites to intricate signaling pathways these silent allies are masters of biological warfare. The world of fungal endophytes is quite fascinating as they engage in a delicate dance with the plant immune system, orchestrating a symphony of defense that challenges traditional notions of plant-pathogen interactions. The journey through the various mechanisms employed by these enigmatic endophytes to combat diseases, will lead to revelational understanding of sustainable agriculture. The review delves into cutting-edge research and promising prospects, shedding light on how fungal endophytes hold the key to biocontrol and the reduction of chemical inputs in agriculture. Their ecological significance, potential for bioprospecting and avenues for future research are also explored. This exploration of the biocontrol mechanisms of fungal endophytes promise not only to enrich our comprehension of plant-microbe relationships but also, to shape the future of sustainable and ecofriendly agricultural practices. In this intricate web of life, fungal endophytes are indeed the unsung heroes, silently guarding our crops and illuminating a path towards a greener, healthier tomorrow.

Biochemical Screening, Fabrication of Green Nanoparticles and Its Antimicrobial, and Antioxidant Studies of Endophytic Fungus Phlebia Species.

Endophytes are organism dwelling totally dynamic and novel biotopes this makes them able to produce novel biochemicals that may become assets to the future. This study aims at understanding the biochemical components of the endophytic fungus Phlebia sp. synthesis of gold and silver nanoparticles from it, and the antimicrobial as well as antioxidant ability of these green synthesised nanoparticles. Aqueous fungal extract was subjected for HRLCMS analysis which revealed 34 biochemicals within the extract. Silver and gold nanoparticles were also produced from the fungal extract. UV-vis analysis revealed a peak at 450 nm for silver nanoparticle and 550 nm for gold nanoparticles. FESEM analysis confirmed the presence of these nanoparticles with its spherical shape. Both of these nanoparticles were able to produce a conspicuous zone of inhibition in the antimicrobial tests against Escherichia coli, Salmonella paratyphi. For both of the organisms under study, a concentration-dependent expansion of the zone of inhibition was discovered in the nanoparticles. However, with silver nanoparticles, a relatively high zone of inhibition and vulnerability of the organism was discovered. Four in vitro free radical scavenging assays, including the DPPH, Hydroxyl, Superoxide, and Nitric oxide radical scavenging assays, were used for antioxidant analysis. The results of every test demonstrated that green synthesised silver nanoparticles had higher activity than gold nanoparticles. All of the tests showed that silver nanoparticles were more active than gold nanoparticles with the maximum value of 86.254 ± 0.296% being discovered at the greatest concentration of superoxide radical scavenging assay.

Exploration of in vitro cytotoxic and in ovo antiangiogenic activity of ethyl acetate extract of Penicillium oxalicum.

Fungal endophytes have established new paradigms in the area of biomedicine due to their ability to produce metabolites of pharmacological importance. The present study reports the in vitro cytotoxic and in ovo antiangiogenic activity of the ethyl acetate (EA) extract of Penicillium oxalicum and their chemical profiling through Gas Chromatography-Mass Spectrometry analysis. Treatment of the EA extract of P. oxalicum to the selected human breast cancer cell lines (MDA-MB-231 and MCF-7) leads to the reduced glucose uptake and increased nitric oxide production suggesting the cytotoxic activity of EA extract of P. oxalicum. Our results further show that treatment of EA extract of P. oxalicum attenuates the colony number, cell migration ability and alters nuclear morphology in both the human breast cancer cell lines. Furthermore, the treatment of EA extract of P. oxalicum mediates apoptosis by increasing the expression of BAX, P21, FADD, and CASPASE-8 genes, with increased Caspase-3 activity. Additionally, in ovo chorioallantoic membrane (CAM) assay showed that the treatment of EA extract of P. oxalicum leads to antiangiogenic activity with perturbed formation of blood vessels. Overall, our findings suggest that the EA extract of P. oxalicum show in vitro cytotoxic and antiproliferative activity against human breast cancer cell lines, and in ovo antiangiogenic activity in CAM model.

Biocontrol potential of endophytic fungi originated from grapevine leaves for management of anthracnose disease caused by Colletotrichum gloeosporioides.

In the present study, 51 fungal endophytes (FEs) were isolated, purified and identified from the healthy leaf segments of ten grapevine varieties based on the spore and colony morphologies and ITS sequence information. The FEs belonged to the Ascomycota division comprising eight genera viz., Alternaria, Aspergillus, Bipolaris, Curvularia, Daldinia, Exserohilum, Fusarium and Nigrospora. The in vitro direct confrontation assay against Colletotrichum gloeosporioides revealed that six isolates viz., VR8 (70%), SB2 (83.15%), CS2 (88.42%), MN3 (88.42%), MS5 (78.94%) and MS15 (78.94%) inhibited the mycelial growth of test pathogen. The remaining 45 fungal isolates showed 20-59.9% growth inhibition of C. gloeosporioides. Indirect confrontation assay manifested that the isolates MN1 and MN4a showed 79.09% and 78.18% growth inhibition of C. gloeosporioides followed by MM4 (73.63%) and S5 (71.81%) isolates. Isolate S5 and MM4 were found to produce azulene and 1,3-Cyclopentanedione, 4,4-dimethyl as antimicrobial volatile organic compounds, respectively. The 38 FEs showed PCR amplification using internal transcribed spacer universal primers. The BLAST search revealed highest similarity with the existing sequences in the database. The phylogenetic analysis revealed the occurrence of seven distinct clusters each corresponding to single genus. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03675-z.

The Epichloë festucae Antifungal Protein Efe-AfpA Has Activity against Numerous Plant Pathogens.

Fungal plant pathogens can present major problems for most crop species. Currently, control of fungal diseases relies heavily on the use of fungicides. However, there are problems associated with fungicide use, including potential toxicity to non-target organisms and the development of resistance in the target fungus. New strategies are being sought to reduce fungicide use. One area of active research is the potential use of antifungal proteins from various fungal species as alternatives or complements to traditional fungicides. An antifungal protein, Efe-AfpA, from the fungal endophyte Epichloë festucae was previously found to protect plants from the pathogen Clarireedia jacksonii, the causal agent of dollar spot disease. Here we report that Efe-AfpA also has inhibitory activity against other important plant pathogens. These results suggest that it may be possible to develop Efe-AfpA as a biofungicide to target a broad range of destructive plant pathogens.

Stable coexistence or competitive exclusion? Fern endophytes demonstrate rapid turnover favouring a dominant fungus.

Fungal endophytes are critical members of the plant microbiome, but their community dynamics throughout an entire growing season are underexplored. Additionally, most fungal endophyte research has centred on seed-reproducing hosts, while spore-reproducing plants also host endophytes and may be colonized by unique community members. In order to examine annual fungal endophyte community dynamics in a spore-reproducing host, we explored endophytes in a single population of ferns, Polystichum munitum, in the Pacific Northwest. Through metabarcoding, we characterized the community assembly and temporal turnover of foliar endophytes throughout a growing season. From these results, we selected endophytes with outsized representations in sequence data and performed in vitro competition assays. Finally, we inoculated sterile fern gametophytes with dominant fungi observed in the field and determined their effects on host performance. Sequencing demonstrated that ferns were colonized by a diverse community of fungal endophytes in newly emerged tissue, but diversity decreased throughout the season leading to the preponderance of a single fungus in later sampling months. This previously undescribed endophyte appears to abundantly colonize the host to the detriment of other microfungi. Competition assays on a variety of media types failed to demonstrate that the dominant fungus was competitive against other fungi isolated from the same hosts, and inoculation onto sterile fern gametophytes did not alter growth compared to sterile controls, suggesting its effects are not antagonistic. The presence of this endophyte in the fern population probably demonstrates a case of repeated colonization driving competitive exclusion of other fungal community members.

Does the Degree of Mutualism between Epichloë Fungi and Botanophila Flies Depend upon the Reproductive Mode of the Fungi?

Epichloë (Ascomycota: Clavicipitaceae) fungi can form an intriguing interaction with Botanophila flies. The fungi live within above-ground shoots of grasses. Some species (type I) only reproduce sexually by forming stromata on all host culms (choke disease). Stromata produce haploid spores (spermatia) that fertilize stromata of opposite mating type to form dikaryotic cells. A second category of Epichloë species (type II) produces stromata on only some of the host culms; culms without choke produce flowers and seeds. These Epichloë can reproduce asexually by invading host seed, as well as sexually. Female Botanophila flies visit stromata for feeding and oviposition. Spermatia pass through the gut of Botanophila intact and viable. Flies can cross-fertilize the fungus during defecation after egg laying. Hence, we described the interaction as a mutualism similar to pollination. Yet, subsequent work by others and ourselves showed that visitation by Botanophila flies was not necessary for cross fertilization of Epichloë. We believe these contradictory results can be reconciled from an evolutionary perspective, if one takes into account the reproductive mode of the fungus. We explore a novel hypothesis to reconcile this contradiction, its predictions and discuss ways in which to test them.

The culturable seed mycobiome of two Banksia species is dominated by latent saprotrophic and multi-trophic fungi.

Seed fungal endophytes play an important beneficial role in the formation of the seedling mycobiome and contribute to plant establishment, but can also occur as latent pathogens and saprotrophs. Current knowledge on the function and diversity of seed fungal endophytes has been gained through studies in agricultural systems whilst knowledge from natural systems is relatively less. We used two co-occurring species from the genus Banksia from four sites in Australia's Sydney Basin Bioregion to investigate the abundance and diversity of seed fungal endophyte communities present in natural ecosystem hosts. Based on results from culturing and DNA sequence analysis of multiple loci, we found that Banksia seeds house a diverse range of fungal endophyte species, that when assigned to functional guilds belonged to multiple trophic modes. Thirty-one of the fungal taxa identified had not been previously reported as endophytes. Amongst the 58 Operational Taxonomic Units identified, Leotiomycetes and Sordariomycetes were the dominant classes and Banksiamyces (Leotiomycetes) and Penicillium (Sordariomycetes) the dominant genera, with many of the species isolated recorded in the literature as having a limited distribution. The two Banksias shared few fungal endophyte species, which were not always present across all study sites. We revealed a 'hidden diversity' within seeds of Banksia from natural ecosystems and provided insights into the influence host species can have on the seed mycobiome.

Epichloë seed transmission efficiency is influenced by plant defense response mechanisms.

Asexual Epichloë are endophytic fungi that form mutualistic symbioses with cool-season grasses, conferring to their hosts protection against biotic and abiotic stresses. Symbioses are maintained between grass generations as hyphae are vertically transmitted from parent to progeny plants through seed. However, endophyte transmission to the seed is an imperfect process where not all seeds become infected. The mechanisms underpinning the varying efficiencies of seed transmission are poorly understood. Host gene expression in response to Epichloë sp. LpTG-3 strain AR37 was examined within inflorescence primordia and ovaries of high and low endophyte transmission genotypes within a single population of perennial ryegrass. A genome-wide association study was conducted to identify population-level single nucleotide polymorphisms (SNPs) and associated genes correlated with vertical transmission efficiency. For low transmitters of AR37, upregulation of perennial ryegrass receptor-like kinases and resistance genes, typically associated with phytopathogen detection, comprised the largest group of differentially expressed genes (DEGs) in both inflorescence primordia and ovaries. DEGs involved in signaling and plant defense responses, such as cell wall modification, secondary metabolism, and reactive oxygen activities were also abundant. Transmission-associated SNPs were associated with genes for which gene ontology analysis identified "response to fungus" as the most significantly enriched term. Moreover, endophyte biomass as measured by quantitative PCR of Epichloë non-ribosomal peptide synthetase genes, was significantly lower in reproductive tissues of low-transmission hosts compared to high-transmission hosts. Endophyte seed-transmission efficiency appears to be influenced primarily by plant defense responses which reduce endophyte colonization of host reproductive tissues.

The Epichloë festucae Antifungal Protein Efe-AfpA Protects Creeping Bentgrass (Agrostis stolonifera) from the Plant Pathogen Clarireedia jacksonii, the Causal Agent of Dollar Spot Disease.

Dollar spot disease, caused by the fungal pathogen Clarireedia jacksonii, is a major problem in many turfgrass species, particularly creeping bentgrass (Agrostis stolonifera). It is well-established that strong creeping red fescue (Festuca rubra subsp. rubra) exhibits good dollar spot resistance when infected by the fungal endophyte Epichloë festucae. This endophyte-mediated disease resistance is unique to the fine fescues and has not been observed in other grass species infected with other Epichloë spp. The mechanism underlying the unique endophyte-mediated disease resistance in strong creeping red fescue has not yet been established. We pursued the possibility that it may be due to the presence of an abundant secreted antifungal protein produced by E. festucae. Here, we compare the activity of the antifungal protein expressed in Escherichia coli, Pichia pastoris, and Penicillium chrysogenum. Active protein was recovered from all systems, with the best activity being from Pe. chrysogenum. In greenhouse assays, topical application of the purified antifungal protein to creeping bentgrass and endophyte-free strong creeping red fescue protected the plants from developing severe symptoms caused by C. jacksonii. These results support the hypothesis that Efe-AfpA is a major contributor to the dollar spot resistance observed with E. festucae-infected strong creeping red fescue in the field, and that this protein could be developed as an alternative or complement to fungicides for the management of this disease on turfgrasses.

Correlation in endophytic fungi community diversity and bioactive compounds of Sophora alopecuroides.

Sophora alopecuroides L. is a traditional Chinese medicine used for the treatment of several different disease states including bacillary dysentery and enteritis. But importantly, it also plays a role as an anti-tumor agent. That said, little is known about the role endophytes play regarding the clinically bioactive metabolites in S. alopecuroides. In order to explore the effects of endophytic fungi on the accumulation, quality, and correlation in the content of the medicinal compounds, the structural diversity of endophytic fungi in S. alopecuroides was analyzed. The relationship between endophytes and quinolizidine alkaloids (QAs), housed within the seeds of S. alopecuroides, which were interpreted based on established methods of high-throughput sequencing and high-performance liquid chromatography. A total of 1,034,418 effective sequence reads and 257 operational taxonomic units (OTUs) were obtained from 33 samples which were sourced from 11 different sampling sites and further classified into 9 phyla, 20 classes, 45 orders, 85 families, and 118 genera. Ascomycota was found to be the dominant phylum of endophytic fungi in S. alopecuroides, with a relative abundance ranging from 60.85 to 98.30%. Alternaria, Cladosporium, Filobasidium, and an unidentified Ascomycota were the core-shared endophytes, accounting for 49.96, 27.12, 14.83, and 7.88%, respectively. Correlation analysis showed that the Simpson's diversity index of endophytic fungal community in S. alopecuroides was significantly positively correlated with the Oxymatrine (OMA) content in different areas, while the Chao and Shannoneven indexes were significantly negatively correlated with OMA. The endophytic fungi of Alternaria were positively correlated with the content of OMA, Oxysophocarpine (OSC), and total QAs. This study has mastered the endophytic fungi resources of S. alopecuroides, explored potential functional endophytic fungi, and provided a scientific basis for using biological fertilization strategies to improve the quality of S. alopecuroides.

Germicide Fenaminosulf Promots Gall Formation of Zizania latifolia without directly affecting the growth of endophytic fungus Ustilago esculenta.

Zizania latifolia is a popular aquatic vegetable in China because of its enlarged edible stems resulting from persistent infection by a fungal endophyte, Ustilago esculenta. Fenaminosulf (FM) is a germicide that can be used to improve agricultural crop yields. In Z. latifolia fields, appropriate spraying of FM not just controls diseases, but also promotes an earlier harvest of Z. latifolia. In this study, we show that the timing of gall formation was advanced and the plant's yield was increased significantly under a high concentration treatment of FM. Yet FM had a strong inhibitory effect on the growth of U. esculenta in vitro, while the transcript levels of mating-type alleles, cell metabolism-related genes and chitin synthase genes were all substantially downregulated. Through a transcriptome analysis, we investigated changes in gene expression of the host Z. latifolia and fungal endophyte U. esculenta in response to FM. FM directly affected the growth of Z. latifolia by altering the expression level of genes involved in plant-pathogen interactions, plant hormone signal transduction and some metabolism pathways. By contrast, FM had little effect on U. esculenta growing inside of Z. latifolia. Collectively, our results provide a more in-depth understanding of the molecular processes that promote gall formation in Z. latifolia, while also identifying potential targets for genetic manipulation to improve the yield and quality of Z. latifolia, in a safer and more effective way.

Leaf photosynthesis: do endophytes have a say?

Endophytes, both bacterial and fungal, constitute an integral component of the leaf ecosystem. Here we argue that the respiratory metabolism of endophytes in the intercellular spaces of leaves could have a significant role in enhancing leaf photosynthesis by enriching the internal CO2 concentration, especially in C3 plants.

Endophytic fungi and their bioactive secondary metabolites in medicinal leguminosae plants: nearly untapped medical resources.

There are many species of Chinese traditional leguminosae family plants that are well known for their medicinal applications, such as Astragalus membranaceus, Catsia tora, Glycyrrhiza uralensis, Sophora flavescens and Albacia acacia. Their unique bioactive composition and internal phenological environment contribute to the formation of specific and unique endophytic fungal communities, which are important resources for new compounds used in a variety of pharmacological activities. Nonetheless, they have not been systematically studied. In the last decade, nearly 64 genera and thousands of species of endophytic fungi have been discovered from leguminosae plants, as well as 138 secondary metabolites (with 34 new compounds) including flavonoid, alkaloids, phenol, anthraquinone, macrolide, terpenoid, phytohormone and many more. These were shown to have diverse applications and benefits, such as antibacterial, antitumor, antioxidative, immunoregulatory and neuroprotective properties. Here, we provide a summarized overview with the aim of raising awareness of endophytic fungi from medicinal leguminosae plants and providing a comprehensive review of the discoveries of new natural products that may be of medicinal and pharmaceutical importance.