Surface sterilization for isolation of endophytes: Ensuring what (not) to grow.

Endophytic microbiota opens a magnificent arena of metabolites that served as a potential source of medicines for treating a variety of ailments and having prospective uses in agriculture, food, cosmetics, and many more. There are umpteen reports of endophytes improving the growth and tolerance of plants. In addition, endophytes from lifesaving drug-producing plants such as Taxus, Nothapodytes, Catharanthus, and so forth have the ability to produce host mimicking compounds. To harness these benefits, it is imperative to isolate the true endophytes, not the surface microflora. The foremost step in endophyte isolation is the removal of epiphytic microbes from plant tissues, called as surface sterilization. The success of surface sterilization decides "what to grow" (the endophytes) and "what not to grow" (the epiphytes). It is very crucial to use an appropriate sterilant solution, concentration, and exposure time to ensure thorough surface disinfection with minimal damage to the endophytic diversity. Commonly used surface sterilants include sodium hypochlorite (2%-10%), ethanol (70%-90%), mercuric chloride (0.1%), formaldehyde (40%), and so forth. In addition, the efficiency could further be improved by pretreatment with surfactants such as Triton X-100, Tween 80, and Tween 20. This review comprehensively deals with the various sterilants and sterilization methods for the isolation of endophytic microbes. In addition, the mechanisms and rationale behind using specific surface sterilants have also been elaborated at length.

Seasonal variation of potentially toxic metal contamination in Yamuna riverine ecosystem, Delhi, India.

The River Yamuna in Delhi region, the National Capital Territory (NCT) of India, carries potentially toxic metals such as Cr, Pb, Mn, Mg, Hg, Fe and Zn. These contaminants are discharged mainly from industrial wastes, agricultural and household activities and domestic sewage. A total of 12 stations (2.5 to 3.5 km apart from each other) were selected for the study, covering the upstream and downstream areas of river Yamuna in Delhi. The investigated sites were evaluated for significant difference between upstream and downstream locations of river Yamuna in three different time periods (June, October, February). Metal contamination were measured in water, sediments (2 µm) and nearby agriculture soil of the river Yamuna, and found with high metal loads as compared with the international standards, chiefly in the downstream sites as the river flows through the Delhi stretch. The multivariate statistical analysis revealed spatial and temporal variations in the metal concentrations which suggest seasonal variation and common point source of some metals while different sources of other metals. The contamination of the river water and adjoining agriculture soils points towards possible entry of these metals into the food chain. The study indicates that considering the current status of metal pollution, the surface water is not in good conditions for use as drinking purpose because of the high concentrations of few potentially toxic metals. Our study recommends regular monitoring of toxic metals in Yamuna river water and sediments, strict ban on the domestic, agriculture and industrial waste disposal for the restoration of the river to its natural state.

Fungal Endophytes as Efficient Sources of Plant-Derived Bioactive Compounds and Their Prospective Applications in Natural Product Drug Discovery: Insights, Avenues, and Challenges.

Fungal endophytes are well-established sources of biologically active natural compounds with many producing pharmacologically valuable specific plant-derived products. This review details typical plant-derived medicinal compounds of several classes, including alkaloids, coumarins, flavonoids, glycosides, lignans, phenylpropanoids, quinones, saponins, terpenoids, and xanthones that are produced by endophytic fungi. This review covers the studies carried out since the first report of taxol biosynthesis by endophytic Taxomyces andreanae in 1993 up to mid-2020. The article also highlights the prospects of endophyte-dependent biosynthesis of such plant-derived pharmacologically active compounds and the bottlenecks in the commercialization of this novel approach in the area of drug discovery. After recent updates in the field of 'omics' and 'one strain many compounds' (OSMAC) approach, fungal endophytes have emerged as strong unconventional source of such prized products.

IMA Genome - F13: Draft genome sequences of Ambrosiella cleistominuta, Cercospora brassicicola, C. citrullina, Physcia stellaris, and Teratosphaeria pseudoeucalypti.

Draft genomes of the fungal species Ambrosiella cleistominuta, Cercospora brassicicola, C. citrullina, Physcia stellaris, and Teratosphaeria pseudoeucalypti are presented. Physcia stellaris is an important lichen forming fungus and Ambrosiella cleistominuta is an ambrosia beetle symbiont. Cercospora brassicicola and C. citrullina are agriculturally relevant plant pathogens that cause leaf-spots in brassicaceous vegetables and cucurbits respectively. Teratosphaeria pseudoeucalypti causes severe leaf blight and defoliation of Eucalyptus trees. These genomes provide a valuable resource for understanding the molecular processes in these economically important fungi.

Harnessing the Phytotherapeutic Treasure Troves of the Ancient Medicinal Plant Azadirachta indica (Neem) and Associated Endophytic Microorganisms.

Azadirachta indica, commonly known as neem, is an evergreen tree of the tropics and sub-tropics native to the Indian subcontinent with demonstrated ethnomedicinal value and importance in agriculture as well as in the pharmaceutical industry. This ancient medicinal tree, often called the "wonder tree", is regarded as a chemical factory of diverse and complex compounds with a plethora of structural scaffolds that is very difficult to mimic by chemical synthesis. Such multifaceted chemical diversity leads to a fantastic repertoire of functional traits, encompassing a wide variety of biological activity and unique modes of action against specific and generalist pathogens and pests. Until now, more than 400 compounds have been isolated from different parts of neem including important bioactive secondary metabolites such as azadirachtin, nimbidin, nimbin, nimbolide, gedunin, and many more. In addition to its insecticidal property, the plant is also known for antimicrobial, antimalarial, antiviral, anti-inflammatory, analgesic, antipyretic, hypoglycaemic, antiulcer, antifertility, anticarcinogenic, hepatoprotective, antioxidant, anxiolytic, molluscicidal, acaricidal, and antifilarial properties. Notwithstanding the chemical and biological virtuosity of neem, it has also been extensively explored for associated microorganisms, especially a class of mutualists called endophytic microorganisms (or endophytes). More than 30 compounds, including neem "mimetic" compounds, have been reported from endophytes harbored in the neem trees in different ecological niches. In this review, we provide an informative and in-depth overview of the topic that can serve as a point of reference for an understanding of the functions and applications of a medicinal plant such as neem, including associated endophytes, within the overall theme of phytopathology. Our review further exemplifies the already-noted current surge of interest in plant and microbial natural products for implications both within the ecological and clinical settings, for a more secure and sustainable future.

Chrysophanol: A Natural Anthraquinone with Multifaceted Biotherapeutic Potential.

Chrysophanol is a unique anthraquinone having broad-spectrum therapeutic potential along with ecological importance. It is the first polyketide that has been reported to be biosynthesized in an organism-specific manner. The traditional Chinese and Korean medicinal systems provide evidence of the beneficial effects of chrysophanol on human health. The global distribution of chrysophanol encountered in two domains of life (bacteria and eukaryota) has motivated researchers to critically evaluate the properties of this compound. A plethora of literature is available on the pharmacological properties of chrysophanol, which include anticancer, hepatoprotective, neuroprotective, anti-inflammatory, antiulcer, and antimicrobial activities. However, the pharmacokinetics and toxicity studies on chrysophanol demand further investigations for it to be used as a drug. This is the first comprehensive review on the natural sources, biosynthetic pathways, and pharmacology of chrysophanol. Here we reviewed recent advancements made on the pharmacokinetics of the chrysophanol. Additionally, we have highlighted the knowledge gaps of its mechanism of action against diseases and toxicity aspects.

Coating with Microbial Hydrophobins: A Novel Approach to Develop Smart Drug Nanoparticles.

Microbial hydrophobin (MH)-based surface coating is emerging as a novel protein engineering approach for drug nanoparticles to enhance the solubility and stability of therapeutic agents. These hydrophobins are amphiphilic proteins that can form self-assembled monolayers on hydrophobic materials and can coat nanoparticles for efficient drug delivery.

Isolation and Characterization of "Terrein" an Antimicrobial and Antitumor Compound from Endophytic Fungus Aspergillus terreus (JAS-2) Associated from Achyranthus aspera Varanasi, India.

The present study aimed at characterizing biological potentials of endophyte Aspergillus terreus JAS-2 isolated from Achyranthus aspera. Crude extracted from endophytic fungus JAS-2 was purified and chemically characterized by chromatographic and spectroscopic studies respectively. Spectral assignment of NMR (nuclear magnetic resonance) data, 1H proton and 13C carbon analysis along with FTIR data elucidated the structure of compound as 4,5-Dihydroxy-3-(1-propenyl)-2-cyclopenten-1-one. After purification and identification a set of experiment was conducted to explore efficacy of compound. Results revealed that on accessing the antifungal activity of compound, growth diameter of tested phytopathogenic fungi was reduced to 50% at higher concentration taken (10 µgµl-1). Compound exhibited in-vitro bacterial cell inhibition at 20 µgml-1 concentration along with moderate antioxidant behavior. Evaluation of anticancer activity against human lung cancer cell line (A-549) exhibited its IC50 value to be 121.9 ± 4.821 µgml-1. Further cell cycle phase distribution were analyzed on the basis of DNA content and evaluated by FACS (Fluorescence Activated Cell Sorting) and it was revealed that at 150 µgml-1 of compound maximum cells were found in sub G1 phase which represents apoptotic dead cells. Terrein (4, 5-Dihydroxy-3-(1-propenyl)-2-cyclopenten-1-one) a multi-potential was isolated from endophytic fungus JAS-2, from well recognized medicinal herb A. aspera. To best of our knowledge, this is the first report of "Terrein" from endophytic derived fungus. This compound had also exhibited anticancer and antifungal activity against human lung cancer cell line A-549 and Bipolaris sorokiniana respectively which is causal organism of many plants disease. Hence endophytes are serving as alternative sources of drug molecules.

Induction of Cryptic and Bioactive Metabolites through Natural Dietary Components in an Endophytic Fungus Colletotrichum gloeosporioides (Penz.) Sacc.

Grape skin and turmeric extracts having the major components resveratrol and curcumin, respectively, were used for the induction of cryptic and bioactive metabolites in an endophytic fungus Colletotrichum gloeosporioides isolated from Syzygium cumini. The increase in total amount of crude compounds in grape skin and turmeric extract treated cultures was 272.48 and 174.32%, respectively, compared to the untreated control. Among six human pathogenic bacteria tested, the maximum inhibitory activity was found against Aeromonas hydrophila IMS/GN11 while no inhibitory activity was observed against Enterococcus faecalis IMS/GN7. The crude compounds derived from turmeric extract treated cultures showed the highest DPPH free radicals scavenging activity (86.46% inhibition) followed by compounds from grape skin treated cultures (11.80% inhibition) and the control cultures (1.92% inhibition). Both the treatments significantly (p ≤ 0.05) increased the antibacterial and antioxidant activities of crude metabolites compared to the control. HPLC profiling of crude compounds derived from grape skin and turmeric extract treated cultures revealed the presence of additional 20 and 14 cryptic compounds, respectively, compared to the control. These findings advocate the future use of such dietary components in induced production of cryptic and bioactive metabolites.

Diversity of endophytic mycobiota of tropical tree Tectona grandis Linn.f.: Spatiotemporal and tissue type effects.

Fungal endophytes were isolated from leaf, bark and stem of Tectona grandis Linn.f. sampled at four geographical locations in winter, summer and monsoon seasons. The recovered 5089 isolates were assigned to 45 distinct morphotypes based on morphology. The sequences of the internal transcribed spacers (ITS) of the nrDNA of some morphotypes were identical, but morphological differences were strong enough to consider these morphotypes as separate species. Forty-three morphotypes were assigned to ascomycotina and two to basidiomycotina. Ascomycotina was the predominating group with 99.7% of total isolates followed by basidiomycotina with only 0.3% of total isolates. Diaporthe (Phomopsis) species dominated the communities independently on tissue type, location or season. More than 60% of the examined tissue pieces were colonized by members of this species complex. While these endophytes are ubiquitous others were tissue or location specific. Tissue type had the strongest effect on the species evenness of the endophytic assemblage followed by geographical location and season. However, Shannon-Wiener index (H') significantly (p ≤ 0.001) varied with all three factors i.e. season, location and tissue type. Leaves supported the highest diversity across all the seasons and locations. In conclusion, all the three factors together determined the structure of endophytic mycobiota assemblage of T. grandis.

Trichoderma asperellum (T42) and Pseudomonas fluorescens (OKC)-Enhances Resistance of Pea against Erysiphe pisi through Enhanced ROS Generation and Lignifications.

Plant signaling mechanisms are not completely understood in plant-fungal biotrophic pathogen interactions. Further how such interactions are influenced by compatible rhizosphere microbes are also not well-studied. Therefore, we explored the pea-Erysiphe pisi (obligate biotroph) system to understand the interaction and applied compatible rhizospheric bio-agents Trichoderma asperellum (T42) and Pseudomonas fluorescens (OKC) singly or in combination to assess their influence on the host while under the pathogen challenge. Transcript accumulation pattern of some vital genes in the lignin biosynthetic pathway in pea under E. pisi challenge indicated enhanced activation of the pathway. Interestingly, transcript accumulations were even higher in the bio-agent treated plants compared to untreated plants after pathogen inoculation particularly in co-inoculated treatments. Further, down regulation of the lignifications-associated ABC transporter gene in the pathogen challenged plants possibly is an indication of passive diffusion of monolignols across the membrane from symplast. Additionally, up regulation of NADPH oxidase gene revealed ROS generation in the challenged plants which was confirmed through spectrophotometric estimation of H2O2. Up regulation of laccase and peroxidase along with higher H2O2 generation points out their involvement in lignifications which was further confirmed through cross section analysis of pea stems that showed increased lignifications in pathogen challenged plants co-inoculated with the bioagents. Interestingly, pathogen responsive MAPK homologs MAPK3/MAPK6 and the enzyme serine threonine kinase that activates MAPKs were down regulated and the results possibly indicate non-participation of the MAPK cascade in this interaction. Therefore, it can be concluded that the microbial treatments enhanced pea resistance to E. pisi by generation of ROS and lignifications.

Pseudomonas fluorescens and Trichoderma asperellum Enhance Expression of Gα Subunits of the Pea Heterotrimeric G-protein during Erysiphe pisi Infection.

We investigated the transcript accumulation patterns of all three subunits of heterotrimeric G-proteins (Gα1 and 2, Gß, and Gγ) in pea under stimulation of two soil-inhabiting rhizosphere microbes Pseudomonas fluorescens OKC and Trichoderma asperellum T42. The microbes were either applied individually or co-inoculated and the transcript accumulation patterns were also investigated after challenging the same plants with a fungal biotrophic pathogen Erysiphe pisi. We observed that mostly the transcripts of Gα 1 and 2 subunits were accumulated when the plants were treated with the microbes (OKC and T42) either individually or co-inoculated. However, transcript accumulations of Gα subunits were highest in the T42 treatment particularly under the challenge of the biotroph. Transcript accumulations of the other two subunits Gß and Gγ were either basal or even lower than the basal level. There was an indication for involvement of JA-mediated pathway in the same situations as activation of LOX1 and COI1 were relatively enhanced in the microbe co-inoculated treatments. Non-increment of SA content as well as transcripts of SA-dependent PR1 suggested non-activation of the SA-mediated signal transduction in the interaction of pea with E. pisi under the stimuli of OKC and T42. Gα1 and 2 transcript accumulations were further correlated with peroxidases activities, H2O2 generation and accumulation in ABA in pea leaves under OKC and T42 stimulations and all these activities were positively correlated with stomata closure at early stage of the biotroph challenge. The microbe-induced physiological responses in pea leaves finally led to reduced E. pisi development particularly in OKC and T42 co-inoculated plants. We conclude that OKC and T42 pretreatment stimulate transcript accumulations of the Gα1 and Gα2 subunits of the heterotrimeric G protein, peroxidases activities and phenol accumulation in pea during infection by E. pisi. The signal transduction was possibly mediated through JA in pea under the stimulus of the microbes and the cumulative effect of the co-inoculated microbes had a suppressive effect on E. pisi conidial development on pea leaves.

Season and tissue type affect fungal endophyte communities of the Indian medicinal plant Tinospora cordifolia more strongly than geographic location.

A total of 1,151 endophytic fungal isolates representing 29 taxa were isolated from symptom-less, surface-sterilized segments of stem, leaf, petiole, and root of Tinospora cordifolia which had been collected at three locations differing in air pollution in India (Ramnagar, Banaras Hindu University, Maruadih) during three seasons (summer, monsoon, winter). Endophytes were most abundant in leaf tissues (29.38% of all isolates), followed by stem (18.16%), petiole (10.11%), and root segments (6.27%). The frequency of colonization (CF) varied more strongly among tissue type and season than location. CF was maximal during monsoon followed by winter and minimal during summer. A species each of Guignardia and Acremonium could only be isolated from leaves, whereas all other species occurred in at least two tissue types. Penicillium spp. were dominant (12.62% of all isolates), followed by Colletotrichum spp. (11.8%), Cladosporium spp. (8.9%), Chaetomium globosum (8.1%), Curvularia spp. (7.6%), and Alternaria alternata (6.8%). Species richness, evenness, and the Shannon-Wiener diversity index followed the same pattern as the CF with the tissue type and the season having the greatest effect on these indices, suggesting that tissue type and season are more influential than geography. Dissimilarity of endophyte communities in regards to species composition was highest among seasons. Colletotrichum linicola occurred almost exclusively in winter, Fusarium oxysporum only in winter and summer but never during monsoon and Curvularia lunata only in winter and during monsoon but never in summer. Emissions of NO(2), SO(2), and suspended particulate matter were negatively correlated with the CF. Ozone did not have any effect. The frequency of most species declined with increasing pollution, but some showed an opposite trend (e.g., Aspergillus flavus). Five unnamed taxa (sterile mycelia) were identified as Aspergillus tubingensis, Colletotrichum crassipes, Botryosphaeria rhodina, Aspergillus sydowii, and Pseudofusicoccum violaceum, using molecular tools. Fifteen of the 29 endophyte taxa exhibited antibacterial activity. B. rhodina (JQ031157) and C. globosum showed activity against all bacterial human pathogens tested, with the former showing higher activity than the latter.

Endophytic Fungal Flora from Roots and Fruits of an Indian Neem Plant Azadirachta indica A. Juss., and Impact of Culture Media on their Isolation.

Azadirachta indica A. Juss. (neem), native to India, is well known worldwide for its insecticidal and ethanopharmacological properties. Although endophytic microbes are known from this plant as only leaves and stems were the subjects of past reports. Now, a variety of procedures and a number of different media were used to isolate the maximum number of endophytic fungi from unripe fruits and roots. A total of 272 isolates of 29 filamentous fungal taxa were isolated at rate of 68.0% from 400 samples of three different individual trees (at locations-Az1, Az2, Az3). Mycological agar (MCA) medium yielded the highest number of isolates (95, with a 14.50% isolation rate) with the greatest species richness. Mycelia Sterilia (1, 2, 3) accounted for 11.06%, Coelomycetes 7.25%, while Hyphomycetes showed the maximum number of representative isolates (81.69%). Mycelia-Sterilia (1, 2, 3), based on their 5.8S ITS 1, ITS2 and partial 18S and 28S rDNA sequences were identified as Fusarium solani (99%), Chaetomium globosum (93%) and Chaetomium globosum (93%) respectively. Humicola, Drechslera, Colletotrichum, and Scytalidium sp. were some of the peculiar fungal endophytes recovered from this plant.

Biosynthesis of antimicrobial silver nanoparticles by the endophytic fungus Aspergillus clavatus.

AIM: To induce the biosynthesis of silver nanoparticles (AgNPs) using Aspergillus clavatus and evaluate their antimicrobial potential. MATERIALS & METHODS: Aspergillus clavatus (AzS-275), an endophytic fungus isolated from sterilized stem tissues of Azadirachta indica A. Juss., was challenged with 1 mM AgNO(3) solution. The characterization of the AgNPs was carried out by x-ray diffraction spectrometry, transmission-electron microscopy and atomic force microscopy. RESULTS & DISCUSSION: The synthesized AgNPs were found to be extracellular, polydispersed spherical or hexagonal particles ranging from 10 to 25 nm in size. Antimicrobial activity was performed using a disc-diffusion method against Candida albicans, Pseudomonas fluorescens and Escherichia coli. The results showed an average minimum inhibitory concentration of 5.83 microg ml(-1) and minimum fungicidal concentration of 9.7 microg ml(-1) against C. albicans. CONCLUSIONS: AgNPs can be mycosynthesized extracellularly using A. clavatus as the fungal system, which is highly advantageous over chemical synthesis not only because it can be synthesized on a large scale, but because of the ease of downstream processing and its biomedical application in antimicrobial activity.

Chemical and functional diversity of natural products from plant associated endophytic fungi.

This review describes examples of naturally occurring bioactive compounds obtained from fungal endophytes from various host plants. The main topics addressed are sources, identification, biological activity, biosynthesis, and ecological and chemosystematic significance of those bioactive compounds whose sources were well defined.

Endophytic actinomycetes from Azadirachta indica A. Juss.: isolation, diversity, and anti-microbial activity.

Endophytic actinomycetes from Azadirachta indica A. Juss. were screened and evaluated for their anti-microbial activity against an array of pathogenic fungi and bacteria. A total of 55 separate isolates were obtained from 20 plants, and 60% of these showed inhibitory activity against one or more pathogenic fungi and bacteria. Actinomycetes were most commonly recovered from roots (54.5% of all isolates), followed by stems (23.6%), and leaves (21.8%). The dominant genus was Streptomyces (49.09% of all isolates), while Streptosporangium (14.5%), Microbispora (10.9%), Streptoverticillium (5.5%), Sacchromonospora sp. (5.5%), and Nocardia (3.6%) were also recovered. Streptomyces isolates AzR 006, 011, and 031 (all from roots) had acute activity against Pseudomonas fluorescens, while AzR027, 032, and 051 (also all from roots) showed activity against Escherichia coli. Meanwhile, an isolate of Nocardia sp. from leaves (AzL025) showed antagonism against Bacillus subtilis. Overall, 32 of the 55 were found to have broad spectrum significant antimicrobial activity, while about 4% of them showed strong and acute inhibition to pathogenic fungi and bacteria. Isolates of Streptomyces AzR031, 008, and 047, Nocardia sp. AzL025, and Streptosporangium sp. AzR 021 and 048 are of particular interest because they showed significant antagonistic activity against root pathogens, including Pythium and Phytophthora sp. Thus, many of the isolates recovered from A. indica in this study may be used in developing potential bio-control agents against a range of pathogenic fungi and bacteria and in the production of novel natural antimicrobial compounds. These results not only further our understanding of plant-microbe interactions but also indicate that there is an untapped resource of endophytic microorganisms that could be exploited in the biotechnological, medicinal, and agricultural industries.

Javanicin, an antibacterial naphthaquinone from an endophytic fungus of neem, Chloridium sp.

The endophytic fungus Chloridium sp. produces javanicin under liquid and solid media culture conditions. This highly functionalized naphthaquinone exhibits strong antibacterial activity against Pseudomonas spp., representing pathogens to both humans and plants. The compound was crystallized and the structure was elucidated by X-ray crystallography. The X-ray structure confirms the previously elucidated structure of the compound that was done under standard spectroscopic methods. The importance of javanicin in establishing symbiosis between Chloridium sp. and its host plant, Azadirachta indica, is briefly discussed.