Browsing by Author "Ravindra N. Kharwar"

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A comparative study of endophytic and epiphytic fungal association with leaf of Eucalyptus citriodora Hook., and their antimicrobial activity
(2010) Ravindra N. Kharwar; Surendra K. Gond; Anuj Kumar; Ashish Mishra
Eucalyptus citriodora Hook, is frequently cultivated tree in India for its wood and medicinal usages. The endophytic and epiphytic fungi were estimated from healthy leaves of E. citriodora growing in the premise of Banaras Hindu University, Varanasi, India. A total of 33 fungal species were isolated from leaf segments. Of 33 taxa, 20 were recorded as endophytes, while 22 as epiphytes. Nine, out of 33 species were found to be common in leaf tissues and surfaces (Alternaria alternata, Aspergillus fumigatus, A. terreus, Cladosporium cladosporioides, Drechslera rostrata, Humicola grisea, Nigrospora oryzae, Penicillium cristata, and Pestalotia sp.). Out of 478 fungal isolates, 279 were epiphytic while only 199 were endophytic. Most isolates were anamorphic filamentous fungi which often don't produce sexual spores. The Sorensen's index of similarity between endophytes and epiphytes (leaf surface colonizers) was found to be at 0.300. Diversity index of fungal species was higher in endophytes than epiphytes. The frequency of colonization differs greatly in both myco-populations. Cladosporium cladosporioides (26%) was dominant species on leaf surfaces while Botrytis cinerea (10.5%) was dominant in leaf tissues. Out of 16 endophytic isolates evaluated for antagonistic test, 8 (50%) gave the antagonistic activity against variety of fungi representing pathogens to both humans and plants. © 2010 Springer Science+Business Media B.V.
A new species of Zasmidium (Mycosphaerellaceae) from India
(Verlag Ferdinand Berger und Sohne GmbH, 2014) Archana Singh; Ravindra N. Kharwar; Raghvendra Singh; Shambhu Kumar
A new species ot Zasmidium smilacis-proliferae on living leaves ot Smilax prolifera (Smilacaceae), from the Chawk forest in the Tarai Region of Uttar Pradesh, India is described, illustrated and compared with other Zasmidium species on Smilax.
A Review of Marine Natural Product Resources with Potential Bioactivity Against SARS-COV-2
(Faculty of Pharmacy, University of Benin, 2023) Neha Mishra; Ena Gupta; Angelo Mark P. Walag; Ravindra N. Kharwar; Priyangka Singh; Pragya Mishra
The emergence of new pathogenic viruses and the constant outbreak of viral diseases have created an upsurge in novel antiviral agents. Marine natural products are the most unexplored reservoir of novel, biologically active, chemically diverse compounds. A systematic literature review was conducted using PRISMA guidelines, accessing four major databases; PubMed, Science Direct, Scopus, and Google Scholar. Numerous studies supported the robust antiviral activity of marine resources against drug-resistant viruses such as SARS, Ebola, Influenza, and HIV. However, adequate research on marine resources for developing anti-covid therapy is lacking. The aim of the review was to explore the marine resources and their compounds that could lead to developing an effective antiviral drug. We also highlighted the current status of novel compounds against different species of corona family and discussed the future prospects of marine resources against COVID-19 management. © 2023 the authors.
Advances in extraction technologies: Isolation and purification of bioactive compounds from biological materials
(Elsevier, 2020) Nishi Srivastava; Arti Singh; Puja Kumari; Jay Hind Nishad; Veer Singh Gautam; Monika Yadav; Rajnish Bharti; Dharmendra Kumar; Ravindra N. Kharwar
Natural bioactive compounds (NBCs) are receiving more popularity because of their various medicinal properties and huge exploitation in various commercial sectors such as food, chemical, and pharmaceutical industries. The extraction of NBCs begins with the selection of a suitable protocol, sample preparation, and extensive literature survey. During the extraction of NBCs from biological materials, the major concern of researchers is on minimizing the interference of unwanted compounds that may coextract with the focused compounds. Numerous extraction procedures have been introduced along with existing classical extraction methodology (conventional extraction methodology), but still, scientists are in search of the development of a standard single method for extracting NBCs from biological materials. There are several critical parameters such as understanding the nature of the plant matrix and chemistry of bioactive compounds and scientific expertise that impact efficiencies of conventional and nonconventional extraction. This chapter focuses on the mechanism of different extraction techniques, extraction instrumentation, and common problems encountered and their elimination. For efficient extraction of NBCs from biological materials, there is an urgent need for advancement in selective extraction procedures, from extraction to fractionation and subsequent purification that will speed up and scale up the isolation of purified compounds from biological materials. © 2021 Elsevier Inc.
Anticancer compounds derived from fungal endophytes: Their importance and future challenges
(2011) Ravindra N. Kharwar; Ashish Mishra; Surendra K. Gond; Andrea Stierle; Donald Stierle
This is a review of anticancer agents isolated from endophytic fungi from 1990-2010. Endophytic fungi are defined as fungi that live asymptomatically within the tissues of higher plants. The designation 'anticancer' is based on the assessment of the authors of the paper of the cytotoxicity of each compound against specific cancer cell lines. Many of the compounds reported here were isolated exclusively from endophytes in culture, while other compounds had been previously reported as chemical constituents of higher plants. The uniqueness of the endophytic community of fungi is stressed as a promising source of novel compounds with anticancer activity, or as an alternative source of compounds originally isolated from higher plants. Endophytes represent a dependable source of specific secondary metabolites, and can be manipulated both physicochemically and genetically to increase yields of desired metabolites and to produce novel analogues of active metabolites. © 2011 The Royal Society of Chemistry.
Assessment of diversity, distribution and antibacterial activity of endophytic fungi isolated from a medicinal plant Adenocalymma alliaceum Miers
(2011) Ravindra N. Kharwar; Satish K. Verma; Ashish Mishra; Surendra K. Gond; Vijay K. Sharma; Talat Afreen; Anuj Kumar
A study was conducted for isolation, identification and antibacterial potential of fungal endophytes of Adenocalymma alliaceum Miers., (Bignoniaceae), a medicinal shrub vine plant which has long history for its usages in curing various disorders. A total of 149 isolates of endophytic fungi representing 17 fungal taxa were obtained from 270 segments (90 from each stem, leaf and petiole) of this plant. Hyphomycetes (77.85%) were the most prevalent, followed by Ascomycetes (8.05%) and Coelomycetes (4.03%) respectively. A considerable amount of fungal isolates was kept under (10.07%) Mycelia-Sterilia (MS). Leaf harboured maximum colonization of endophytic fungi (72.22%) which was greater than stem (67.78%) and petiole (25.54%). The Jc similarity index was maximum (0.619) between stem vs leaf followed by leaf vs petiole (0.571) and stem vs petiole (0.428). The dominant endophytic fungi were Alternaria alternata, Aspergillus niger, Stenella agalis, Fusarium oxysporum, Curvularia lunata and Fusarium roseum. Among twelve endophytic fungi tested for antibacterial activity, crude extracts of nine endophytic fungi (75%), showed antibacterial potential against one or more clinical human pathogens. Alternaria alternata, Curvularia lunata, Penicillium sp. and Chaetomium globosum exhibited significant antibacterial activity against 4 of 5 tested pathogens, showing broad spectrum activity. This investigation explains the value of sampling from different tissues of a host plant for the greater species diversity, and additionally, the antibacterial screening of some endophytic fungi from this specific medicinal plant may represent a unique source for many of the useful antibacterial compounds. © 2011 Springer Science+Business Media B.V.
Bacterial endophytes from rice cut grass (Leersia oryzoides L.) increase growth, promote root gravitropic response, stimulate root hair formation, and protect rice seedlings from disease
(Springer International Publishing, 2018) Satish K. Verma; Kathryn Kingsley; Marshall Bergen; Camille English; Matthew Elmore; Ravindra N. Kharwar; James F. White
Background and Aims: Leersia oryzoides, a wild relative of rice (Oryza sativa), may carry potential seed-borne bacterial endophytes which could be used to enhance growth of rice. We hypothesized that seed-associated bacteria from L. oryzoides would be compatible with rice and promote seedling growth, development, and survival. Methods: We isolated bacteria from seed of L. oryzoides and checked compatibility with rice as well as Bermuda grass seeds for seedling growth promotion. Internal colonisation of bacteria into root cells was observed by ROS staining and microscopic observation. Growth promoting bacteria were evaluated for IAA production, phosphate solubilization and antifungal activities. Results: Overall, ten bacteria were found to be growth promoting in rice seedlings with effects including restoration of root gravitropic response, increased root and shoot growth, and stimulation of root hair formation. All bacteria were identified by 16S rDNA sequencing. Six bacteria were found to become intracellular in root parenchyma and root hairs in rice and in Bermuda grass seedlings. Six bacteria were able to produce IAA in LB broth with highest (47.06 ± 1.99 μg ml−1) by LTE3 (Pantoea hericii). Nine isolates solubilized phosphate and inhibited at least one soil borne fungal pathogen. Conclusions: Seed bacteria of L. oryzoides are compatible with rice. Many of these bacteria become intracellular, induce root gravitropic response, increase root and shoot growth, and stimulate root hair formation in both rice and Bermuda grass seedlings. Presence of bacteria protects seedlings from soil pathogens during seedling establishment. This research suggests that bioprospecting microbes on near relatives of rice and other crop plants may be a viable strategy to obtain microbes to improve cultivation of crops. © 2017, Springer International Publishing AG.
Biosynthesis of antimicrobial silver nanoparticles by the endophytic fungus Aspergillus clavatus
(2010) Vijay C. Verma; Ravindra N. Kharwar; Alan C. Gange
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 AgNO3 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 5.83μg ml-1 and minimum fungicidal concentration of 9.7 μg 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. © 2010 Future Medicine Ltd.
Chemical and functional diversity of natural products from plant associated endophytic fungi
(Natural Product Incorporation, 2009) Vijay C. Verma; Ravindra N. Kharwar; Gary A. Strobel
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.
Chrysophanol: A natural anthraquinone with multifaceted biotherapeutic potential
(MDPI AG, 2019) Prateeksha; Mohd Aslam Yusuf; Brahma N. Singh; Surya Sudheer; Ravindra N. Kharwar; Saba Siddiqui; Ahmed M. Abdel-Azeem; Leonardo Fernandes Fraceto; Kavya Dashora; Vijai K. Gupta
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. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.
Coating with Microbial Hydrophobins: A Novel Approach to Develop Smart Drug Nanoparticles
(Elsevier Ltd, 2018) Brahma N. Singh; Braj R. Singh; Vijai K. Gupta; Ravindra N. Kharwar; Lorenzo Pecoraro
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. © 2018 Elsevier Ltd
Correction to Biofabrication of anisotropic gold nanotriangles using extract of endophytic Aspergillus clavatus as a dual functional reductant and stabilizer (Nanoscale Research Letters, (2011), 6, (261))
(2011) Vijay C. Verma; Ravindra N. Kharwar; Santosh K. Singh; Ravindra Solanki; Satya Prakash
[No abstract available]
Diversity and antimicrobial activity of endophytic fungi isolated from Nyctanthes arbor-tristis, a well-known medicinal plant of India
(Elsevier B.V., 2012) Surendra K. Gond; Ashish Mishra; Vijay K. Sharma; Satish K. Verma; Jitendra Kumar; Ravindra N. Kharwar; Anuj Kumar
Endophytic fungi from Nyctanthes arbor-tristis were isolated and evaluated for their antimicrobial activity. A total of 19 endophytic fungi were isolated from 400 segments of healthy leaf and stem tissues of N. arbor-tristis. Eighteen endophytic fungi were obtained from leaf, while only ten from stem. Alternaria alternata had the highest colonization frequency (15. 0%) in leaf, whereas Cladosporium cladosporioides ranked first in stem with a colonization frequency of 12%. The diversity and species richness were found higher in leaf tissues than in stem. The similarity indices between leaf and stem were 0. 473 for Jaccard's and 0. 642 for the Sorenson index, respectively. Of 16, 12 (75%) endophytic fungal extracts showed antibacterial activity against either one or more pathogenic bacteria. The endophytic Nigrospora oryzae showed maximum inhibition against Shigella sp. and Pseudomonas aeruginosa. The leaf endophytes Colletotrichum dematium and Chaetomium globosum exhibited a broad range of anibacterial activity and were active against Shigella flexnii, Shigella boydii, Salmonella enteritidis, Salmonella paratyphi, and P. aeruginosa. Nine out of 16 (56. 25%) endophytic fungi exhibited antifungal activity to one or more fungal pathogens. Colletotrichum dematium inhibited 55. 87% of the radial growth of the phytopathogen Curvularia lunata. The antimicrobial activity of these endophytic microorganisms could be exploited in the biotechnological, medicinal, and agricultural industries. © 2011 The Mycological Society of Japan and Springer.
Diversity of endophytic mycobiota of tropical tree Tectona grandis Linn.f.: Spatiotemporal and tissue type effects
(Nature Publishing Group, 2017) Dheeraj K. Singh; Vijay K. Sharma; Jitendra Kumar; Ashish Mishra; Satish K. Verma; Thomas N. Sieber; Ravindra N. Kharwar
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. © The Author(s) 2017.
Draft genome sequences of ambrosiella cleistominuta, cercospora brassicicola, C. citrullina, physcia stellaris, and teratosphaeria pseudoeucalypti
(BioMed Central Ltd, 2020) P. Markus Wilken; Janneke Aylward; Ramesh Chand; Felix Grewe; Frances A. Lane; Shagun Sinha; Claudio Ametrano; Isabel Distefano; Pradeep K. Divakar; Tuan A. Duong; Sabine Huhndorf; Ravindra N. Kharwar; H. Thorsten Lumbsch; Sudhir Navathe; Carlos A. Pérez; Nazaret Ramírez-Berrutti; Rohit Sharma; Yukun Sun; Brenda D. Wingfield; Michael J. Wingfield
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. © The Author(s).
Effect of bacterial endophyte on expression of defense genes in Indian popcorn against Fusarium moniliforme
(Springer Netherlands, 2015) Surendra K. Gond; Marshall S. Bergen; Mónica S. Torres; James F. White; Ravindra N. Kharwar
An endophytic Bacillus amyloliquefaciens subsp. subtilis was isolated from surface sterilized seedlings of tropical corn. To evaluate the hypothesis that this endophyte is defensive in nature and determine the mechanisms of host defense we examined the effects of the endophyte on pathogens and host disease resistance genes. The bacterial endophyte showed antifungal activity against fungal pathogens Fusarium moniliforme, Colletotrichum gloeosporioides and Aspergillus flavus. Plant protection activity was also observed in Indian popcorn seedlings inoculated with the endophyte against F. moniliforme. Gene expression analysis was conducted and demonstrated that up-regulation of several defense genes in corn was greater after 48 hours of pathogen challenge than 24 hours. Plant defense pathways triggered by the endophytic bacterium appeared to be SA (salicylic acid) independent. The abundance of the zm Jasmonic acid-induced gene expression was greater in pathogen challenged plants pre-treated with the endophytic bacterium than in the plants challenged only with pathogen. Pre-treatment with the endophytic bacterium resulted in a more intense induction of maize pathogenesis related protein genes PR-1 and PR-10 during interaction with F. moniliforme compared to plants that were treated only with the fungal pathogen. © 2015, Springer Science+Business Media Dordrecht.
Effect of Vermiculite Soil Amendment on Immobilization of Selected Heavy Metals of Rhizospheric Zone of Maize
(Taylor and Francis Ltd., 2022) Shobhika Parmar; Vir Singh; Vijay K. Sharma; Brijesh K. Yadav; Ravindra N. Kharwar
The elevated level of metals in the soil increases the assimilation of these metals by plants causing a serious health risk to humans. A series of pot experiments were conducted to study Pb, Cu, and Zn uptake by Zea mays to investigate the role of vermiculite for metals immobilization in the soil root zone. The metals accumulation was decreased (13.17% to 29.21% in above-ground parts and 4.18% to 69.74% in roots) and total plant biomass increased (1.43% in control soil and 10.54% in polluted soil) in vermiculite amended soils. The effective immobilization of metals in vermiculite amended soil was revealed by decreased values of translocation factors for root-stalk and subsequently for stalk-leaves under varying metal concentrations. Vermiculite amendment to soil had no observable effect on the chemical structure while there were few changes in the functional group of the soil structure. The study reveals that vermiculite can be amended to the soil for reducing phytoavailability of toxic metals to plant uptake and in return flow processes for controlling food-chain contamination and groundwater pollution without compromising with crop production. © 2021 Taylor & Francis Group, LLC.
Endophytic actinomycetes from azadirachta indica A. Juss.: Isolation, diversity, and anti-microbial activity
(2009) Vijay C. Verma; Surendra K. Gond; Anuj Kumar; Ashish Mishra; Ravindra N. Kharwar; Alan C. Gange
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. © 2008 Springer Science+Business Media, LLC.
Endophytic Fungal Flora from Roots and Fruits of an Indian Neem Plant Azadirachta indica A. Juss., and Impact of Culture Media on their Isolation
(2011) Vijay C. Verma; Surendra K. Gond; Anuj Kumar; Ravindra N. Kharwar; Lori-Ann Boulanger; Gary A. Strobel
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. © 2011 Association of Microbiologists of India.
Exploring endophytic communities of plants: Methods for assessing diversity, effects on host development and potential biotechnological applications
(Springer International Publishing, 2019) Satish K. Verma; Ravindra N. Kharwar; Surendra K. Gond; Kathryn L. Kingsley; James Francis White
Endophytic microbes colonize plants growing in diverse habitats and play important roles in modulating development and improving fitness of host plants. Endophytes may be major components of undiscovered microbial diversity. Further, endophytes may have applications in growth promotion of crop plants and protectors of plants from biotic and abiotic stresses. Endophytes have been a source of bioactive molecules of pharmaceutical importance. Major focus areas in the investigation of endophytes include (1) assessment of endophyte diversity, (2) determining the roles played by endophytes in modulation of host plant development and (3) assessing the biotechnological potentials of endophytes. The study of endophytes is particularly challenging because endophytic microbes often go unobserved in plants, many endophytes cannot be isolated, and plants free of endophytes sometimes cannot be obtained, making it difficult to conduct experiments. In this chapter we discuss some of the methodologies that are being used to overcome challenges to the study of endophytic microbes. © Springer Nature Switzerland AG 2019.