Secrets of plants : Endophytes

Endophytic fungi are an important component that colonizes in healthy tissues of living plants and can be readily isolated from any microbial or plant growth medium. They act as reservoirs of novel bioactive secondary metabolites, such as alkaloids, phenolic acids, quinones, steroids, saponins, tannins, and terpenoids that serve as a potential candidate for antimicrobial, anti-insect, anticancer and many more properties. Their huge diversity and particular habituation, they can provide a good area for research in the field of making new medicines and novel drug-like molecules. Because of the impact of endophytes on host plant by enhancing their growth or increasing their fitness, also making them tolerant to abiotic and biotic stresses and holding the secondary metabolites, endophytes are gaining attention as a subject for research. This review aims to comprehend the contribution and uses of endophytes and relationships between endophytic fungi and their host medicinal plants.


Introduction
Endophytes are microorganisms that reside within study plant tissues intercellularly and/or intracellularly but usually remain asymptomatic and does not show any noticeable damage to the host.Endophytic fungi inhabit plant tissues without destroying or producing substances that cause an infection in the host cell.They exhibit complex interactions with their hosts, which involves mutualism and antagonism. 1,2Plants strictly limit the growth of endophytes, and these endophytes use many mechanisms to gradually adapt to their living environments. 3Unlike mycorrhizal fungi that colonize plant roots and grow into the rhizosphere, endophytes reside entirely within plant tissues and may grow within roots, stems and/or leaves, emerging to sporulate at plant or host tissue senescence. 1,4Symbioses have been responsible for the origin of major ecological and evolutionary transitions in the history of life.Plants are hosts to complex communities of endophytes that colonize the interior of both below-and above-ground tissues.The endophytic fungus grows asymptomatically in aerial plant tissues and is vertically transmitted from the plant to its offspring via seeds and tillers.Endophytes are thought to interact mutualistically with their host plants mainly by increasing host resistance to herbivores 1 and have been termed acquired plant defenses. 5In return, plants provide spatial structure and protection from desiccation, nutrients, and photosynthate and, in the case of vertical-transmission, dissemination to the next generation of hosts.Endophytic fungi represent an important and quantifiable component of fungal biodiversity in plants that impinge on plant community diversity and structure.
They produce a wide range of compounds useful for plants for their growth, protection to environmental conditions, and sustainability, in favor of a good dwelling place within the hosts.Due to its ability of producing bioactive secondary metabolites similar or same compounds as their host plant, promoting growth, good yield, inducing host plants to tolerate both biotic and abiotic stresses and disease resistance, endophytes is currently attracting a lot of research interest and therefore, can be used as a potential source of novel natural products for food, industrial, medicinal and agricultural industries.

Classification of endophytes
There are mainly two classes in which endophytic fungi are classified as Class I Clavicipitaceae and Class II Nonclavicipitaceous.The Class 1 endophytes are the clavicipitaceous endophytes associated with warm-and cool-season grasses.They are host specific, mainly in the grass family poaceae and rarely in cyperaceae, and are often vertically transmitted through seeds.Transmission of Class 1 endophytes is primarily vertical, with maternal plants passing fungi on to offspring via seed infections. 6Class 1 endophytes frequently increase plant biomass, confer drought tolerance, and produce chemicals that are toxic to animals and decrease herbivory. 5However, the benefits conferred by these fungi appear to depend on the host species, host genotype and environmental conditions. 7,8ompared with Class 1 endophytes, Class 2 endophytes are highly diverse and comprised of species from pezizomycotina (ascomycota) to agaricomycotina and pucciniomycotina (basidiomycota). 9They colonize roots, stems, leaves, or the whole plant.They can be vertically or horizontally transmitted.However, NC-endophytes represent three distinct functional classes class 2, 3 and 4, based on host colonization and transmission, in plant biodiversity and fitness benefits conferred to hosts while the C group has just one class.Class 2 endophytes have been the most extensively researched and has been shown to enhance fitness benefits of their plant host as a result of habitatspecific stresses such as pH, temperature, and salinity.Class 3 endophytes are restricted to growth in belowground plant tissues and form in localized areas of plant tissue.Class 4 endophytes are also restricted to plant tissues below ground but can colonize much more of the plant tissue. 10NC-endophytes have been recovered from every major lineage of land plants, and from all terrestrial ecosystems, including both agroecosystems and biomes ranging from the tropics to the tundra. 11he scale of their diversity, their ecological roles, the insights they provide into the evolution of various ecological modes in fungi, their potential applications, and the ability of many fungi to switch between endophytic and free living lifestyles 12,13 are becoming more apparent, engendering growing enthusiasm from mycologists, ecologists, physiologists and applied scientists.

N o n -c o m m e r c i a l u s e o n l y
sues, while the inhabited host tissues remain intact and functional.The diversity and numbers of fungal endophytes vary considerably and their detection depends on biotic, abiotic and experimental factors.Detection and recovery of endophytic fungi have relied heavily on dissection of plant organs into small fragments followed by their surface sterilization and subsequent plating of fragments onto a nutrient-rich agar medium. 4,14raditionally, endophytic fungi inside plant tissues can be recognized by two basic techniques, i.e. direct observation and cultivation-dependent methods.In the direct observation method, endophytic fungal structures within living plant tissues are directly examined under a light and electron microscope, which can show all endophytic mycobiota within the plant tissue. 15In contrast, cultivation-dependent techniques have been routinely employed in endophyte diversity studies. 1 It is important to isolate endophytic fungi for further detailed studies into their characterization, population dynamics, species diversity, or as inoculate to improve plant growth and health, or screening for novel biologically active secondary metabolites (Figure 1).
Endophytes can also be isolated by culturing from ground tissue extract 16 or by direct culturing of plant tissues 17 on media suitable for bacteria or fungi or actinomycetes.Conventionally, identification of endophytes is based on morphological characteristics for bacteria, fungi, and actinomycetes and with the help of biochemical tests for bacteria and actinomycetes.With the development of molecular biology, ribosomal DNA Internal Transcribed Spacer (ITS) sequence analysis is widely used for the identification of microorganisms.Ribosomal DNA (rDNA) ITS was proved to be a valuable source of evidence to resolve phylogenetic relationships at lower levels, such as among genera or species. 18icroorganisms are important mediators of plant-herbivore interactions.The role of endophytic microorganisms in plant associations has been extensively discussed.Endophytes are considered plant mutualists.They receive nutrition and protection from the host plant while the host plant may benefit from enhanced competitive abilities and increased resistance to herbivores, pathogens, and various abiotic stresses. 7As endophytes colonize ecological niches similar to those colonized by phytopathogens, interactions between these two groups are possible, and in fact, many studies have shown that endophytic microorganisms isolated from surface disinfected plant tissues exhibit potential as biocontrol agents against phytopathogens and insects. 19Reports of molecular studies on endophytic bacterial diversity have revealed a large richness of species.Endophytes promote plant growth and yield, suppress pathogens, may help to remove contaminants, solubilize phosphate, or contribute assimilable nitrogen to plants. 20The endophyte-host relationship is believed to be complex and probably varies from host to host and microorganism to the microorganism. 21Many experiments have been conducted to compare how endophyte-infected plants and non-infected plants behave in relation to environmental stress, and attack by insect and animal predators. 22Therefore, search for interesting biological activities within the natural biodiversity has been the basis for the development of various applications in biotechnology, agriculture, production of pharmaceuticals compounds and other fields.

Application of endophytes
Medicinal plants and their endophytes are important resources for the discovery of natural products.Since endophytes residing inside healthy plant tissues without any discernible infectious symptom and exhibit mutualism and antagonism with the host, they could be a potential source of novel natural products for medicinal, agricultural, and industrial uses.Endophytes provide a wide variety of structurally unique bioactive natural products, such as alkaloids, benzopyranones, chinones, flavonoids, phenolic acids, quinones, steroids, terpenoids, tetralones, xanthones, and others. 23

Antioxidants and phenolic content of endophytes
Antioxidants are thought to be highly effective in the management of ROS-mediated tissue impairments.Many antioxidant compounds possess anti-inflammatory, anti-atherosclerotic, antitumor, antimutagenic, anticarcinogenic, antibacterial, and antiviral activities to a greater or lesser extent.Afra et al., in 2015, revealed that endophytic fungus Aspergillus sp. from Trigonella foenum-graecum seeds demonstrated the highest both total phenolic content in term of gallic acid equivalent and antioxidant activity for free radical scavenging assay. 24The antioxidant activity and total phenolic content (TPC) of ethyl acetate extracts of endophytic fungi isolated from Eugenia jambolana were studied by Manila et al., in 2014. 25The study showed that 36% endophytic extracts of Chaetomium sp., Aspergillus sp., Aspergillus peyronelii and Aspergillus Niger was having high phenolic content exhibited potent antioxidant activity.The free radical scavenging ability of phenols is attributed to the occurrence of hydroxyl groups.Phenols and alkaloids were the major phytochemical constituents of endophytes and were reported by Huang et al.Cephalosporium sp., an endophytic fungus isolated from the root of Trachelospermum jasminoides (Apocynaceae) produce a phenolic compound (graphislactone A) with strong free radical scavenging and antioxidant activity. 26Cui et al., in 2015 isolated 315 endophytes from Rhodiola crenulata, R. angusta, and R. sachalinensis and reported that antioxidant assay by showing that the DPPH radical-scavenging rates of 114 isolates (63.33%) were >50%, and those of five isolates (Rct45, Rct63, Rct64, Rac76, and Rsc57) were >90%. 27The culture filtrate of Pestalotiopsis microspora, which was isolated from combretaceaous plant Terminalia morobensis, showed promising antioxidant activity. 28

Antimicrobial activities of endophytes
Many endophytic fungi have the ability to produce antimicrobial substances.An increasing number of compounds with antibacterial activity are being isolated from endophytic fungi, including fumitremorgins B isolated from Phomopsis sp., and periconicins A and B from Periconia sp. 29esearch has shown that the compound ophiopogon japonicus pill has displayed effective inhibition of Staphylococcus aureus and an extract from O. japonicus showed strong inhibition of malic mildew. 30.3% of the strains isolated from Dracaena cambodiana and Aquilaria sinensis showed antimicrobial activity, 31 whereas 27.6% of strains isolated from Camptotheca acuminata displayed antimicrobial activity against some pathogens. 32Antimicrobial activity of endophytic fungi from Lippiasidoides Cham.showed antimicrobial activity in an assay on a solid medium. 33The steroid ergosta-7,9(14),22-triene-3β-ol, produced by the endophytic fungus Nigrospora sphaerica isolated from leaves of Vinca rosea, showed antifungal activity against Cryptococcus neoformans with an IC50 value of 14.81 μg/mL. 34 Beauvericin, isolated from Fusarium oxysporum, which is associated with cinnamomum kanihirai, was reported toxic against methicillin-resistant S. aureus and B. subtilis. 38The endophyte of Magnolia x soulangeana, Fusarium dimerum, was the source of enniatins which exhibited effectiveness against Bacillus subtilis, Candida albicans, Trychosporom cutaneum and Cryptococcus neoformans. 39

Anticancer activities of endophytes
There is some evidence that bioactive compounds produced by endophytes could be substitute approaches for innovation of new drugs, as various natural products of plants, microorganisms, and marine sources were identified as anticancer agents.Firakova et al., in 2007 reported diterpenoid that taxol also known as paclitaxel as a novel drug since it's due to the mode of action compared to other anticancer agents. 40Taxol is beneficial for the cure of advanced breast cancer, lungs cancer, and refractory ovarian cancer.Taxol was from the bark of tree belonging to taxus family (Taxus brevifolia), its main general resource.Another significant anticancer compound is the alkaloid Camptothecin (C 20 H 16 N 2 O 4 ), a powerful antineoplastic agent which was firstly isolated from the wood of Camptotheca acuminate Decaisne (Nyssaceae) in China. 41Ergoflavin (C 30 H 26 O 14 ), a dimeric xanthene linked in position 2, belongs to the compound class called ergochromes and was described as a new anticancer agent isolated from an endophytic fungus rising on the Mimuscop elengi (Sapotaceae) plant leaves 42 Wu et al., in 2015 also reported leaf endophytes from Morinda citrifolia inhibited the growth of human carcinoma cell lines of lungs, prostate, and breast. 43Bioactive compound sclerotiorin has been isolated from an endophytic fungus Cephalotheca faveolata. 44clerotiorin was found to be potent antiproliferative against different cancer cells.An endophytic fungus Colletotrichum gloeosporioides (strain JGC-9) was isolated from Justicia gendarussa, a medicinal plant and screened for taxol production. 45odophyllum hexandrum, which produced podophyllotoxin, podophyllotoxin glycoside and demethoxypodophyllotoxin.The isolated metabolites exhibited cytotoxicity in U-87 cell line. 46The production of podophyllotoxin from endophytic Alternaria tenuissima isolated from Sinopodophyllum emodi (Wall) was also reported by Liang et al. 47

Conclusions
Endophytic fungi comprise a diverse group of species existing in various ecosystems and are capable of synthesizing bioactive compounds, which have proven to be useful for novel drug discovery.This group, which causes no apparent effects on plant performance but lives on the metabolites produced by the host, is presumably the most dominant functional group among endophytes by quantity.Since many microorganisms have developed resistance against the current drugs so it is necessary to search for new drugs.Much more work is essential to understand endophytes physiology, biochemical pathways, defensive role, secondary metabolite production, motivation and encouragement of researcher from life sciences to contribute research related to endophytes.It is vital importance to review and highlight the previous successes, on-going research and latest developments in research associated with endophytic microorganisms to draw the attention of the research community toward this emerging field and possible exploitation of the available sources for their therapeutic uses in various fields, such as the medical, pharmaceutical, food and cosmetics.

Figure 1 .
Figure 1.The interaction between endophytes and plant host.