Abstract: Endophytes are considered as plant mutualists which are living asymptomatically within plant tissues have been found in virtually all plant species. Endophytes 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. This review focuses on the biology of endophytic fungi, their discovery, isolation, identification and diversity and their biological activities in environmental and agricultural sustainability. It also considers and their medicinal applications especially in the production of anticancer, antimicrobial, antioxidant and antiviral compounds. Endophytic fungi are one of the most creative groups of secondary metabolite producers that play important biological roles for human life. They are potential sources of novel natural agents for exploitation in the pharmaceutical industry, agriculture and in environmental applications. So, in this review we summarize their potential role against some important plant pathogens.
INTRODUCTION
Many plant pathogens invade plants, including fungi, bacteria nematode and viruses, which are the most obvious threats to sustainable food production. It is not economically feasible to routinely use chemicals to eradicate the plant disease. Using these chemicals consistently, it leaves harmful residues and can bring resistance to pathogens1. Consequently, knowledge has been developed for safer non-chemical methods to monitor lucrative plant disease, which poses less threat to human health and the environment2. Replacing fungicides with bio-control agents is a choice for controlling plant pests, producing food for protection and significantly reducing pollution3-5. As an alternative for host-plant resistance and pesticide-based pest and pathogen control, the proper application of naturally occurring microorganisms to suppress pathogen populations and increase the production of significant crops. Endophytic microorganisms, which grow in the intercellular spaces of higher plants, are recognized in terms of diversity and pharmaceutical potential as one of the most chemically promising groups of microorganisms6.
In particular, plants are infected with different micro-organisms. Endophytic species are those which colonize the internal tissue of the plant and display no external sign of infection or negative effect on the host7. Endophytic microorganisms are recognized as one of the most chemically promising diversity and pharmaceutical potential groups of microorganisms growing in the intercellular spaces of higher plants6. Beneficial endophytic microorganisms include fungi and bacteria that colonize the internal tissues of plants without causing visible damage to their hosts8,9. Although, disease symptoms of host plant can be caused by endophytes under stress conditions7, 10. Of the nearly 300 000 plant species that exist on the earth, each individual plant is host to one or more endophytes11. In addition, endophytic microorganisms are not known to be saprophytes as they are associated with living tissues and may contribute to the plant’s well-being in some way. Endophytes occur in a number of tissue types in a wide range of plants, actively colonizing the plant with bacterial colonies and biofilms, latently living in intercellular spaces, in the vascular tissue or in cells12. Endophytes are the microorganisms that reside in the tissues of living plants, are relatively unstudied and potential sources of novel natural products for exploitation in agriculture. This comprehensive review is based on critical study of different research works and investigations all around the globe and also depicts endophytic role of various microbes in enhancing crop productivity and maintaining soil health aiming towards sustainability of agriculture in long run.
What is an endophyte?: The word endophytes’ derived from Greek word endon and phyton and the meaning of endon-within and phyton means-plant .The term endophytes was first coined by de Bary (1886). Plants are generally associated with diversified microorganisms. Endophytic are those mico organisms which grow in the intercellular spaces of higher plants and are accepted as in terms of diversity and pharmaceutical potential and displaying no external sign of infection or without causing negative effect on their host6-8. Based on differences in evolution, taxonomy, plants host and ecological functions, endophytes are divided into 2 main groups clavicipitaceous and non-clavicipitaceous. Clavicipitaceous are able to infect only some species of grasses and non-clavicipitaceous are found in the asymptomatic tissues of other higher plants13. Plants comprise huge and diverse niches for endophytic organisms. Of the nearly 300 000 plant species that exist on the earth, each individual plant is host to one or more endophytes11. Fungi and bacteria have been studied for biological control they are different from plant pathogenic microorganisms because they, do not cause diseases to plants and are distinct from epiphytic microorganisms which live on the surface of plant organs and tissues14.
Isolation of endophytic fungi: The most important phase for the isolation of endophytic fungi that are living in plant tissues is surface sterilization and the plant parts under examination should be slice into tiny pieces to assist sterilization and isolation procedures. To accomplish complete surface sterilization, there are numerous techniques to eradicate the majority of the epiphytic fungi from the external tissues and support the growth of the internal mycota, according to the type of tissue as well as its specific location15.
Identification of endophytes: Endophytic fungi can be readily identified on the basis of morphological methods, using characters of the phenotype of the fungal culture, i.e., colony or hyphae, the characters of the spore, or reproductive structure if these features were discernible16-18. The majority of endophytic fungi are supposed to be the ascomycetes and asexual fungi19. These isolates cab be stimulated to sporulate on medium contains stripes or extract of host plant20. Sterile isolates should be examined on a regular basis for fruiting bodies over a period of 3-4 months and the isolates that failed to sporulate are called to as mycelia sterilia, are divided into different morphotypes according to their culture properties. These groups of fungi are significantly common in endophytes studies21,22.
Fungi as producers of biologically active metabolites: Fungi have been used as a tool for producing novel metabolites and more than 20,000 bioactive metabolites are of microbial origin23. Fungi is a good source of producing biologically active secondary metabolites, which are directly used as drugs or function as lead structures for synthetic modifications24-30. A number of antibiotics as shown in Table 1 and many medicinal drugs from microbial origin are recognized such as the antibiotic penicillin from Penicillium sp., the immunosuppressant cyclosporine from Tolypocladium inflatum and Cylindrocarpon lucidum, the antifungal agent griseofulvin from Penicillium griseofulvum fungus, the cholesterol biosynthesis inhibitor lovastatin from Aspergillus terreus fungus and β-lactam antibiotics from various fungal taxa, has shifted the focus of drug discovery from plants to microorganisms.
| Table 1: Antibiotic produced by fungal endophytes | ||||
| Antibiotics | Occurrence of host | Fungal endophyte | Target pathogens | References |
| Pyrrocidines A, B | Maize | Acremonium zeae | Aspergillus flavus, Fusarium verticillioides | Wicklow et al.56 |
| Massariphenone, ergosterol peroxide | Rehmannia glutinosa | Verticillium sp. | Pyricularia oryzae P-2b | You et al.57 |
| Cadinane sesquiterpenes | Cassia spectabilis | Phomopis cassiae | Cladosporium sphaerospermum, Cladosporium cladosporioides | Silva et al.58 |
| Tetrahydrofuran, 2-methyl furan, 2-butanone, aciphyllene | Tropical tree | Muscodor albus | Stachybotrys chartarum | Atmosukarto et al.59 |
| Fusicoccane diterpenes | Taxus cuspidata | Periconia sp. | Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhimurium | Kim et al.60 |
| 3-O-Methylalaternin, altersolanol A | Urospermum picroides | Ampelomyces sp. | Staphylococcus aureus, S. epidermidis, Enterococcus faecalis | Aly et al.61 |
| Aliphatic compounds | Excoecaria agallocha | Phomopsis sp. | Candida albicans, Fusarium oxysporum | Huang et al.62 |
| Ginkgo biloba | Chaetomium globosum | Mucor miehei | Qin et al.63 | |
| Quercus variabilis | Cladosporium sp. | Trichophyton rubrum, Candida albicans, Aspergillus niger, Epidermophyton floccosum, Microsporum canis | Wang et al.64 | |
| Flavonoids | Juniperus cedre | Nodulisporium sp. | Bacillus megaterium, Microbotryum violaceum, Septoria tritici, Chlorella fusca | Dai et al.65 |
| Alkaloids | Garcinia dulcis | Phomopsis sp. | Mycobacterium tuberculosis | Rukachaisirikul et al.66 |
| Ginkgo biloba | Chaetomium globosum | Mucor miehei | Qin et al.63 | |
| Maize | Acremonium zeae | Aspergillus avus, Fusarium verticillioides | Wicklow et al.56 | |
| Peptides | Acrostichum aureurm | Penicillium sp. | Staphylococcus aureus, Candida albicans | Cui et al.67 |
| Pinus sylvestris and Fagus sylvatica | Cryptosporiopsis sp., Pezicula sp. | Yeasts | Noble et al.68 | |
| Tripterigium wiflordii | Cryptosporiopsis quercina | Candida albicans | Strobel et al.69 | |
| Tropical tree and vine species in several of the world's rainforests | Muscodor albus | Candida albicans | Strobel et al.69 | |
| Phenols | Cerbera manghas[ | Penicillium sp. | Staphylococcus aureus | Han et al.70 |
| Saurauia scaberrinae | Phoma species | Staphylococcus aureus | Hoffman et al.71 | |
| Unidentified | Pestalotiopsis adusta | Fusarium culmorum, Gibberella zeae and Verticillium aiboatrum | Li et al.72 | |
| Quinones | Callicarpa acuminate | Edenia gomezpompae | Phythophtora capsici, Phythophtora parasitica, Fusarium oxysporum, Alternaria solani | Macias-Rubalcava et al.73 |
| Unidentified | Pestalotiopsis adusta | Fusarium culmorum, Gibberella zeae, Verticillium aiboatrum | Li et al.72 | |
| Urospermum picroides | Ampelomyces sp. | Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis | Aly et al.61 | |
| Steroids | Artemisia annua | Colletotrichum sp. | Phytophthora capisici, Rhizoctonia cerealis, Gaeumannomyces graminis var. tritici, Helminthosporium sati6um | Lu et al.74 |
| Juniperus cedre | Nodulisporium sp. | Bacillus megaterium, Microbotryum violaceum, Septoria tritici, Chlorella fusca | Dai et al.65 | |
| Terpenoids | Cassia spectabilis | Phomopis cassiae | Cladosporium sphaerospermum, | Silva et al.58 |
| Cladosporium cladosporioides | ||||
| Daphnopsis americana | Not identified | Staphylococcus aureus, Enterococcus faecalis | Brady et al.75 | |
| Daphnopsis americana | Not mentioned | Staphylococcus aureus, Enterococcus faecalis | Brady et al.76 | |
| Taxus cuspidate | Periconia sp. | Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Salmonella typhimurium | Kim et al.60 | |
Mechanisms of diseases control displayed by endophytes: Many studies recently found that endophyte fungal have the ability to protect host from diseases and limit the damage caused by pathogen microorganism31-33. The common methods of these researches were in vitro coculture with pathogens and endophytes or comparison of the survival rate of plant inoculated with fungal endophytes with endophyte-free plant.
Antimicrobials and their activities produced from endophytes
Antifungal activity of endophytes: Endophytic fungi are those that found inside the living plant tissues or organs, without causing them any damaging symptoms8 and provide the greater host plant resistance to biotic or abiotic stresses. The Phylum mainly Ascomycota, Basidiomycota and Zygomycota include fungal species which have been reported to be endophytic in nature for example several studies displayed that endophytes are an alternative to switch synthetic pesticides, considering the increasing incidence of chemical resistance in fungal pathogens and promising environmental and mammalian toxicities4,5. The endophytic fungi seem to produce bioactive compounds, originally isolated from the host plants, as well as bioactive metabolites that are clearly different from other plants and feature that are unique structural characteristics, which may have potential to use in agriculture and medicine5,34,35. Several metabolites, such as alkaloids, terpenoids, steroids, isocoumarins and chromones, phenolics and volatiles isolated and characterized from endophytic fungi display antifungal activity against plant pathogenic fungi4. The culture filtrates of antagonists/endophytes, which were obtained from 2-3 weeks old cultures, produced antibiotics that strongly suppressed conidial germination of the pathogen. This suggests that the antibiotics possibly played an important role in suppressing conidial germination and infection by the pathogen36.
Antibacterial activity of endophytes (against phytobacteria): Bacterial endophytes are well-known as one of bioactive compounds providers, such as the secondary metabolite compounds with various biological activities37. Endophytes live in the intercellular space38. The unique features of bacterial endophytes such as their shorter life cycle than their host plants, which can save production time, using bacterial endophytes. Pseudomonas aeruginosa is bacteria that cause urinary tract infections, meningitis, diarrhea, entrokolitis necrosis and pneumonia39 whereas, Klebsiella pneumonia causes pneumonia, urinary tract infections and sepsis in patients with vulnerable immune system40. Bacillus cereus produces diarrhea-causing enterotoxins. On the other hand, Methicillin-resistant Staphylococcus aureus (MRSA) has a mutated gene that is resistant to almost all beta-lactam antibiotics. Meanwhile, S. aureus can cause septicaemia, pneumonia, endocarditis, osteomyelitis, gastroenteritis and abscesses41. The present study aims to obtain the bacterial endophytes isolates from S. polycephalum, where the most potent isolate could be used as antibacterial agent against bacterial pathogens such as P. aeruginosa, K. pneumoniae, MRSA and B. cereus. This study could be as an alternative to over-used of synthetic antibiotics thus it can be medically accountable (Table 1).
Antiviral activity of endophytes: Endophytic and marine fungi growing in unique environments are being constantly explored for their bioactive natural products possessing cytotoxic, anticancer, antibacterial or antifungal potential since past decade42-44. Fungi also potentially contain and/or produce several effective molecules that could also be used as antivirals for other hosts45. Presently, there is a rather limited understanding of the antiviral mechanisms of fungal products on virus infection. Thus, more detailed knowledge on the actual molecular targets is crucial in order to develop these molecules further to efficiently combat virus infections in the future.
Nematicidal activity of endophytes: Endophytic fungi can protect their host plants by producing natural compounds which are dangerous to nematodes. The first antagonistic activity of endophytic fungi against plant parasitic nematodes was observed in tall fescue (F. arundinacea) infected by Pratylenchus scribneri. The antagonistic activity of fungal endophyte, F. oxysporum was observed; in the roots of tomato plant reduced 60% infection of Meloidogyne incognita successfully. The bacterial endophyte Burkholderia ambifaria, isolated from corn root, produced some toxic metabolites which inhibited egg hatching and mobility of second-stage juveniles of M. incognita46. The successful results of nematicidal activity of an arbuscular mycorrhiza, Glomus coronatum and an endophytic fungus, F. oxysporum, against the M. incognita in tomato plant were also shown47. Similarly another important parasitic nematode Radopholus similis cause on banana and other plants by dual culture techniques the antagonistic effect of endophytic fungi was shown against R. similis population48.
Insecticidal activity of endophytes: Some endophytic fungi can protect their host plants from pathogens and pests31. The foliar endophytes are toxic to insects and vertebrates by producing alkaloids49. Webber firstly reported the endophytic fungi, Phomopsis oblonga to protect elm trees against the beetle Arthrocnemum brevilineum50. The insecticidal activity of endophytic fungi (Acremonium coenophialum) was showed against aphids (Rhopalosiphum padi, Schizaphis graminum) and milkweed bug (Oncopeltus fasciatus)51.
There are different genera of entomopathogenic fungus such As acremonium, beauveria, cladosporium, clonostachys and paecilomyces, were isolated from the coffee plants, among them B. bassiana and Clonostachys rosea were pathogenic to coffee berry borer52. Larvicidal and growth inhibitory activities of B. bassiana against Spodoptera litura was also exhibited by many scientists53. The endophytic fungi Claviceps purpurea possess a significant insecticidal activity against A. gossypii54. The Cladosporium oxysporum showed insecticidal activity against A. fabae 55.
Advantages of the endophytes in agriculture: There are several advantage of endophytes such as: Endophytes encourage the plants growth, increase plant disease resistance, improve the plants ability to withstand environmental stresses and recycle nutrients77. Endophytes are rich source of secondary metabolites with multi-fold importance15. Among the other endophytes microorganism, fungal endophytes produce large number of secondary activities78. Endophytes have strong fungicidal, bactericidal and cytotoxic metabolites64.
Endophytes produce some enzymes which are used for various practical application like degradation and bio-transformation of organic compound79-80. The derivatives of endophytes are used in biotechnological application81. Due to its antimicrobial, anticancer and antiviral activities it has been significance in the field of pharmaceutical science82.
Plant growth stimulants: Endophytes promote the plant growth through a variety of mechanisms, as endophytic metabolites provide a variety of fitness to host plants enhanced by increasing plant resistance to biotic and abiotic stresses, as well as enhance plant growth. Many endophytes are capable of solubilization of phosphate, enhance uptake of phosphorus (P), nitrogen fixation, production of siderophores and plant hormones such as auxin, abscisins, ethylene, gibberellins and indole acetic acid (IAA), which are important for plant growth regulations79,83-88. Gibberellic acid (GA) is a potent phytohormone, that regulates plant growth. Fungal endophyte, Cladosporium sphaerospermum from the plant, Glycine max (L) Merr. produce GA3, GA4 and GA7. It induced plant growth in rice and soybean89. A pestalotin analogue isolated from the Pestalotiopsis microspora exhibited significant gibberellin activity against Distylium chinense seeds and increase germination rate (85.56%)90. The Fusarium tricinctum and A. alternata derivatives of indole acetic acid (IAA) enhanced the plant growth91.
Crop protection: Endophytic fungi are also capable of inducing resistance to diseases and many mechanisms have been proposed for this resistance. The mechanisms of endophyte induced resistance are related to the nutritional status of the host and to increase the fitness of plants by enhancing their tolerance to abiotic stress92. Endophytic fungi Cryptosporiopsis cf. quercina and Colletotrichum sp., are found effective against phytopathogens such as Rhizoctonia cerealis, Phytophthora capsici, Pyricularia oryzae and Gaeumannomyces graminis 93. Trichoderma and Aspergillus are used to manage many soil-borne plant pathogens94. Trichoderma has long been considered as one of the most promising endophyte/bio-control agent for several plant pathogens95,96. Sixteen fungal endophytic species with 2 strains of Aspergillus flavus i.e., brown and green were isolated from physic nut seeds during one year of storage97.
CONCLUSION
Endophytic fungi have wide application in different fields. It has the potential to produce many bioactive compounds. The secondary metabolites produced by the endophytic fungi have the ability to act as bio-control agent. Endophytic fungi isolated from the medicinal plants would be a promising source for many pharmaceutical ingredients. Plants are the major preliminary source for pharmaceutical drug products. Isolating a compound from the plants and large scale production of a product is expensive and time consuming. But endophytic fungi originated from medicinal plants have the capability to produce valuable compounds and can be easily cultured and large scale production is possible through fermentation process. In future the products from the endophytic fungi will be a cheap source for medical, agriculture and other industries. It is sure that the research on endophytic fungi will lead to isolate more novel compounds.
SIGNIFICANCE STATEMENT
The study on different endophytic fungi and bacteria show brilliant prospect for successive studies for sustainability of soils and agriculture ultimately.