A REVIEW OF THE MUTUALISTIC RELATIONSHIP OF ENDOPHYTIC FUNGI FOR THE PRODUCTION OF BIOACTIVE METABOLITES

A REVIEW OF THE MUTUALISTIC RELATIONSHIP OF ENDOPHYTIC FUNGI FOR THE PRODUCTION OF BIOACTIVE METABOLITES

Mohd Azharuddin *, Mohammad Saad and Amol R. Kharat

Department of Pharmacognosy, Government College of Pharmacy, Aurangabad, Maharashtra, India.

ABSTRACT: Endophytes constitute a remarkably multifarious group of microorganisms ubiquitous in plants and maintain an imperceptible association with their hosts for at least a part of their life cycle. Their enormous biological diversity, coupled with their capability to biosynthesize bioactive secondary metabolites, has provided the impetus for a number of investigations on endophytes. There is a need to search new ecological niches for the potential of natural bioactive agents for different pharmaceutical, agriculture, and industrial application; these should be renewable, eco-friendly, and easily obtainable natural products discovery in the search for new drugs and is the most potent source for the discovery of novel bioactive compounds. Therefore, a large number of bioactive compounds are isolated from plants, bacteria, fungi, and many other organisms. Endophytic fungi, the most promising of these, have been a source of various such bioactive compounds. Many of these compounds are being used for the treatment of a number of diseases. This review emphasis on the biology of fungal endophytes, their discovery, isolation, identification by morphological and molecular methods, production, purification and structure elucidation of the bioactive compounds.

Keywords: Fungal endophytes, Endophytic fungi, Antimicrobial, Bioactive compounds, Endophytism, Isolation, Bioactive compounds, Structural elucidation

INTRODUCTION: Fungi are important components in every ecosystem, intimately associated with crucial processes like the decomposition, recycling and transportation of nutrients in different environments. It has been estimated that there may be over a million different fungal species on this Earth, of which only a small fraction [approx. 5%] have been identified ¹. There are also many bacteria that exist as plant endophytes; in most instances, they coexist with endophytic fungi.

The existence of endophytes has been known for over one hundred years. They live as imperfect fungi most of the time and have been described as benign parasites or true symbionts. It has been suggested that they can influence the host plants' distribution, ecology, physiology, and biochemistry ². Botanists have conducted much research about the relationship of plant endophytes, especially for grasses such as tall fescue, where it has been exhibited that endophytes produce toxins that discourage insects and other grazing animals ³.

It wasn’t until the past decade that endophytes were extensively studied for their potential as novel sources of effective new drugs. Microbes, both fungi, and bacteria, have provided modern medicine or drugs with valuable, effective treatments, including penicillin from the fungus Penicillium notatum and bacitracin from Bacillus subtilis, a common bacterium. Additionally, a potent chemotherapeutic agent, taxol is synthesized by an endophyte of the Pacific Yew tree. Endophytes represent a huge diversity of microbial adaptations that have developed in special and sequestered environments. Their diversity and specialized habituation make them an exciting field of study in the search for new medicines or novel drug-like molecules. The hunt for new drugs is particularly important in view of the fact that so many microorganisms are developing resistance to some of the current drugs. Endophytic fungi are a group of fungi that colonize living and internal tissues of plants without causing any immediate, overt negative effects ⁴. Recent studies have revealed these fungi's ubiquity, with an estimated 1 million species of endophytic fungi residing in plants ⁵ and even lichen ⁶. Endophytic fungi represent an important and quantifiable component of fungal biodiversity and are known to affect plant community diversity and structure ⁷. According to ¹, only about 100,000 fungal species have been described out of a conservative estimate of 1.5 million. Recent studies of endophytic fungi from tropical and temperate forests support the high estimates of species diversity ^8-10.

Relationship between Endophytic Fungi and Host Plant: A variety of relationships exist between fungal endophytes and their host plants, ranging from mutualistic or symbiotic to antagonistic or slightly pathogenic ¹¹. Endophytes may produce an overabundance of substances of potential use in agriculture, industry, and modern medicine such as novel antibiotics, antimycotics, immunosuppressant and anticancer compounds. In addition, the studies of endophytic fungi and their relationships with host plants will shed light on the ecology and evolution of both the endophytes and their hosts: the evolution of endophyte plant symbioses; the ecological factors that influence the direction and strength of the endophyte host plant interaction. Since, natural products are likely adapted to a specific function in nature, searching for novel secondary metabolites should concentrate on organisms that inhabit novel biotopes ¹².

TABLE 1: INFLUENCES OF HOST MEDICINAL PLANTS ON THE POPULATION STRUCTURE OF ENDOPHYTIC FUNGI ^13-15

Classification of Endophytic Fungi: Schaechter (2011) ¹⁶ stated that endophytic fungi have frequently been divided into two groups based on differences in taxonomy, host range, colonization transmission patterns, tissue specificity and ecological function. Group one is the Clavicipitaceous endophytes (C-endophytes) which infect some grasses. Group two is the Nonclavicipitaceous endophytes (N Cendophytes). While Rodriguez et al., (2009) ¹⁷.

Clavicipitaceous Endophytes (Class I): The Clavicipitaceae is a family of fungi (Hypocreales; ascomycota) including free living and symbiotic species associated with insects and fungi or grasses, rushes and sedges ¹⁸.

Many of its members produce alkaloids that are toxic to animals and humans. European investigators first noted clavicipitaceous endophytes of grasses in the late 19^th century in seeds of Lolium temulentum, L. arvense, L. linicolum and L. remotum ¹⁸.

Nonclavicipitaceous Endophytes (Class II): Traditionally NC-endophytes treated as a single functional group, while Rodriguez et al., (2009), ^19, showed that NC-endophytes represent three distinct functional groups. Class II endophytes include the hyper diverse endophytic fungi associated with leaves of tropical trees as well as the highly diverse associates of above-ground tissues of nonvascular plants, seedless vascular plants, conifers and woody and herbaceous angiosperms in biomes ranging from tropical forests to boreal and Arctic/Antarctic communities ²⁰.

FIG. 1: BIOTECHNOLOGY APPLICATION OF SECONDARY METABOLITES AND EXTRACELLULAR ENZYMES PRODUCED FROM ENDOPHYTIC FUNGI

TABLE 2: LIST OF SOME BIOACTIVE COMPOUNDS PRODUCED BY ENDOPHYTIC FUNGI POSSESSING BIOLOGICAL ACTIVITIES ^21-26

Identification of Fungal Endophytes: Morphological identification of endophytic fungi by mycologists is a critical step ²⁷.

TABLE 3: FUNGAL ENDOPHYTES ISOLATED FROM VARIOUS PLANTS ^28-32

Methods for Isolation of Fungal Endophytes: Isolation by cutting of selected plant parts.

Isolation by Blender shaft. Isolation by Mortar and Pestle

TABLE 4: MEDIA USED FOR ISOLATION OF FUNGAL ENDOPHYTES ³³

Production and Optimization of Endophyte-Derived Bioactive Compounds ³⁴:

Production of Bioactive Compounds from Fungal Endophytes: The symbiotic relationship among endophytic fungi and plants gives the powerful ability to produce new bioactive compounds. However, there are two main substrate-based methods for producing bioactive compounds: solid-state fermentation and submerged-state fermentation ³⁴.

Solid state Fermentation (SSF): Solid State fermentation is widely used for the production of the bioactive compound from the fungal endophytes ³⁴. These biomolecules are mostly metabolites generated by endophytic fungi grown on solid support selected for this purpose. In this fermentation process, different solid substrates such as Wheat bran, Rice bran, coconut oil cake, vegetable waste, gram husk, orange peel, sugarcane bagasse etc, were used with pure cultures of endophytic fungi ³⁴.

SSF enables the optimal growth of endophytic fungi, permitting the mycelium to spread on the surface of solid compounds through which air can flow ³⁴. SSF uses culture substratum with low water levels. The solid medium contains both the substrates and solid support. After fermentation, fermented media are mixed with effective solvent and further used for purification and analysis ³⁴.

Submerged Fermentation: In submerged fermentation, enzymes and other reactive compounds are submerged in a liquid such as alcohol, oil, or nutrient broth. Endophytic fungi are sited in a small closed flask containing the rich nutrient broth with a high volume of oxygen. The in situ production of enzymes results in the production of bioactive molecules. Batch Fed fermentation method is commonly used, which utilizes the sterilized nutrients under optimized conditions along with fungal endophytes, increasing density. The addition of nutrients maintains the growth rate of fungal endophytes, also reduces risk of an overflow of metabolism ³⁴.

Optimization of Production of Bioactive Compounds from Fungal Endophytes ³⁵: Optimization of both fermentation processes depends on considerations of carbon homes and nitrogen homes, inoculums, phosphorus, organic acids, surfactants, incubation period, temperature, moisture level, and pH level under optimized conditions to achieve the greatest production of bioactive compounds from fungal endophytes.

• Effect of different medium
• Effect of carbon sources
• Effect of nitrogen sources
• Effect of inoculum amount
• Effect of inoculum time
• Effect of pH and temperature ³⁵

TABLE 5: MEDIA USED FOR THE PRODUCTION OF BIOACTIVE COMPOUNDS BY FUNGAL ENDOPHYTES ³⁶

Novel fungal Endophytes Verses Novel Bioactive Compounds ^37-42: Discovering novel bioactive compounds from undiscovered endophytes is the current trend. Not all endophytes are culturable, and these may produce useful bioactive metabolites. Metabolomics of endophyte-infected and endophyte-free plant hosts could reveal intersections in secondary metabolite paths that may be pushed into synthesizing novel chemical species or lead compounds, another possibility of manipulating these chemo-diverse organisms ³⁷.

In fungal endophytes, genes coding for enzymes of secondary metabolic pathways usually occurs as gene clusters being positioned in the same locus and co-expressed. These gene clusters are known to evolve swiftly through multiple rearrangements, duplication, and losses and are capable of interspecific feast through horizontal gene transfer. It is important to screen fungal species for their secondary metabolite assortment under different growing conditions; culture parameters such as composition of growth medium, aeration, pH and the presence of certain enzyme inhibitors change vividly the secondary metabolite profile and even induce the synthesis of several new metabolites ³⁸ is because the synthetic capability of endophytes, like in other organisms, has been fine tuned by natural selection over millions of years. Smith et al. (2008) united sequence analysis with bioassay procedures to explore the endophyte diversity of the tropics. Their results suggest that tropical plants harbour a substantial portion of undiscovered endophytes that may be vested with novel biochemical diversity. Hence, including fungal endophytes in natural product discovery programs is necessary. Testing endophytes isolated from different tissues of plant hosts and plants growing in unusual and less studied habitats will be more productive ^39-42.

CONCLUSION: Isolation of fungal endophytes from medicinal and other plants may result in methods to produce biologically active agents for biological exploitation on a large commercial scale, as they are easily cultured in a laboratory and fermenter instead of harvesting plants and affecting the eco-friendly biodiversity.

ACKNOWLEDGEMENT: NIL

CONFLICTS OF INTEREST: The authors declare no conflict of interest exists.

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    How to cite this article:

    Azharuddin M, Saad M and Kharat AR: A review on mutualistic relationship of endophytic fungi for the production of bioactive metabolites. Int J Pharm Sci & Res 2023; 14(6): 2651-57. doi: 10.13040/IJPSR.0975-8232.14(6).2651-57.

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