Endophytic Fungi: A Source of Potential Antifungal Compounds

The emerging and reemerging forms of fungal infections encountered in the course of allogeneic bone marrow transplantations, cancer therapy, and organ transplants have necessitated the discovery of antifungal compounds with enhanced efficacy and better compatibility. A very limited number of antifungal compounds are in practice against the various forms of topical and systemic fungal infections. The trends of new antifungals being introduced into the market have remained insignificant while resistance towards the introduced drug has apparently increased, specifically in patients undergoing long-term treatment. Considering the immense potential of natural microbial products for the isolation and screening of novel antibiotics for different pharmaceutical applications as an alternative source has remained largely unexplored. Endophytes are one such microbial community that resides inside all plants without showing any symptoms with the promise of producing diverse bioactive molecules and novel metabolites which have application in medicine, agriculture, and industrial set ups. This review substantially covers the antifungal compounds, including volatile organic compounds, isolated from fungal endophytes of medicinal plants during 2013–2018. Some of the methods for the activation of silent biosynthetic genes are also covered. As such, the compounds described here possess diverse configurations which can be a step towards the development of new antifungal agents directly or precursor molecules after the required modification.

Endophytic fungus Xylaria sp. YM 311647 associated with Azadirachta indica from Yuanjiang County, China was also reported to produce five new guaiane sesquiterpenes, (50)(51)(52)(53)(54) (Figure 3). The antifungal activities of 50-54 were evaluated by means of the broth microdilution method against C. albicans, A. niger, P. oryzae, F. avenaceum and H. compactum Compounds 50-54 exhibited average or poor antifungal activities against P. oryzae and H. compactum (MIC values in the range of 32-256 µg/mL). Among them, 53 exhibited the most promising inhibitory activity against P. oryzae with a MIC value of 32 µg/mL. Compounds 52 and 53 showed average antifungal activities against H. compactum with MIC values of 64 µg/mL. In addition, 53 and 54 exhibited the most promising antifungal activities against C. albicans with MIC values of 32 µg/mL. Compound 52 showed average inhibitory activities against C. albicans, A. niger, and H. compactum with MIC values of 64 µg/mL. All compounds showed no notable inhibitory activities against Fusarium avenaceum [33]. Amazonian endophytic fungus X. feejeensis residing in Croton lechleri yielded nonenolide, xyolide (55) (Figure 3). Compound 55 exhibited antifungal activity against oomycetes Pythium ultimum with a MIC value of 425 µM [34]. Endophytic fungus Xylaria sp. XC-16 associated with Toona sinensis was isolated from Yangling, Shaanxi Province, China and was observed to produce a potent antifungal compound Cytochalasin Z28 (56) (Figure 3), displaying enhanced activity with an MIC of 12.5 µM as opposed to the antifungal activity possessed by hymeaxszol possessing an MIC value of 25 µM against the plant pathogen Gibberella saubinetti [35].
Two eicosanoic acids, 2-amino-3,4-dihydroxy-2-25-(hydroxymethyl)-14-oxo-6,12-eicosenoic acid (79) and myriocin (80) (Figure 4), were isolated from Mycosphaerella sp. an endophytic fungus of Eugenia bimarginata DC. (Myrtaceae) collected in Brazil (Savannah). These compounds displayed antifungal activities against several isolates of C. neoformans and C. gattii, with MIC values for compound 79 ranging from 1.3 to 2.50 µg/mL and for compound 80 was 0.5 µg/mL [47]. Both compounds exhibited antifungal activities against several isolates of C. neoformans and C. gattii, with MIC values ranging from 0.49 to 7.82 µM for compound 79 and 0.48-1.95 µM for compound 80 in another study. When checked by the checkerboard microtiter assay, both compounds exhibited synergistic activity against C. gattii with amphotericin B. Ultrastructural analysis divulges various signs of damage in C. gattii and C. neoformans cells treated with compounds 79 and 80, including deformities in cell shape, depressions on the surface, and withered cells. Compounds 79 and 80 showed less loss of cellular material in cells of C. gattii compared to those treated with amphotericin B.
The difference in cellular material loss increased in a test compound concentration-dependent manner. Compound 80 also induced the formation of several pseudohyphae, suggesting that it could reduce virulence in C. gattii cells [48].
Endophytic fungus Guignardia sp., associated with Euphorbia sieboldiana collected from Nanjing, Jiangsu, China was the source of guignardone N (81) and guignardic acid (82) (Figure 4). Both compounds were evaluated for their inhibitory effects alone and with fluconazole on the growth and biofilms of Candida albicans. At 6.3 µg/mL combined with 0.031 µg/mL of fluconazole, compounds 81 and 82 were found to have prominent inhibition on the growth of C. albicans with fractional inhibitory concentration (FIC) index values of 0.23 and 0.19, respectively. Combined with fluconazole, both (40 µg/mL for (81) and 20 µg/mL for (82) could also inhibit C. albicans biofilms and reverse the tolerance of C. albicans biofilms to fluconazole [49].
Altenusin (120) (Figure 6), a biphenyl derivative, was isolated from an endophytic fungus, Alternaria alternata Tche-153 of Terminalia chebula, collected from Bangkok, Thailand. Employing disk diffusion method and the microdilution checkerboard technique, altenusin (120) in amalgamation with each of three azole drugs, ketoconazole, fluconazole or itraconazole at their low sub-inhibitory concentrations displayed potent synergistic activity against C. albicans with the fractional inhibitory concentration index range of 0.078 to 0.188 [64]. It is reported that Schizosaccharomyces pombe cells treated with altenusin were more rounded in shape than untreated cells which suggest that altenusin could act through the inhibition of cell wall synthesis or assembly in S. pombe [65].
Trichoderma koningiopsis YIM PH30002 collected at Wenshan, Yunnan Province of China was the source of two new metabolites koninginins R and S (138-139) ( Figure 7). These isolated compounds showed certain antifungal activities against phytopathogens, Fusarium flocciferum and Fusarium oxysporum. Compound 138 possess the weak activity against F. oxysporum and F. flocciferum with the MICs at 128 µg/mL, while compound 139 displayed the poor activity against F. oxysporum with the MIC at 128 µg/mL [75].
Fusaripeptide A (149) (Figure 7), a new cyclodepsipeptide, was isolated from the culture of the endophytic fungus Fusarium sp. associated with roots of Mentha longifolia growing in Saudi Arabia. Its structure was elucidated based on 1D and 2D NMR and HRESI and GC-MS experiments. The absolute configuration of the amino acid residues of 149 was assigned by chiral GC-MS and Marfey's analysis after acid hydrolysis. Compound 149 exhibited potent antifungal activity toward C. albicans, C. glabrata, C. krusei, and A. fumigates with IC 50 values of 0.11, 0.24, 0.19, and 0.14 µM, respectively. Under similar condition control amphotericin B exhibited antifungal activity toward C. albicans, C. glabrata, C. krusei, and A. fumigates with IC 50 values of 0.3, 0.6, 0.5, 0.7 µM, respectively [81].
Fusarithioamide A, a new benzamide derivative (150) (Figure 7) was isolated from Fusarium chlamydosporium associated with the leaves of Anvillea garcinii collected from Al-Azhar University, Saudi Arabia. Compound 150 exhibited good antifungal activity against C. albicans with inhibition zone diameters (IZD 16.2 mm and MIC 2.6 µg/mL which is comparable to the positive control substance clotrimazole (IZD 18.5 mm and MIC 3.7 µg/mL) [82].
Two new isoaigialones, B (165) and C (166) (Figure 8), along with aigialone (167) (Figure 8), were obtained from Phaeoacremonium sp., an endophytic fungus associated with the leaves of Senna spectabilis was collected in the Araraquara Cerrado area, in June 2001, Araraquara, Sao Paulo state, Brazil. These compounds were evaluated against Cladosporium cladosporioides and C. sphaerospermum using direct bioautography. Compounds 165 and 167 exhibited antifungal activity, with a detection limit of 5 µg, for both fungi, while compound 166 displayed weak activity (detection limit > 5 µg), with a detection limit of 25 µg. Nystatin was used as a positive control, showing a detection limit of 1 µg [86].

Antifungal Potential of Volatile Organic Compounds (VOCs) from Endophytic Fungi
Volatile organic compounds (VOCs) are generally carbon compounds which exist in the gaseous phase at normal/ambient temperatures and pressures. Over 250 different VOCs produced by fungi comprising different chemical classes such as aldehydes, ketones, alcohols, phenols, thioesters, and so forth, have been identified in the context of the deterioration of fruits, vegetables, indoor environments (sick building syndrome); as chemotaxonomic markers; and in the morphogenesis and development of fungi.
However, bioprospecting fungal endophytes for the production of volatile antimicrobials came into the limelight with the discovery of Muscodor albus from the plant Cinnamomum zeylanicum, from Honduras. M. albus was found to produce an admixture of VOCs which could effectively kill a variety of pathogenic bacteria and fungi associated with plants and animals. This research garnered much attention and drove people to explore the volatile antibiotic properties of endophytic fungi for varied applications [90,91].
The genus Muscodor comprises of an endophytic fungi which is predominantly sterile, does not possess true reproductive structures like other fungi, and emanates a characteristic smell which is largely attribute to its VOC composition [92]. Since the report of M. albus in the late 1990s, to date, 20 species have been added to this genus, which have largely been identified based on their volatile signatures, molecular phylogeny, and morphological characteristics ( Table 2). The characteristic VOC profile, therefore, is helpful in delineating the species, as well as playing a significant role in its anti-fungal and anti-bacterial properties. In this section, we only be highlight the anti-fungal potential of VOCs produced by these endophytic fungi.
The majority of the VOCs produced by the endophytic fungi comprises of a mixture of volatile components which generally has either a synergistic effect or an additive effect that enhances their bioactivity against pathogenic microbes. However, in a couple of studies, the major components of the volatile mixture were independently evaluated to understand their true antimicrobial/anti-fungal potential. These are generally synthetically generated and converted into a volatile form and subsequently evaluated for their bioactivity against the test microorganisms. For instance, Sclerotina sclerotiorum was completely inhibited by 2-methyl-1-butanol and 3-methyl-1-butanol with an EC 50 value of 0.8 µL/mL. 2-methyl-1-butanol also inhibited Penicillium digitatum with an EC 50 value of 0.48 µL/mL and B. cinerea with a value of 1.38 µL/mL. However, the volatile admixture of the M. albus VOC exhibited an IC 50 range between 0.08 and 1.13 µL/mL, which clearly confirms the hypothesis of the synergistic/additive effects of the volatile components [93].
Recently, ethyl acetate has been reported to be the main VOC of yeasts Wickerhamomyces anomalus, Metschnikowia pulcherrima, and Saccharomyces cerevisiae, which inhibit the decay causing mold, as well as B. cineria. All three yeasts exhibit excellent biological control properties and were used for checking the mold and pathogenic attack in sweet cherries and strawberries. W. anomalus induced the highest killing activity amongst the three which was attributed to the higher production of Ethyl acetate. The role of the ethyl acetate was re-affirmed by using synthetic ethyl acetate from strawberry fruits to affirm the anti-fungal action [94].
Similarly, Phaeosphaeria nodorum, which existed as an endophyte in plum leaves (Prunus domestica) was found to inhibit the pathogen Monilinia fruticola. The major component of the VOC produced by Phaeosphoran odorum comprised of 3-methyl-1-butanol, acetic acid, 2-propyn-1-ol, and 2-propenenitril [95]. Similarly, six VOCs from the endophytic fungus Hypoxylon anthochroum (that is, phenylethyl alcohol), 2-methyl-butanol and 3-methyl-1-butanol, eucalyptol, ocimene, and terpenoline were tested against Fusarium oxysporum. The results indicated that these compounds exhibited concentration-dependent anti-fungal activity individually but have better action and control synergistically. Thus, the mixture of six VOCs may be used for the control of Fusarium oxysporum in tomatoes [96].
The genus Muscodor is one of the best studied endophytic fungus which produces a synergistic mixture of VOCs having lethal effects against a wide variety of plant and human pathogenic fungi, nematodes, and bacteria as well as certain insects [97][98][99][100]. The volatility of the Muscodor species has been used to replace methyl bromide (MeBr)-a traditional soil fumigant-which has been globally banned as it causes the depletion of ozone layer. Different species of Muscodor, their major VOCs, and their anti-fungal spectrum are given in Table 2. Geographically, each Muscodor species has a characteristic signature volatility. For instance, the Indian Muscodor species invariably has 4-Octadecylmorpholine as a marker compound while 2-methyl propanoic acid is generally found in Muscodor isolated from North and South America.
The majority of the VOCs from the fungal endophytic fungi are used as biological control agents to prevent the fungal deterioration of crops, fruits, and vegetable, under both pre-and post-harvest conditions. However, the exploitation of these fungally volatile organic compounds (FVOCs) from endophytic fungi are not being actively applied to humans for the prevention of fungal infections.
There exists a huge scope in evaluating these FVOCs from endophytic fungi since they could be helpful in curing superficial skin infections, the sanitization of public toilets, and in night soil. They can also find applications in personal care products such as for the aroma/fragrance in deodorants and sprays. They could presumably be helpful in the development of sprays for inhalation to treat fungal diseases like Aspergillosis in lungs.

Methods Used for Activation of Silent Biosynthetic Genes
Several research studies confirm that most of the biosynthetic gene clusters are observed to be silent or expressed at a low (minimal) level upon employing conventional culturing conditions for growth/propagation of microorganisms [113]. To activate such silent biosynthetic genes, numerous strategies have been employed, such as the one strain many compounds (OSMAC) approach (activation mediated through modification in composition of medium, aeration, temperature or shape of culturing flask), co-culturing method (facilitating activation through interspecies crosstalk) and genomics based approaches (expression of orphan biosynthesis genes in a heterologous host). In recent times, the use of chemicals as modifiers to alter the epigenetic makeup/constitution of a microorganism to improve its biosynthetic potential has become a beneficial tool. The method uses a chemical that acts as DNA methyltransferase inhibitors (DNMTi) or histone deacetylase inhibitors (HDACi), thereby stimulating the transcription previously silent gene clusters and fostering the production of a spectrum of natural products. A comprehensive description of some of these methods are given below/highlighted in the subsequent section.

Epigenetic Modification
Endophytes have proven to be the prolific source of bioactive metabolites and offer a substitute and untapped reserve for the discovery of novel metabolites. Studies have led to findings that tell biosynthetic gene clusters of microorganisms are mostly silent or expressed at very low levels under standard culture conditions and are least expressed, but under stress condition may it be biological, chemical or physical their expression takes place. Epigenetic modulators lead to the expression of these silent or cryptic genes. Epigenetic gene regulation is mediated by covalent histone modification, DNA methylation chromatin modeling basically induced by DNA methyl transferase inhibitors such as 5-aza-2-deoxycytidine, 5-azacytidine, hydralazine, procaine and histone deacetylase [114]. Chromatic modification in fungi to enhance gene transcription has led to secondary metabolite production of anthraquinones, cladochromes, lunalides, mycotoxins, and nygerones [115]. Structural genes that control transcriptional factor regulates the synthesis of secondary metabolites in fungi, these genes mediate factors occupied in environmental signals like pH, nitrogen and carbon sources, temperature, light, etc. [116]. In lab condition, these gene clusters are mostly silent. Under which natural conditions these clusters become activated is still unexplained. As per genetic sequencing studies carried so far, it is estimated that the clusters of genes responsible for secondary metabolites have not yet been deciphered completely [117].
To express silent biosynthetic pathways, molecules such as HDAC and DNMT are used to enhance the fungal metabolites production. Different studies suggest an increase in chemical diversity of metabolites by induction with these epigenetic modifiers. For growth and acclimatization with the environment fungus are known to produce diverse secondary metabolites. Cross talk between microbes and plant lead to the expression of these pathways which stays silent in in vitro conditions. Metabolic profiles shift led by SMs induced modifier is due to expression of cryptic genes [126].

The Co-Culture Strategy
Interspecific interaction among different species leads to evolution and biodiversity, organism combines their genetic information for better adaptability. The cohabitation of different microorganisms that share similar niches competes with growth, morphology, adaptation, and development patterns [127,128]. The increased productions of metabolites in co-culture which are not produced in axenic culture are the result of competition or antagonism faced by the microorganism that leads to activation of cryptic genes. [129]. Co-cultivation is a way to provide natural habitat to fungi so that gene clusters become activated. In Aspergillus nidulans, the cryptic gene has been successfully activated leading to isolation of novel compounds [130].

The Co-Culture Strategy
Interspecific interaction among different species leads to evolution and biodiversity, organism combines their genetic information for better adaptability.
The cohabitation of different microorganisms that share similar niches competes with growth, morphology, adaptation, and development patterns [127,128]. The increased productions of metabolites in co-culture which are not produced in axenic culture are the result of competition or antagonism faced by the microorganism that leads to activation of cryptic genes. [129]. Co-cultivation is a way to provide natural habitat to fungi so that gene clusters become activated. In Aspergillus nidulans, the cryptic gene has been successfully activated leading to isolation of novel compounds [130].
Chaetomium sp. was isolated from Sapium ellipticum the Cameroonian medicinal plant. When Chaetomium sp. was cultured axenically on solid rice medium, average yields per culture flask were 2.8, 13.9, 132.7 and 14.6 mg of acremonisol A (246), SB236050 (247) (Figure 12), and SB238569 (248), respectively, and 1:1 mixture of 3-and 4-hydroxybenzoic acid methyl esters (249-250), respectively, ( Figure 13) was observed. When Co-cultivation of Chaetomium sp. was undertaken with viable or autoclaved cultures of Bacillus subtilis there was a strong accumulation of the 1:1 mixture of (249), and (250), was observed, accounting for an 8.3 and 7.4-fold increase, respectively, compared to axenic fungal controls in both cases. SB236050 (247) and SB238569 (248), two major polyketides of Chaetomium sp., were not detected in co-cultures. Five new compounds, Shikimeran A (251), Bipherin A (252), Chorismeron (253), Quinomeran (254), and Serkydayn (255), and two known compounds, isosulochrin (218) and protocatechuic acid methyl ester (256) (Figure 12), were only detected in cocultures of Chaetomium sp. with viable or autoclaved B. subtilis cultures, but were lacking in both fungal or bacterial controls when cultured axenically [124].  These studies indicate that co-culture generates a complex and promising environment to obtain new secondary metabolites as a response to the interaction between endophytic fungi. The above also indicates that the production of new natural products depends on stimuli.

Conclusions
Endophytic fungi are the ubiquitous source of novel chemical compounds having the potential to display antifungal activities. Interestingly, the active metabolites from endophytic fungi possess excellent antifungal activity not only against human fungal pathogens but also on plant fungal pathogens. In addition, the volatile organic compounds (VOCs) from genus Muscodor displayed significant antifungal as well as antibacterial properties and, therefore, they are used to prevent fungal deterioration of crops, fruits and vegetables. However, their application to control human fungal infection has not been explored. Fungal VOCs can be investigated for the development of sprays for inhalation to treat fungal diseases such as Aspergillosis in lungs, curing superficial skin infections and sanitization. Endophytic fungi are being studied to produce natural compounds which are originally produced from their host plants and, thus, emerging as an alternative and sustainable source of valuable natural products. It is important to investigate the interactions between endophytic fungi with the host plant and other endophytes which are very sensitive to the culture conditions and hence, provide an opportunity to tune the in vitro culture conditions to produce the desired range of secondary metabolites. It is possible to produce a compound of interest by varying the culture conditions such as media composition, aeration rate and temperature. In addition, cultivation of endophytic fungi in presence of bacteria or other fungi (co-cultivation) yield novel compounds which otherwise do not appear when fungi or bacteria are cultivated alone. Therefore, considerable research on endophytic fungi is required for the development of suitable co-culture system for the sustained production of the desired secondary metabolite.
Author Contributions: S.K.D., V.P., S.S. and M.K.G. reviewed the contents critically. V.P. and M.K.G. drew chemical structures and assisted in the preparation of Table 1. S.S. wrote the Antifungal potential of Volatile organic compounds of review. The manuscript has been read and approved by all named authors.
Funding: This research received no external funding.