Exploring endophytes for in vitro synthesis of bioactive compounds similar to metabolites produced in vivo by host plants

1.   Importance of medicinal plants

Different types of microbial species as symbionts of a plant, living most of their lifetime within the tissues showing no symptoms, are recognized as endophytes [1]. Normally plants have always been a primary source of food and medicine since time immemorial. Medicinal plants have always remained a primary source for treating common ailments and diseases in some parts of the world lacking basic healthcare facilities. Several allopathic drugs are either transformed or derived directly from plant parts thus putting pressure on already depleting plant resources. Alternative source of some of the metabolites commonly derived from plants would eventually reduce our dependence on plant-based bio-resources. The herbal medicines derived from plants have been well documented since ancient civilizations of India, Egypt, China, Central Asia, Greece, etc. These civilizations, over several centuries, have played a considerable role in exploring and reporting beneficial properties of diverse group of plant species [2]–[4]. Medicinal plants and their derivatives remain a major source of medicine for regular ailments in developing countries as they are reasonably priced and easily accessible [5].

Last few decades have again received a considerable interest towards the search for unique metabolites from natural sources [6]. Several components of drugs are still derived directly from plant parts while few others are transformed from the molecules obtained from various plants. Even after exploring for natural compounds all these years, plants continue to hold treasure house of unknown metabolites [7]. Demand for Ayurvedic and Chinese herbal medicines are very high due to inadequate facilities for allopathic treatment and poor healthcare system in these regions [8].

About 70% of people across the globe continue to rely on herbal medicines as remedies and for treating numerous diseases [9]. There is a considerable growth in consumption of medicines derived from plants even in Western and European countries [10]. Herbal products occupy fair share in overall drug market across the globe which will continue to grow steadily [11],[12]. Medicinal plants continue to hold a significant place in various therapeutics and health care systems leading to massive demand for plant-based bio-resources [13].

2.   Characteristics of endophytes

The microorganisms such as fungi, bacteria including actinomycetes and viruses that reside within plant tissues are known as endophytes [14]. The endophytes have been classified as true endophytes or transient endophytes depending upon their diversity, biological nature, classification and method of transmissions [15]. Endophytes were further classified by Rodriguez et al. into clavicipitaceous (class 1) and non-clavicipitaceous (classes 2, 3 & 4) based upon the narrow or broad range of hosts, types of tissues colonized, pattern of colonization in plants that is either extensive or limited, in planta bio-diversity that could be high, low or unknown, vertical or horizontal types of transmission through different generations and habitat or non-habitat adapted fitness benefits. Tolerance to drought conditions and enhancement of growth are common non-habitat adapted benefits, irrespective of origin of habitat, whereas benefits of habitat adapted are specific to the habitat with selective pressures that include salinity, pH and temperatures [16].

Considerable attention in the extensive investigation of beneficial microorganisms from the plant tissues fully demonstrate their unique abilities to produce secondary metabolites of the host plant and collection of functionalities (Figure 1) with their possible applications in agriculture, pharmaceutical and industrial sectors [17]–[20]. Importance of endophytes came into light only after the demonstration of toxic syndrome in cattle caused by endophytes of pasture grasses [21],[22]. Endophytes are abundant in nature and have been found in all those plant species that have been studied so far. These microorganisms share an obligate or facultative relationship with the plant while causing no harm to their host [23]. Endophytes have characteristic of producing bioactive compounds, as they have been isolated from the tissues of roots, leaves and stems of their host plant, which produce similar metabolites [24],[25].

Identification of fungal endophytes has been carried out by studying the morphological characteristics after sporulation. However, classification of non-sporulating fungi is problematic and it is carried out through phylogenetic analyses of rDNA-ITS sequences after the amplification of DNA extracted from the fungal mycelia [26],[27]. Similarly, phylogenetic analyses of the 16s sequences obtained after the amplification of rDNA would help to identify the bacterial endophytes [28].

Figure 1.  Possible applications of metabolites and functionalities derived from endophytes in different sectors.

DownLoad: Full-Size Img PowerPoint

3.   Metabolites and activities of endophytes

Microbial endophytes are well-known for their ability to produce a wide range of pharmacologically important compounds with enormous therapeutic potentials; which have been identified as antiviral, antifungal, antibacterial, antitumor and anticancer agents. A number of endophytes are prospective source of plant growth promoting factors, and plant hormones. They can synthesize compounds of applications in the field of agriculture, iron chelating agents, compounds with nematocidal, insecticidal activities and abiotic stress tolerant properties. Some endophytes have shown their ability to secrete wide range of extracellular enzymes, such as phosphatase enzyme to convert insoluble phosphates to soluble form for its easy assimilation by plants. Endophytes produce molecules suitable for the production of bio-fuels and degrade complex organic and inorganic substances with suitable use in industrial sectors. The useful properties of endophytes are listed below with their potential significance in respective sectors.

3.1.   Potential significance of endophytes with respect to agriculture

Published work state that endophytes are definitely an excellent source of metabolites and desired functions that could prove to be beneficial in organic farming system. Some of the endophytes could be used as bio-pesticides against phytopathogens due to their antimicrobial, nematocidal and insecticidal qualities.

3.1.1.   Pesticidal properties of endophytes

The extracts from a perennial grass native to most of Europe Phleum pratense, demonstrated myco-toxic properties, which were secreted by a systemic grass symbiont fungal endophyte Epichloe typhina. The antifungal properties of the extracts were detected against Cladosporium herbarum [29]. A strain of fungus, L1930, obtained from Larix laricina displayed insecticidal property against larvae of spruce budworm. Chitinase, known to degrade chitin polymers that are essential part of a fungal cell wall, was produced by bacteria, an endophyte of Sinapis arvensis. The bacterial endophyte was identified as Bacillus cereus strain [30] and was known to play a defensive role against a phytopathogen Rhizoctonia solani [31].

Strain of Neotyphodium sp. (AR601) producing large quantities of alkaloids such as loline and ovaline inoculated into a cultivar ‘Jackal’ of turf tall fescue have shown birds deterring ability [32]. Several endophytes have consistently shown to induce effective resistance in plants against common phytopathogens, by producing proteins related to pathogenesis. Fungal endophytes found from the leaves of trees typically growing in Indian states of Western Ghats, and Tamil Nadu, were able to secrete chitinase and chitosanase, which could increase defenses in host plant against phytopathogens, by initiating host defenses and increasing resistance [33],[34].

3.1.2.   Plant growth promotion by endophytes

Endophytes have been identified to solubilize phosphates, produce siderophores, secrete plant growth promoting factors and increase soil nutrition by degrading complex organic molecules. It was observed that ericoid plants were able to thrive in extreme conditions due to the presence of an endophyte, Hymenoscyphus ericae, that produced several enzymes along with phosphate solubilization properties [35]. Lu et al isolated Colletotrichum sp. B501 from the healthy stems of Artemisia annua L, secreted IAA and 3β-hydroxy-ergosta-5-ene, these compounds that showed properties for plant growth [36]. The production of a range of factors and plant hormones have been reported from both fungal and bacterial endophytes [37]–[41]. Some endophytes have been found to increase tolerance of plants in soils contaminated with heavy metals [39],[42],[43]. In vitro investigation of endophyte-plant interaction in Echinacea purpurea demonstrated that colonization potential of bacterial strains belonging to Pseudomonas and Arthrobacter genus were tissue specific in host plants from which they were originally obtained but did not show similar specificity in non-host plants [44]. Further, plant growth promotion (PGP) was observed in inoculated plants due to the secretion of Indoleacetic acid by endophytic bacteria. Physiology of plants were influenced by compounds secreted by endophytes and plant metabolites, in turn, they regulated the growth of endophytes. Similarly, endophytic strains of Bacillus sp. isolated from Thymus vulgaris demonstrated plant growth promoting traits in Solanum lycopersicum L under salt stress along with showing antagonistic activity against Fusarium oxysporum and reduced the antioxidant stress on plants [45]. Antagonistic properties against human pathogens were observed in cultivable bacteria obtained from different segments viz. roots, stem, leaf and flower of Origanum vulgare L [46]. Pseudomonas and Bacillus were the most represented genera of endophytes in Lavandula dentata that demonstrated multiple PGP traits [47]. All these aspects make these endophytes a potential source of bio-fertilizer, bio-pesticide, plant growth promoter and maintain overall growth and development of the plants.

Some of the bacterial and fungal endophytes and their potential applications in agriculture are listed in Table 1 (1.1 and 1.2).

3.2.   Potential significance of endophytes with respect to pharmaceuticals

Products derived from natural sources are a major area of research for discovering the range of their functions, that could be used in pharmaceutical industries [66],[67]. Microorganism from different biotypes have repeatedly proven to be a constant source of secondary metabolites with novel and unique properties, which have found a major place in medical sector [68]. Since the discovery of endophytes and their ability to produce plant secondary metabolites and other bioactive compounds, several reports are available on mining of novel secondary metabolites [69],[70]. Different saponins showing antagonism were extracted from Fusarium sp. PN8 isolated from Panax notogensing [71].

3.2.1.   Antimicrobial properties of endophytes

Some species of endophytes are known to produce antimicrobial compounds. Phomopsichalasin (11) an antimicrobial agent was extracted from Phomopsis sp., isolate no. MF6031 obtained from the twigs of Salix gracilostyla var. melanostachys. The compound 11 exhibited antibacterial activity against Bacillus subtilis, Salmonella gallinarium and Staphylococcus aureus with some amount of antagonism towards Candida tropicalis [72]. Findlay et al isolated an endophytic fungus from the needles of Larix laricina (Du Roi) K. Koch [53]. which produced 6-oxo-2-propenyl-3, 6-dihydro-2H-pyran-3-yl ester (12) showing antibacterial activity against Vibrio salmonicida, S. aureus and Pseudomonas aeruginosa.

In another study, a Colletotrichum sp. isolated from internal stem tissues of Artemisia annua L. showed antifungal, antibacterial and fungistatic properties. Its metabolites 6-isoprenylindole-3-carboxylic acid (19), 3β,5α-dihydroxy-6β-phenylacetyloxy-ergosta-7,22-diene (21), 3β-hydroxy-ergosta-5-ene (15), 3-oxo-ergosta-4,6,8(14),22-tetraene (16) and 3β,5α-dihydroxy-6β-acetoxy-ergosta-7,22-diene (20) showed antibacterial activity against Gram-positive and Gram-negative bacteria. Compounds 15, 20 and 21 had antifungal properties, compound 15, 19 and 20 demonstrated fungistatic property [36]. Several other researchers have also studied endophytes possessing antimicrobial properties [71],[73]–[77].

Table 1.1.  Bacterial endophytes with potential significance in agriculture sector.

Sl. No.	Functionalities	Endophytes	Properties	Host plant	Ref
1.	Chitinase	Bacillus cereus strain 65	Antifungal	Sinapis arvensis L.	[31]
2.	Jasmonates, Abscisic acid and phosphate solubilization	Bacillus sp., Achromobacter sp., Alcaligenes sp.	Plant growth and development	Helianthus annuus L.	[38]
3.	Leu-surfactin (8)	Bacillus mojavensis RRC 101	Biocontrol of Fusarium verticillioides	Bacopa monnieri L.	[48]
4.	Nitrogen fixation	Rhizobium leguminosarum	Biofertilization, increase rice yield.	Oryza sativa L.	[40]
5.	Phosphatases, Siderophore, Nitrogen fixation	Rahnella sp. and Pseudomonas sp.	Bio-fertilization	Musa L.	[49]
6.	Siderophore	Streptomyces sp. GMKU 3100	Promote plant growth	Oryza sativa L	[50]
7.	Plant growth promoting factors	Enterobacter sp. FD17	Enhancement of maize yield	Zea mays L.	[51]
8.	IAA, Siderophore, Phosphate solubilization	Serratia sp., Enterobacter sp., Acinetobacter sp., Pseudomonas sp., Stenotrophomonas sp., Agrobacterium sp., Ochrobactrum sp., Bacillus sp. and Tetrathiobacter sp.	Plant growth promotion in Zea mays.	Zingiber officinale Roscoe	[41]

---

 | Show Table

DownLoad: CSV

Table 1.2.  Fungal endophytes with potential significance in agriculture sector.

Sl. No.	Functionalities	Endophytes	Properties	Host plant	Ref
Clavicipitaceous
1.	Ethyl trans-9.10-epoxy-ll-oxoundecanoate (1), Ethyl 9-oxononanoate (2), Ethyl azelate (3), Hydroxydihydrobovolide (4).	Epichloe typhina	Antifungal	Phleum pratense L.	[52]
2.	8,1′,5′-trihydroxy-3′,4′ dihydro-1′H-[2,4′]binapthalenyl-1,4,2′-trione (5)	Fungus L1930 (unidentified)	Insecticide	Larix laricina (Du Roi) K. Koch	[53]
3.	Phosphatase, Protease, Cellulase, Hemicellulases, Pectinolytic enzymes, Ligninase	Hymenoscyphus ericae	Phosphate solubilization, Protein breakdown, Cell wall lysis.	Ericoid plants	[35]
4.	Indole-3-acetic acid (IAA) and 3β-hydroxy-ergosta-5-ene (6)	Colletotrichum sp. B501	Plant growth hormone	Artemisia annua L.	[36]
5.	Phosphate solubilization	Penicillium sp.	Bio-fertilization	Triticum aestivum L.	[54]
6.	3-Hydroxypropionic acid (7)	Phomopsis phaseoli and Melanconium betulinum strains	Nematicidal	Broad leaved tree of tropical rainforest, Betula pendula Roth. And Betula pubescens Ehrh.	[55]
7.	Volatile organic compounds	Muscodor albus	Mycofumigation	Cinnamomum zeylanicum Blume	[56]
8.	Protease amylase, lipase, laccase, cellulase and pectinase.	Various fungal species	Enhance resistance of grasses to multiple stresses.	Catharanthus roseus L. (G. Don.), Calophyllum inophyllum L., Bixa orellana L., and Alpinia calcarata. Roscoe	[57]
9.	Gibberellins	Penicillium sp. M5.A and Aspergillus sp. M1.5	Promote plant growth and development.	Monochoria vaginalis (Burm.f.) C. Presl ex Kunth	[37]
10.	Siderophore	Phaeotheca sp. Fusarium sp., Penicillium sp. and Arthrinium sp.	Antibacterial	Pinus sylvestris L. and Rhododendron tomentosum Harmaja	[58]
11.	1,8-cineole (monoterpene) (9)	Hypoxylon sp.	Antimicrobial	Persea indica (L.) Spreng.	[59]
12.	Chitosanase, chitinase.	Xylariaceae sp., Aureobasidium pullulans, Colletotrichum sp., Lasiodiplodia theobromae, Phomopsis sp. and Fusarium sp., Botrytis sp., Trichoderma sp., Alternaria sp., Nodulisporium gregarium, Nigrospora oryzae, Drechslera sp., Pithomyces sp. Sordaria sp. and Pestalotiopsis sp.	Pathogenesis related proteins, phytoalexins and proteinase inhibitors in plants. Acts against phytophagous nematodes and plant pathogenic fungi.	Leaves of different tree species of Western Ghats.	[33]
13.	Phosphate solubilization	Penicillium sp.	Bio-fertilization	Camellia sinensis (L.) Kuntze	[60]
14.	Gibberellins and Indole acetic Acid	Penicillium sp. LWL3 and Phoma glomerataLWL2	Promote plant growth	Cucumis sativus L.	[61]
15.	Plant growth promoting factors	Phoma sp.	Bio-fertilizatzion	Tinospora cordifolia (Thunb.) Miers and Calotropis procera (Aiton) W.T. Aiton	[62]
16.	Trichodemin	Trichoderma brevicompactum	Antifungal against phytopathogens	Allium sativum L.	[63]
17.	Indole acetic acid, Gibberellins and Reactive oxygen species.	Galactomyces geotrichum WLL1	Promote growth of plants in heavy metal contaminated soil.	Trapa japonica Flerov	[42]
18.	Not identified (ethyl acetate extract)	Aspergillus sp. and Emericella sp.	Insecticidal properties	Rhizophora mucronata Lam.	[64]
19.	Plant Growth promotion and Resistance to heavy metals	Phialocephala fortinii, Rhizodermea veluwensis, and Rhizoscyphus sp.	Growth enhancement, Nutrient uptake, Decrease Heavy metal concentration	Clethra barbinervis Sieb. Et Zucc.	[43]
20.	Not identified (ethyl acetate extract)	Several fungal isolates belonging to Ascomycota and few Zygomycota.	Antifungal properties against root rot pathogens.	Panax notoginseng (Burkill) F. H. Chen ex C. Y. Wu & K. M. Feng	[34]
Non clavicipitaceous
21.	Indole Acetic Acid (IAA)	Rhodotorula sp. and Rhodosporidium sp.	Plant growth	Populus L.	[65]
22.	Plant growth promoting factors and reduce cadmium toxicity	Piriformospora indica	Enhance plant growth in cadmium toxic soil.	Triticum aestivum L.	[39]

---

 | Show Table

DownLoad: CSV

3.2.2.   Other medicinal properties of endophytes

Few endophytes show the medicinal properties as anticancer and antitumor in their metabolites. An endophyte isolated from Taxus brevifolia Nutt., Taxomyces andreanae, was able to produce Taxol, the host secondary metabolite, in a broth culture medium [78]. Similarly, different metabolites with anticancer properties were obtained from the microbial species isolated from different plant species [79],[80]. A metabolite, Hypericin, with anti-viral, antimicrobial and anti-inflammatory properties was produced from a microbial strain isolated from Hypericum perforatum L. [81]. Lovastatin was produced in significant amount by an endophyte Phomopsis vexans isolated from Solanum virginianum L. [82].

The secondary metabolites and other functions from endophytes could have potential applications in therapeutics without causing damage to the respective plant species. The bacterial and fungal endophytes suitable for therapeutic purposes are listed in Table 2 (2.1 and 2.2).

Table 2.1.  Bacterial endophytes with potential significance in therapeutic sector.

Sl. No.	Functionalities/ Metabolites/Compounds	Endophytes	Properties	Host plant	Ref
1.	Xiamycin (62), methyl ester of Xiamycin (63)	Streptomyce sp. GT2002/1503	Antiviral	Bruguiera gymnorrhiza (L.) Savigny	[83]
2	Agarwood	Bacillus pumilus.	Antimicrobial, Laxative, sedative, digestive, etc.	Aquilaria species	[84]

---

 | Show Table

DownLoad: CSV

Table 2.2.  Fungal endophytes with potential significance in therapeutic sector.

Sl. No.	Functionalities/Metabolites/Compounds	Endophytes	Properties	Host plant	Ref
Clavicipitaceous
1.	Taxol (10)	Taxomyces andreanae	Antitumor	Taxus brevifolia Nutt.	[78]
2.	Phomopsichalasin (11)	Phomopsis sp. isolate no. MF6031	Antimicrobial	Salix gracilostyla var. melanostachys	[72]
3.	Cryptocandin (13)	Cryptosporiopsis quercina	Antimycotic	Tvipterigeum wilfordii Hook. f.	[85]
4.	3β,5α,6β-trihydroxyergosta-7,22-diene (14), 3β-hydroxy-ergosta-5-ene (15), 3-oxo-ergosta-4,6,8(14),22-tetraene (16), 3β-hydroxy-5α,8α-epidioxy-ergosta-6,22-diene (17), 3β-hydroxy-5α,8α-epidioxy-ergosta-6,9(11),22-triene, 3-oxo-ergosta-4-ene (18), 6-isoprenylindole-3-carboxylic acid (19), 3β,5α-dihydroxy-6β-acetoxy-ergosta-7,22-diene (20) and 3β,5α-dihydroxy-6β-phenylacetyloxy-ergosta-7,22-diene (21).	Colletotrichum sp.	Antibacterial, antifungal and fungistatic.	Artemisia annua L.	[36]
5.	7-butyl-6,8-dihydroxy-3(R)-pent-11-enylisochroman-1-one (22), 7-but-15-enyl-6,8-dihydroxy-3(R)-pent-11-enylisochroman-1-one (23), 7-butyl-6,8-dihydroxy-3(R)-pentylisochroman-1-one (24)	Geotrichum sp. Ccre7	Antifungal, antituberculous and antimalarial	Crassocephalum crepidioides (Benth.) S. Moore	[74]
6.	Asperfumoid (25), Asperfumin (26), Monomethylsulochrin (27), Fumigaclavine C (28), Fumitremorgin C (29), Physcion (30), Helvolic acid (31), 5α,8α-epidioxy-ergosta-6,22-diene-3β-ol (32), Ergosta-4,22-diene-3β-ol (33), Ergosterol (34), Cyclo (Ala-Leu) (35) and Cyclo (Ala-Ile) (36).	Aspergillus fumigates CY018	Antimycotic	Cynodon dactylon (L.) Pers.	[73]
7.	Brefeldin A (37)	Cladosporium sp.	Antimicrobial	Quercus variabilis Blume	[75]
8.	Ampelopyrone (38), macrosporin (39), 3-O-methylalaternin (40), methyltriacetic lactone (41), citreoisocoumarin, macrosporin (42), 3-O-methylalaternin (43), desmethyldiaportino (44), desmethyldichlorodiaportin (45), ampelanol (46), altersolanol A (47), alterporriols D (48), alterporriols E (49) and altersolanol J (50).	Ampelomyces sp.	Cytotoxic and antimicrobial	Urospermum picroides (L.) Scop. ex F.W. Schmidt	[86]
9.	Paclitaxel (51)	Fusarium solani	Anticancer	Taxus celebica (Warb.) H. L. Li	[79]
10.	Usnic acid (52), Cercosporamide (53), Phomodione (54).	Phoma sp. isolate No. 2323	Antibacterial	Saurauia scaberrinae Hemsley	[77]
11.	Phomopsin A (55), Phomopsin B (56), Phomopsin C (57), Cytosporone B (58), Cytosporone C (59)	Phomopsis sp. ZSU-H76	Antifungal	Excoecaria agallocha L.	[87]
12.	Not identified	Fusarium sp. DF2	Antimicrobial	Taxus wallichiana Zucc.	[88]
13.	Deoxypodophyllotoxin (61)	Aspergillus fumigatus Fresenius	Anticancer	Juniperus communis L. Horstmann	[80]
14.	Benquinol (64), Benquoine (65)	Phomopsis sp. CMU-LMA	Antibacterial and cytotoxic	Alpinia malaccensis (Burm. f.) Roscoe	[89]
15.	Terpene (66)	Phomopsis sp.	Antibacterial	Allamanda cathartica L.	[90]
16.	8-octadecanone (67), 1-tetradecene (68), 8-pentadecanone (69), octylcyclohexane (70) and 10-nonadecanone (71).	Fusarium solani	Antimicrobial	Taxus baccata L.	[91]
17.	Emerimidine A (72), Emerimidine B (73), Emeriphenolicins A (74), Emeriphenolicins D (75), Aspernidine A (76), Aspernidine B (77), Austin (78), Austinol (79), Dehydroaustin (80), and Acetoxydehydroaustin (81)	Emericella sp. (HK-ZJ)	Antiviral	Aegiceras corniculatum (L.) Blanco	[92]
18.	Guignardin A (82), Guignardin B (83), Guignardin C (84), Guignardin D (85), Guignardin E (86), Guignardin F (87), Palmarumycin C1 (88), BG1 (89) and JC1(90).	Guignardia sp. KcF8	Antimicrobial, Cytotoxic, Protein inhibitor	Kandelia candel (L.) Druce	[93]
19.	Lovastatin (91)	Phomopsis vexans	Lower blood cholesterol	Solanum xanthocarpum	[82]
20.	Unknown	Luteibacter sp. NORREL-Li2	Bio convert major ginsenosides into minor ginsenoside	Platycodong randiflorum (Jacq.) A. DC.	[94]
21	Saponins	Fusarium sp. PN8 and Aspergillus sp. PN17	Antimicrobial	Panax notoginseng (Burkill) F. H. Chen ex C. Y. Wu & K. M. Feng	[71]
Not identified
22.	Protocatechuic acid (92) and acropyrone (93).	Fungal endophyte	Antibacterial	Citrus jambhiri Lush.	[76]
23.	Hypericin (60)	INFU/Hp/KF/34B	Antibiotic, antiviral, anti-inflammatory, seasonal effective disorder, relief from sinusitis	Hypericum perforatum L.	[81]
24.	6-oxo-2-propenyl-3,6-dihydro-2H-pyran-3-yl ester (12)	L1930 (unidentified)	Antimicrobial	Larix laricina (Du Roi) K. Koch	[53]

---

 | Show Table

DownLoad: CSV

Table 3.1.  Bacterial endophytes with potential significance in industrial sectors.

Sl. No.	Functionalities	Endophyte	Properties	Host plant	Ref
1.	Pectinase	Paenibacillus amylolyticus	Pectin lyase	Coffea Arabica L.	[95]
2.	Thermostable α-amylase	Nocardiopsis sp.	Starch degradation	Pachyrhizus erosus L.	[96]
3.	Thermostable glucoamylase	Streptosporangium sp.	Starch degradation	Zea mays L.	[97]
4.	Protease	Bacillus halotolerans strain CT2	Alkaline protease	Solanum tuberosum L.	[98]

---

 | Show Table

DownLoad: CSV

Table 3.2.  Fungal endophytes with potential significance in industrial sectors.

Sl. No.	Functionalities	Endophyte	Properties	Host plant	Ref
Clavicipitaceous
1.	Amylase, cellulase, xylanase and ligninase.	Fusarium sp., Phomopsis sp. Phoma sp., Colletotrichum sp.,	Wood degradation	Brucea javanica (L.) Merr.	[99]
2.	Microbial oil and cellulase	Phomopsis, Cephalosporium, Microsphaeropsis, and Nigrospora.	Production of bio-fuel	Taxus chinensis var. mairei Mast, Cupressus torulosa D. Don, Keteleeria davidiana varchienpeii, Sabina chinensis cv. Kaizuca and Keteleeria evelyniana Mast.	[100]
3.	Myco-diesel	Gliocladium roseum (NRRL 50072)	Energy production and utilization	Eucryphia cordifolia Cav.	[101]
4.	1,4-Cyclohexadi-ene (94)	Hypoxylon sp.	Oxidizes to benzene (component of crude oil)	Persea indica (L.) Spreng.	[59]
5.	Polyurethanases	Pestalotiopsis microspora E2712A	Degrade polyester polyurethane	Ecuadorian Amazonian plant	[102]
6.	Lipase	Candida guillermondi	Synthesis of methyl oleate	Ricinus communis L.	[103]
7.	Amylase	Alternaria sp., Phoma sp., Nigrospora sp.	Starch hydrolysis at alkaline pH and low temperature	Eremophilia longifolia (R.Br.) F. Muell.	[104]
8.	Bio-pigment	Phoma sp.	Bio-pigment production	Clerodendrum viscosum L.	[105]
9.	Xylanases	Trichoderma harzianum	Xylan degrading enzyme	Sargassum wightii	[106]
10.	Laccase	Hormonema sp. and Pringsheimia smilacis	Degrade Lignin	Eucalyptus globules Labill.	[107]
11.	Cellulase and Xylanase	Acremonium sp. Aspergillus sp.	Degrade cellulose and Xylan	Memecylon excelsum Blume, Glochidion borneese Mull. Arg.) Boerl.	[108]
Non clavicipitaceous
12.	Lignocellulolytic enzymes	Bjerkandera sp.	Wood degradation	Drimys winteri J. R. Forst. & G. Forst. and Prumnopitys andina (Poepp. ex Endl.) de Laub.	[109]
13.	Microbial oil and cellulase	Sclerocystis	Production of bio-fuel	Taxus chinensis var. mairei Mast, Cupressus torulosa D. Don, Keteleeria davidiana varchienpeii, Sabina chinensis cv. Kaizuca and Keteleeria evelyniana Mast.	[100]

---

 | Show Table

DownLoad: CSV

3.3.   Potential significance of endophytes in industries

Microorganisms and their derivatives play a significant role in processing of substrate into several products for use in industrial sectors. There are many reports of enzymes being produced by endophytes isolated from different plant species. Enzymes like amylase, pectinase and lipases obtained from different endophytes have been known to hydrolyze starch, pectin and oils, respectively. Other enzymes include cellulases, xylanases, amylase, laccase, and proteases, which have application in various industrial sectors [98],[104],[106],[107]. An endophyte, Nocardiopsis sp., isolated from Pachyrhizus erosus L., was found to secrete a thermostable α-amylase, which is useful for starch degradation [96]. Similarly, Candida guillermondii from Ricinus communis L. produce lipase and helps in the synthesis of methyl oleate [103].

Some endophytes are also known to produce bio-fuels, as alternate source of conventional fuels. A fungal isolate, Hypoxylon sp. from Persea indica (L.) Spreng. was found to secrete 1,4-Cyclohexadi-ene (94). The compound 94 readily oxidizes to benzene, which is a main component of crude oil [59]. In another work, an endophyte Gliocladium roseum (NRRL 50072) isolated from Eucryphia cordifolia Cav. produced a bio-fuel known as myco-diesel [101].

While some of the endophytes are known to degrade polyurethane which are of great value to the industrial sector [102], others are known to produce pigment suitable for use in food industry [105]. Bacterial and fungal endophytes with their ability to produce bioactive compounds with their potential applications in industries are listed in Table 3 (3.1 and 3.2).

3.4.   Understating the potential of endophytes through genome mining

Some microorganisms are known to synthesize only a limited number of secondary metabolites (SMs) as compared to the ones estimated through genome mining [110]. SMs are synthesized through pathways that utilize multiple enzymes. Biosynthetic gene cluster (BGC) comprises set of genes that encode for proteins required during a pathway. Diverse methods could be employed to activate the cluster of genes that remain silent under in vitro conditions. The genome mining approach could reduce the time taken to identify the putative genes required for the synthesis of secondary metabolites [110]. The sequencing of genes have helped in the identification of genes related to SMs and enhanced the characterization process [111]. Nielsen and Nielsen have suggested three approaches for understanding the unknown BGCs that include targeted approach: where the similar BGCs are compared to form a probable BGCs, untargeted approach involves the use of different databases to mine for information and lastly through the use of metabolomics techniques [112]. Wang et al. [113] developed bacteriophage recombinases to quickly identify and stimulate BGCs that are cryptic in strains of Burkholderia species. Poplar trees augmented with a modified strain of endophyte, Pseudomonas putida W619-TCE, showed increased reduction (90%) of trichloroethylene evapotranspiration under field tests [114].

4.   Progress and developments

In an effort to meet the increasing demand of food and feeds, chemical fertilizers and pesticides have been commonly used in agricultural system for improving soil fertility and controlling pests, respectively. The adverse effect of use of toxic chemicals in agriculture has resulted in increasing interest in sustainable farming practices [115],[116]. Biofertilizers and Biopesticides derived from microorganisms have been effective in dealing with phytopathogens as well as Biofertilization of the soil. Bacterial and fungal endophytes have shown positive effects in plant growth promotion, pest management and improving soil health [117],[118]. Numerous endophytes have shown their ability to promote plant growth and antagonism against phytopathogens under in vitro conditions. Some of the strains have found their place in modern agricultural practices, such as perennial ryegrass (PRG) due to its endophyte, Neotyphodium lolii, was able to protect the host plant from Argentine stem weevil infection without producing any toxic compounds harmful to livestock. A product of Rye grass, AR1, infected with the endophyte has been beneficial for livestock production in places with lesser number of black beetles. Another strain of PRG, Endosafe, has shown better survival response in places dominated with black beetles but with decreased biomass production compared to AR1 [119].

Adaptive Symbiotic Technologies in Seattle, USA, have developed several products under the brand name BioEnsure® using a combination of beneficial endophytes. The products are able to induce tolerance in crops to drought, high salt concentration and temperature; it improves water utilization by plants and is fairly stable in different climate and soil types. The microbial formulations can easily be applied to fields along with other agriculture inputs and are non-competitive against other normal microbial flora of the soil. The products have a viability of more than two years at 4 °C. BioEnsure® products not genetically modified are classified as organic products by Organic Material Review Institute, Eugene, USA [120]. Muscodor albus isolated from cinnamon tree, has shown properties related to bio-fumigation and it may replace the use of methyl bromide for fumigation of soils [121]. Though the effects of endophytes cannot substitute chemical inputs altogether, combination of different methods and suitable endophyte-plant combinations could be considered for integrated pest management programs [122].

5.   Constrains in commercialization of endophytes

There have been numerous reports on the production of plant secondary metabolites by endophytes outside its host but there are no products as such that have successfully been produced in mass scale and commercialized. Production of Taxol by endophytic fungus in the early 90s was thought to develop the process of obtaining metabolites from endophytes with eventual decrease in over-use of plants. However, apart from the use of few endophytes in agricultural system, not a single product from endophytes has made it to the market with a significant advance in secondary metabolite industry [123].

The reason for the production of host metabolites by the endophytes could be hidden in their genes that must have undergone genetic recombination during the time of their evolution [124]. Our inability to understand the mechanisms by which these endophytes function inside the host, and as stated by Bailey et al their evolutionary significance [125] has limited our knowledge.

Some of the constraints involved in the production of secondary metabolites under laboratory conditions include: the low-yield of secondary metabolites, optimization of growth-conditions involving variety of abiotic factors and silent gene clusters, synthesis of metabolites with unidentified functions, unclear understanding of pathways involved in the production of metabolites, role of secondary metabolites in different pathways and lack of a complete knowledge on secondary metabolites [110]. The cellular relationship between the host and its endophyte limits our ability to understand the mechanism of host-secondary metabolite production by an endophyte, and the eventual reduction in synthesis when outside its host in vitro system.

6.   Future perspectives

Endophytic microorganisms have convincingly demonstrated their remarkable ability to typically produce an abundance of pharmacological metabolites with possible usage in drug manufacturing. Extensive search for newer metabolites is important to deal with multi-drug resistant microorganisms and to find alternative therapeutic drugs for several diseases. Secretion of plant growth-promoting factors and antagonistic agents against phytopathogens could easily substitute chemical inputs in sustainable agriculture practices with suitable endophytes. Novel enzymes with better specific-activity obtained from endophytes could be valuable in fermentation industries. However, most of the published findings are from controlled experiments and similar results from in vivo trials could satisfactorily establish the practical possibility of endophytes commercialization. Specific mechanisms involved in the complex interactions, types of selection pressures that properly govern the crosstalk between endophytes and their suitable host, efficient production of host secondary metabolites, and possible ways to effectively manipulate the biochemical-pathways, would undoubtedly require comprehensive understanding before the successful commercialization of bioactive metabolites from endophytes.

7.   Conclusions

It is estimated that there are more than quarter million species of plants in this planet with a possibility of obtaining more than one million endophytes from these plants. Very few of these microorganisms from their diverse group have been isolated and studied so far. Apart from producing array of metabolites and functions advantageous to its host plant, these microbial resources have proven to secrete similar secondary metabolites even outside its host using in vitro systems. These properties of endophytes not only make them suitable candidates for exploring their ability to produce various bioactive compounds, enzymes, and biopigments, etc., but it may also reduce the dependency of humans on endangered plant species for their secondary metabolites, thus resulting in sustainable use of plant-based bio-resources. The necessary factors controlling growth of endophytes for biosynthesis of host secondary metabolites in vitro, are required to be optimised for commercial-scale production of plant-derived natural compounds employing these endophytes.