key: cord-0060808-ghpzs37z
authors: Saxena, Sanjai
title: Biologically Active Secondary Metabolites from Endophytic Alternaria Species
date: 2021-03-26
journal: Endophytes
DOI: 10.1007/978-981-15-9371-0_1
sha: 78b41a5fbd181f68e091868cfcb3ae518ef54445
doc_id: 60808
cord_uid: ghpzs37z

Microbial secondary metabolites have contributed immensely in the development of a variety of medicines, namely antibiotics, metabolic inhibitors, immunomodulatory agents, antioxidants and anticancer agents. Endophytism in the past decade has further opened avenues of exploration and exploitation of new chemical entities produced during the plant–microbe interaction for pharmaceutical as well as agricultural applications. Alternaria is a genus belonging to ascomycete and generally comprise of members which cause agricultural spoilage, involved in decay and decomposition and some exist as opportunistic human pathogens. The genus is a prolific producer of secondary metabolites, which are finding applications in the agrochemical as well as pharmaceutical industry. The present review is an update on specialized members of Alternaria genus existing as an endophyte and the new chemical entities produced by them possessing agrobiological and pharmacological properties.

Endophytes are microorganisms which reside within the plant tissues without any symptomatic signatures of their existence, and their relationship with the host fluctuates from a saprobe to a latent pathogen or commensalistic/mutualistic symbiont (Stone et al. 2000) . Endophytes play an imperative role in combating with the abiotic and biotic stresses encountered by the host plant apart from improving the growth and yield (Rodriguez et al. 2009 ). During the course of their interactions within the plant system, there is a lot of chemical communication happening which is mediated by a variety of signal molecules which have largely remained underexplored (Saikkonen et al. 2010; Paramanantham et al. 2019) .

The concept of endophytism evolved with the discovery of taxol-producing endophytic fungus Taxomyces andreanae from the taxol-producing plant Taxus brevifolia (Stierle et al. 1995) . This discovery raised questions on the ability of endophytic fungus as a producer of the host phytochemicals due to horizontal gene transfer from the host to the endophyte during the course of evolution or vice versa? (Suryanarayanan 2013) . However, in due course many other endophytic isolates were found to produce taxol which were isolated from unrelated plants suggesting the biogenetic capabilities of the endophytic fungus being far more than their hosts (Porras-Alfaro and Bayman 2011). Subsequently a spectrum of endophytic fungi was explored from the medicinal plants to assess the possibility of fermentative production of phytomedicines. Today, endophytic fungi producing phytomedicines such as camptothecin, podophyllotoxin, huperzine and vincristine have been well studied (Aly et al. 2011b; Nisa et al. 2015) . Apart from producing the phytomedicines, the endophytic fungi exist under a variety of biotic and abiotic stress which render them metabolically active thereby producing novel chemical entities which help in sustaining themselves in the torrid internal environment of the plants (Braga et al. 2016; Rana et al. 2020) . Over the last two decades, endophytic fungi have been recognized as a lucrative and prolific source of novel chemistries which could be developed into newer medicines as well as environment and userfriendly agrochemical (Zhang et al. 2006; Gouda et al. 2016; Martinez-Klimova et al. 2017; Al-Ani 2019) . However, this is just the tip of the iceberg since every plant on the earth, under different ecological niches, harbours a novel set of endophytic microorganisms (fungi as well as bacteria). Thus, exploration and exploitation of endophytes are alive and well in the present millennium.

The genus Alternaria was established in 1817 with Alternaria alternata as a type isolate. Alternaria belongs to the family Dematiaceae and commonly exists as a saprophyte deriving energy through cellulolytic activity. Generally, Alternaria species are inhabitants of soil or decaying (plant based) organic matter. However, some members of this genus are opportunistic pathogens and cause diseases in economically important plants such as ornamentals, vegetables like broccoli, cauliflower, tomatoes citrus, apples, and oil crops (Thomma 2003) . They have also been identified as post-harvest pathogens. Secondary metabolites produced by the members of the genus Alternaria existing as a saprophyte or a pathogen have been largely explored and studied; however, bioactive compounds produced by endophytic species of Alternaria are relatively a newer aspect (Lou et al. 2013) . Existence of Alternaria species as an endophyte has been recorded in tropical forests of South India wherein it was isolated from trees belonging to different families.

Some compelling examples of Alternaria species existing as an endophyte in different plant families and reported to exhibit potential bioactivities include Lamiaceae, Polygonaceae (Aly et al. 2008) , Rhodomelaceae ), Lythraceae (Kjer et al. 2009 ), Rubiaceae, Sapindaceae, Vitaceae, Euphorbiaceae, Cupressaceae (Soltani and Hosseyni Moghaddam 2014) , Fabaceae, Calophyllaceae, Cactaceae and from plants in the 'restingas' area of Brazil (Das et al. 2017) . Approximately 268 secondary metabolites have been reported from different Alternaria species till 2013. In this chapter we shall discuss the bioactive metabolites produced by the members of the genus Alternaria existing as an endophyte for their presumptive use as pharmaceuticals and agrochemicals.

The discovery of drugs from microbes began with the serendipitous discovery of Penicillin from the blue mould Penicillium notatum by Sir Alexander Fleming which marked the beginning of the golden era of antibiotics in the pharmaceutical research and development (Aly et al. 2011a) . Ever since the discovery of Penicillin, fungi have been extensively explored for bioactives which have been developed into different drug classes such as antibiotics, antifungal, immunomodulatory, antihypercholesterolemic, anticancer, anti-obesity agents and in treating neurodegenerative diseases by producing inhibitors which could hinder in the formation of toxic protein aggregates (Saxena et al. 2019) . Fungal endophytes are also a prolific source of novel bioactive compounds which could propel the development into newer drugs (Aly et al. 2010 ). The bioactive compounds which have been produced by endophytic Alternaria species can be classified as antimicrobial agents, cytotoxic agents (anticancer agents), anti-diabetic agents, anti-parasitic agents and enzyme inhibitors. In the upcoming paragraphs, an attempt has been made to discuss these promising bioactive compounds on their pharmacological actions.

With the burgeoning incidences of human pathogenic microorganisms encountering resistance to the current armamentarium of antimicrobials, clinicians are finding it difficult to treat drug-resistant chronic infections caused by these superbugs. Hence there is a demand for novel chemical entities with either a unique mode of action or a novel chemical scaffold against which these superbugs have not developed a resistance mechanism. Alternaria sp. Samif01 was isolated as an endophyte existing in the roots of Salvia miltiorrhiza Bunge, produced alternariol-9-methyl ether (1) which exhibited a potential in vitro antibacterial activity against the test panel of microorganisms with a Minimal Inhibitory Concentration (MIC) range between 25 and 75 μg/mL. Other compounds with antibacterial activities produced by Alternaria sp. Samif01 are altenuisol (2), 4-hydroxy alternariol-9-methyl ether (3) and alternariol (4). Altenuisol exhibited a potent activity against Staphylococcus aureus, while alternariol is active against both Bacillus subtilis and S. aureus, i.e. exhibits a broad-spectrum antibacterial activity (Jin et al. 2017) . Another compound reported from Alternaria alternata Tche-153 was altenusin (5) which was isolated from ethyl acetate extract of the liquid culture. Alternaria alternata Tche-153 was isolated from the Thai medicinal plant Terminalia chebula. Altenusin exhibited a potent azole synergistic activity against Candida albicans of which ketoconazole exhibited the highest synergism. As Candida species are encountering resistance to azole class of antifungals, Altenusin holds a promise to be used as an antifungal combination with ketoconazole and other members of the azole family for combating refractory candidiasis (Phaopongthai et al. 2013) . Paracoccidioides brasiliensis is a human pathogenic fungus which causes systemic mycosis known as Paracoccidioiodomycosis. This was being treated by drugs like sulphonamides, amphotericin B and itraconazole. However, in the recent past these drugs have been found to become ineffective which is getting evident by the extension in the duration of the antifungal course or relapses. This has created a demand for new antifungals for this disease. Altenusin isolated from Alternaria sp. UFMGCB55 exhibited a potent antifungal activity against 11 clinical isolates of Paracoccidioides brasiliensis. All the test isolates were highly susceptible to altenusin with MIC values between 1.9 and 3.9 μg/mL. Thus, altenusin hold a promise as a lead for developing a new antifungal agent. Yet another promising antifungal compound reported from Alternaria brassisicola MP-408 which is existing as an endophyte in Malus halliana is Herbarin A (6). Herbarin exhibits a pronounced antifungal activity against Trichophyton rubrum and Candida albicans with a MIC of 15.6 μg/mL. Alternaria brassisicola MP-408 also produced Alterchromone A (7) which exhibited antibacterial as well as anti-candidal activity. The MIC for antibacterial activity was 3.9 μg/mL for E. coli and Bacillus subtilis, while it was 1.8 μg/mL for Pseudomonas florescence. Similarly, for anti-candidal activity the MIC was 3.9 μg/mL (Gu 2009 ). Recently an endophyte of Ocimum tenuiflorum L., Alternaria tenuissima OE7 has exhibited a potential antifungal activity against human pathogenic fungi including Microsporum gypseum, Trichophyton rubrum, Candida albicans and C. tropicalis. Thus, Alternaria tenuissima OE7 appears to be a promising candidate for exploration of antifungal compounds in the drug development programme (Chatterjee et al. 2020) . Solanopyrone P (8), solanopyrone A (9) and solanopyrone C (10) were isolated from the fermentation broth of Alternaria tenuissima SP07 which existed as an endophyte in the roots of Chinese medicinal plant Salvia przewalskii. These compounds exhibited a potent antibacterial activity against Gram-positive human pathogens including methicillin-resistant Staphylococcus aureus (MRSA). Another known metabolite alternariol methyl ether (11) was also produced by A. tenuissima SP07 and exhibited a similar antibacterial activity against the Gram-positive bacteria (Wang et al. 2014b ). Another promising antifungal secondary metabolite is (3R,6R)-3-benzyl-6-isopropyl-4methylmorpholine-2,5-dione (12), which exhibits a potent activity against Alternaria solani, Colletotrichum gloeosporioides and Phyricularia grisea. It was isolated from Alternaria atrans MP-7 which is existing as an endophyte in Psidium guajava. Alternaria atrans MP-7 also produces a derivative of fusaric acid, Atransfusarin (13) which exhibited a remarkable antifungal activity against Alternaria solani, P. grisea and C. gloeosporioides with an MIC of 6.25 μM as compared to the positive controls, carbendazium and fusaric acid (Yang et al. 2019c) . Another compound possessing a strong antibacterial activity against Gram-positive pathogenic microbes is altersetin (14) isolated from Alternaria sp. P0506, existing as an endophyte in Vinca minor. This compound has exhibited an appreciable activity in the murine sepsis model wherein it was intraperitonially injected within a concentration range of 10/25 mg/kg body weight. Staphylococcus aureus was used as the test organism to develop the murine sepsis model (Hellwig et al. 2002) . Fermentation broth of Alternaria sp. MHE 68 isolated from Pelargonium sidoides yielded cyclodecasiloxane (15) which exhibited a potent broadspectrum antibacterial activity including drug-resistant enterococcal strains, viz. Enterococcus faecium and Enterococcus gullinarum (Manganyi et al. 2019) . Another endophytic isolate of Alternaria isolated from Schinus terebinthifolius, Alternaria alternata LGMF626 exhibited a potent anti-MRSA activity with an MIC of 18.52 μg/mL of the cell-free extract. Further fractionation of the extract yielded E-2-hexyl-cinnamaldehyde (16) and 3-benzylhexahydropyrrolo-[1,2-α] pyrazine-1,4-dione (17) and 3-isobutylhexahydropyrrolo-[1,2-α]pyrazine-1,4dione (18) (Tonial et al. 2016 ). E-2-hexyl-cinnamaldehyde exhibited an MIC of 18.52 μg/mL against Staphylococcus aureus while the isolated pyrrolopryazine alkaloids showed a combined MIC of 55.55 μg/mL.

Viruses are the most enigmatic microbes as they are living inside a host and non-living outside the host. Essentially, viruses require a host to replicate or reproduce and, in this process, they affect the host. The human body has mechanisms to combat the viral intruders; however, it depends on the extent and nature of the viruses and the immune status of the host. Over past two decades, there has been a spurt of viral infections across the globe either as major or minor outbreaks, epidemics and pandemics, the most recent being SARS-CoV-2 apart from influenza, herpes, dengue, etc. Getting vaccinated is one of the most probable methods to develop resistance to viral infections; however, it really takes a long time to develop a suitable vaccine. Chemotherapeutic interventions also exist in treating viral diseases which largely act on their replication system inside the host. However, owing to their evolutionary mechanism, they evolve into the drug-resistant versions, thereby making it more difficult for the clinicians to deal with these diseases. With this premise, it is pertinent as well as a necessity to explore novel chemical structures which are effective as well as potent to combat viruses and could be possibly converted into a therapeutic entity.

Fungi have already been recognized as fountainheads of novel chemical entities possessing diverse pharmacological activities including anti-viral activity which primarily is assessed using different in vitro assays. However, fungal endophyte producing anti-viral agents is fairly a newer resource. The premise of fungal endophytes producing anti-viral agents could be hypothesized as a mechanism to help in overcoming the biotic stress encountered by the host plant when attacked by a plant virus. Apart from developing them into therapeutic entities, they can be used in developing topical anti-viral disinfectants. Alternaria tenuissima QUE1se exists as an endophyte in the stem of Quercus emoryi and found to produce Altertoxins, viz. altertoxin V (19), altertoxin I (20), altertoxin II (21) and altertoxin III (22) which inhibit HIV-1 virus at a concentration of 0.5, 2.20, 0.3 and 1.5 μM, respectively. All the compounds appear to be promising for being developed into anti-HIV therapeutics after carrying out Quantitative Structural Activity Relationships (QSAR) studies (Bashyal et al. 2014) . Alternaria solani residing in the roots of Aconitum transsectum Diels. has furnished a metabolite, 7-dehydroxyl-zinniol (23), which has a moderate anti-hepatitis B virus activity (Ai et al. 2012) . Similarly, another endophytic isolate Alternaria alternata PGL-3 was isolated from the peel of Puncia granatum. The ethyl acetate extract of the culture broth of this fungus exhibited highly potent inhibition of HCV NS3/4a protease with an IC 50 value of 17 μg/mL and yielded two previously known compounds alternariol (4) and alternariol-9-methyl ether (11). Thus, it could be concluded that the anti-HBV of Alternaria alternata PGL-3 is attributed to these compounds (El-Kassem et al. 2019).

Tropical diseases largely comprise of pathogenic parasites which are vector or nonvector-borne and predominate in populations where there is poor hygiene and sanitation; the living conditions are poor and have close proximity with animals. It is estimated that more than 1 billion people across the globe are suffering from these tropical parasitic diseases. The pathogenic parasites in tropical regions generally comprise of protozoans, helminths, bacteria and viruses. However, we shall more emphasize on the parasitic diseases caused by the protozoans and helminths which have limited options for the treatment unlike bacteria and viruses for which vaccines can be designed and developed. As these parasitic organisms are difficult to cultivate inside the laboratory, have multicellular complex organization and complete their life cycle in multiple hosts, hence developing vaccines is very difficult. Therefore, small molecule drugs appear to be the best option in treating these diseases. Endophytic fungi existing in tropical plants are highly diverse and can probably be the best source in screening and isolation of compounds which possess anti-parasitic activity. Alternaria sp. UFMGCB 55 existing as an endophyte in Trixis vauthieri (Asteraceae family) collected from Brazil exhibited anti-trypanocidal activities. Further isolation of bioactive extract of this endophytic isolate yielded a biphenyl, altenusin (5). Further it was found that the trypanocidal activity of altenusin was mediated through inhibition of the enzyme trypanothione reductase (TR). The IC 50 value of altenusin for TR was found to be 4.3 AE 0.3 μM. Thus, altenusin is a promising lead for developing a novel lead/therapeutic drug for the treatment of trypanosomiasis and leishmaniasis (Cota et al. 2008) . Another isolate, Alternaria alternata P1210 existing as an endophyte in roots of halophyte Salicornia sp. in Spain furnished two compounds (AE)-alternarlactones A (24) and alternarlactones B (25). These compounds exhibited anti-parasitic activity against Leishmania donovani and Plasmodium falciparum (Shi et al. 2019) . Two perylenequinone class of compounds, namely 3, 6a,9,10-tetrahydroxy-7,8-epoxy-4-oxo-4,5,6,6a,6b,7,8,9-octahydroperylene (26) and 3,6a,7,10-tetrahydroxy-4,9-dioxo-4,5,6,6a,6b,7,8,9-octahydroperylene (altertoxin I) (20), have been isolated from Alternaria sp. DC401 existing as an endophyte in Pinus ponderosa. Both these perylenequinone compounds have exhibited anti-leishmanial activity with an IC 50 and IC 90 values 3.14 and 14.69 mg/mL, respectively, against Leishmania donovani. However, L. donovani exhibited a higher susceptibility to Altertoxin I with an IC 50 of 1.4 mg/mL and IC 90 of 5.754 mg/mL. This also showed antimalarial activity against chloroquine-resistant and -sensitive clones of Plasmodium falciparum with IC 50 of 2.459 and 3.164 mg/mL (Idris et al. 2015) . Thus, possibilities exist where bioactive agents of endophytic Alternaria species could be exploited as anti-parasitic agents directly or indirectly.

Cancer is a generic term given to a set of diseases which can affect any part of the human body. They generally result due to formation of abnormal cells which exhibit uncontrolled growth and proliferation. As these abnormal cells have higher demand for energy due to their continuous proliferative tendency, they invade in other parts of the body thereby affecting the organs and organ systems. This eventually leads to failure of the organ systems and proves fatal. Today, cancer is the second leading cause of global deaths with about 9.6 million deaths registered in 2018.

One of the major strategies to prevent the spread of these abnormally proliferating cells is chemotherapy. Chemotherapy entails the use of a chemical compound of natural or synthetic origin which eventually kills the abnormal cell to prevent further proliferation. This action of the therapeutic chemical is referred to as cytotoxicity. However, challenges do exist in the treatment of cancer. The chemotherapeutic agents used do not have a mechanism of differentiating between healthy and abnormal cells which results in deleterious side effects. Apart from the deleterious side effects due to chemotherapy, the other major challenge is the cost of chemotherapy as well as refractory behaviour of the cancerous cells. Hence, natural product chemists, medicinal chemists and cancer biologists are in a continuous foray to explore novel direct acting new chemical entities from biological matrices which have a directed action on the target cell with minimal side effects. Endophytic fungi have been in centre stage for production of plant-based anticancer agents ever since the discovery of Taxomyces andreanae from Taxus brevifolia which produced taxol in the free fermenting conditions (Stierle et al. 1995) . Since then a number of endophytic fungi have been explored, documented which have the inherent potential to produce anticancer phytomedicines such as camptothecin, podophyllotoxin, vincristine and vinblastine via fermentative route (Aly et al. 2010; Kharwar et al. 2011; Rana et al. 2020) . As endophytic fungi have been recognized as a treasure trove of new chemical entities with a spectrum of bioactivities, it becomes relevant to explore their cytotoxic effects using in vitro assays. Alternaria alternata existing as an endophyte in Paeonia lactiflora was found to produce alternate C (27) which exhibited cytotoxic activities against MDA-MB-231 and MCF-7 cell lines with an IC 50 of 20.1 and 32.2 μM, respectively (Wang et al. 2019) . Bianthraquinone derivatives, i.e. alterporriol family of compounds were first reported from Alternaria porri. An isolate, Alternaria sp. Z69-6B existing as an endophyte in fruit of a mangrove plant Aegiceras corniculatum, from China, has been found to produce three new bianthraquinone derivatives named as alterporriol K, alterporriol L and alterporriol M. Alterporril K (28) and alterporriol L (29) exhibited a moderate cytotoxic activity towards MDA-MB-435 and MCF-7 cells with an IC 50 values in range of 13.1-29.1 μM (Huang et al. 2011) . Xanalteric acid I (30) and Xanalteric acid II (31) were isolated from the solid rice culture of Alternaria sp. existing as a leaf endophyte in Sonneratia alba and exhibit a moderate activity against L5178Y cells at a concentration of 10 μg/mL (Kjer et al. 2009 ). Crude ethyl acetate extract of Alternaria phargmospora, an endophyte isolated from leaves of Vinca rosea, yielded 5-butyl-6-(hydroxymethyl)-4-methoxy-2H-pyran-2-one (32) and 4-methoxy-6-methyl-5-(3-oxobutyl)-2H-pyran-2-one (33) which exhibited appreciable anti-leukaemic activity against HL60 cells with an IC 50 of 2.2 and 0.9 μM, respectively. The activity of these compounds was also found against the immortalized myelogenous leukaemia cell lines K562 with an IC 50 of 4.5 and 1.5 μM, respectively (Metwaly et al. 2014) . Alternariol (4), altenusin (5) and altertoxin II (21) have also been isolated from the culture broth of endophytic Alternaria sp. EK10.4.5W existing in the seeds of Ziziphusjujuba. Alternariol, Altenusin and Altertoxin II have exhibited a potential cytotoxic activity against mouse lymphoma cell lines L5178Y with an IC 50 of 1.7, 6.8 and 6.2 μM which was comparable to the positive control kahalalide F having an IC 50 of 4.3 μM (Orfali et al. 2017) . Alternaria sp. R6, a mangrove plant endophytic fungus has been found to produce derivatives of resveratrol-resveratroaldehyde A (34), resveratroaldehyde B (35) and resveratroaldehyde C (36). Resveratroaldehyde A and C have exhibited an in vitro broad-spectrum inhibitory activity against three human cancer cell lines, MDA-MB-435, human liver Hep G2 and human colon HCT-116 by MTT assay with IC 50 <50 μM. However, resveratroaldehye A and resveratroaldehyde B exhibited potential activity against the cell lines MDA-MB-435 and HCT-116 with an IC 50 <10 μM (Wang et al. 2014a) . Tenuazonic acid (37) and Vivotoxin II (38) were for the first time reported from the endophytic fungus Alternaria sp. 28, from the bark of medicinal plant Ginkgo biloba. Both these compounds exhibited significant cytotoxic effect in the brine shrimp assay with mortality rate of 73.6% and 68.9%, respectively, at a concentration of 10 μg/mL (Quin et al. 2009 ).

One of the most sought out targets for drug development are enzymes. All the metabolic processes occurring in humans are driven by the catalytic power of the enzymes. They animate life. Despite the fact that enzymes are indispensable for existence of life, sometimes alterations in their expression levels or activity may lead to diseases.

A significant percentage of available oral therapeutic agents used clinically today inhibit enzymes. Thus, enzyme inhibitors form an important drug class (Copeland et al. 2007 ). Enzyme inhibitors from endophytic fungi are also being explored for developing new drugs. Xanthine oxidase (XO) is an important enzyme involved in metabolism of purines and converting them into uric acid which is eventually excreted in urine. However, hyperuricemia results when the uric acid concentration in blood serum goes beyond 6-7 mg/dL. More recently XO has also been implicated in oxidative stress during a stroke or a heart attack. Hence XO inhibitors are playing a critical role in combating oxidative stress and hyperuricemia (Kapoor and Saxena 2014) . Alternariol (4) from Alternaria brassisicola ML-P08 exhibits a potential XO inhibitory activity with an IC 50 of 15.5 μM (Gu 2009 ). Diabetes is a chronic metabolic disorder when the pancreas does not produce enough insulin, or the insulin is not effectively used in controlling the blood sugar level. There are several enzymes which convert the complex carbohydrates into glucose in our body during the process of digestion. Thus, these enzymes play a critical role in the input of glucose in blood. However, if these enzymes are inhibited, the breakdown of complex carbohydrates would drastically reduce and help in the management of blood glucose levels. Two α-glucosidase inhibiting compounds, prenylcephalochromin A (39) and prenylcephalochromin B (40), were isolated from Alternaria sp. ZG22, symbiotically residing in Dasymaschalon rostratum. Prenylcephalochromin A exhibited an IC 50 of 2.9 mM while for prenylcephalochromin B the value was 2.8 mM for α-glucosidase inhibition when compared to the positive control acarbose (a clinically approved drug) which exhibited an IC 50 value of 2.3 mM (Song et al. 2019a) . Another anti-diabetic agent reported was 2,4,6-triphenylaniline (41) which was isolated from Alternaria longipes strain VITN14G existing as a mutualistic symbiont in Avicenna officinalis, a mangrove plant. Both α-amylase and β-glucosidase inhibitory activities were exhibited by 2,4,6-triphenylaniline with an IC 50 value of 27.05 and 39.02 μg/mL, respectively (Ranganathan and Mahalingam 2019) . Hence these compounds hold a promise to be developed into anti-diabetic agents for blood-glucose-level management.

Similarly, for the treatment of Alzheimer's dementia (AD), acetylcholine esterase is a suitable enzyme target. Due to presence/overproduction of this enzyme, the acetylcholine levels are drastically reduced in AD. Thus, inhibiting this enzyme would help in restoring the acetylcholine levels in brain, hence acetylcholine esterase is a promising drug target (Guizior et al. 2015) . Aromatic polyketide dimer bialternacin E (42) has been isolated from Alternaria sp. NF2128 which exists an endophyte in Chinese medicinal plant Maianthemum bifolium and found to inhibit acetylcholine esterase with an IC 50 of 15.5 μM (Yang et al. 2019a ). Alternaria alternata existing as an endophyte in Catharanthus roseus has been found to produce altenuene (43) (Fig. 1.1) which exhibited a significant butyrylcholine esterase inhibitory activity and holds promise for therapeutic development for AD (Bhagat et al. 2016 ).

Endophytic Alternaria species have also been reported for other pharmacological actions such as anti-platelet/anti-thrombin activity and radical scavenging activity. Some endophytic isolates of Alternaria have been reported to produce high titres of known polyfunctional compounds such as resveratrol, swainsonine and taxol. Alternaria alternata MT-47, an endophyte of Huperzia serrata (Chinese club moss), produces four secondary metabolites, viz. alternatin D (44), alternariol (4), (2)-alternarlactam (45) and stemphyperylenol (46), which exhibit the potential to be developed into anti-platelet and anti-coagulants by inhibiting the release of ATP from thrombin-activated platelets (Yang et al. 2019b ). Alternaria sp. R6 was isolated as an endophyte from the root of Myoporum bontioides and produced two new compounds, viz. (AE)-(4R*,5S*,6S*)-3-amino-4,5,6-trihydroxy-2-methoxy-5methyl-2-cyclohexen-1-one (47) and (AE)-(4S*,5S*)-2,4,5-trihydroxy-3-methoxy-4-methoxycarbonyl-5-methyl-2-cyclopenten1-one (48). Both of these compounds exhibited a very potent ABTS [2,2 0 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] radical scavenging activity (Wang et al. 2014b ). More recently, altenusin has been identified as an agonist of farnesoid X receptor (FXR). FXRs are responsible for maintaining homeostasis of bile acids, glucose and lipids. Hence FXR agonists play an important role in treatment of non-alcoholic fatty liver disease (NAFLD). NAFLD is a chronic liver disease and is responsible for type 2 diabetes and obesity globally. The EC 50 values of altenusin to act as a novel agonist for treatment of NAFLD is 3.2 AE 0.2 μM. Hence, altenusin represents the first is class of non-steroidal FXR agonist with a promise to treat NAFLD and associated metabolic syndrome (Zheng et al. 2017 ). Alternaria oxytropis exists as an endophyte in Locoweed (Oxytropis lambertii) and produces Swainsonine (49) which already possesses multiple biological activities such as anti-viral, antibacterial and antitumour (Song et al. 2019b) . Similarly, Alternaria tenuissima TER995 existing as an endophyte in the bark of Terminalia arjuna was recognized as one of the highest Taxol (Paclitaxel) (50) (Fig. 1.1) producing strains, i.e. 124.32 μg/mL, reported till date (Ismaiel et al. 2017) . Alternaria sp. MG1, an endophyte of Vitis vinifera L. cv. Merlot, was found to produce resveratrol[3,5,4-trihydroxy stilbene] (51) (Shi et al. 2012) . Resveratrol is a novel molecule with multiple bioactivities involved in using human diseases with particular reference to cancer, cardiovascular diseases and ischaemic injuries (Saxena and Srivastava 2014) . 

Global agriculture is undergoing a renaissance with adoption of environmentally benign and sustainable technologies, directed towards drastic reduction in input of xenobiotics of pure synthetic origin. One of the major aspects of making the world food secure is through reduction in the pre-and post-harvest losses drastically apart from carrying out crop improvement programmes. Agrochemicals thus have played a significant role as pesticides comprising of herbicides, fungicides, insecticides, nematicides and rodenticides. However, their indiscriminate use has led to severe 1 Biologically Active Secondary Metabolites from Endophytic Alternaria Species ecological and health implications. This demands for alternatives which could be less toxic or benign to the chemical alternatives being used currently.

Plant-microbe interactions could possibly provide safer alternatives safer/benign chemicals as compared to synthetic chemical compounds. As endophyte co-evolve with their host and also help in combating with biotic and abiotic stressors, they could possibly be the sources of biorational compounds produced as a result of this interaction and possibly be developed into next generation of safer agrochemicals (Segaran and Sathiavelu 2019) . The members of the genus Alternaria have been responsible for pathogenesis of major economically important crops with their relationship ranging from a saprobe to an endophyte (Woudenberg et al. 2015) . Alternaria species produce secondary metabolites which can be classified as phytotoxins and mycotoxins while they are interacting with the host in the capacity of a pathogen. The phytotoxins produced by Alternaria sp. can be classified as hostspecific and non-host-specific (Meena and Samal 2019) . Many of these phyto/ mycotoxins have been developed into biorational agrochemicals (Saxena and Pandey 2001; Copping and Duke 2007; Saxena 2014) . However, exploiting the relationship of this genus, as an endophyte, is relatively a newer aspect but gaining threshold with the exploration of bioactivities of the signal molecules involved during the endophytic relationship.

Endophytic Alternaria J46 was isolated from the stem of medicinal plant Platycladus orientalis. The culture filtrate of Alternaria J46 exhibited a significant inhibition in seed germination of large crabgrass, bromegrass and barnyard grass. Further purification of the extract led to the isolation of tenuazonic acid (37), vivotoxin II (38) and cyclo(L-leucine-trans-4-hydroxy-L-proline) (52) (Fig. 1.1) . Tenuazonic acid holds a promise towards the development of a biobased herbicide for controlling weeds in cotton crop. In a pot trial tenuazonic acid exhibited an EC 50 between 119 and 795 μg/mL against 14 weeds but did not have any toxicity or damage to cotton (Gossypium hirsutum), Tobacco (Nicotianatabacum) and Asian Copperleaf (Acalyphaaustralis), even at a concentration of 1000 μg/mL. The field trial of tenuazonic acid controlled the two weeds, Amaranthusretroflexus and Digitariasanguinalis without affecting the Cotton (Hao et al. 2015) . Alternariol-9-methyl ether (1) which was isolated from endophytic Alternaria sp. Samif01 was also found to exhibit anti-nematode activity with IC 50 values of 98.17 and 74.62 μg/ mL, against Bursaphelenchus xylophilus and Caenorhabditis elegans respectively (Jin et al. 2017) . Plasmopara viticola Berl. et De Toni is one of the most destructive pathogens which causes grapevine downy mildew disease in the tropics. Antifungal compounds produced by endophytic fungi can be used as biorational pesticides replacing synthetic chemical-based fungicides (Musetti et al. 2007 ). Diketopiperazines are dipeptides which have been previously implicated with antifungal activity. Recently three diketopiperazines have been reported from an endophytic Alternaria alternata (Fr.) Keissel which reportedly have been found to control this fungal pathogen in green house trial. These promising compounds are cyclo-(L-phenylalanine-trans-4-hydroxy-L-proline) (53), cyclo(L-leucine-trans-4-hydroxy-L-proline) (53) and cyclo(L-alanine-trans-4-hydroxy-L-proline) (54) (Musetti et al. 2007 ). These further warrants field trials for their development into commercial biorational fungicide to prevent grapevine from the infection of Plasmopara viticola.

Another isolate of Alternaria alternata existing as an endophyte in Azadirchta indica A. Juss. was found to have insecticidal activity when its ethyl acetate extract was tested against Sodoptera litura, a phytophagous insect causing economic losses to crops globally (Kaur et al. 2013) . Similarly chloroform extract of Alternaria sp. Cas1, an endophytic isolate of Ricinus communis, also exhibits lethal effects on S. litura (Singh et al. 2012) . Endophytic Alternaria species are being explored for their anti-phyto pathogenic activity for possible use as plant antibiotics or biofungicide. Alternaria sp. FL25 exists as an endophyte in the leaves of Ficuscarica and produced fumitremorgin B (55), fumitremorgin C (56) and helvolicacid (57) which was isolated from the ethyl acetate extract of the culture filtrate. All the three compounds exhibited potential antifungal activity against the test panel of phytopathogenic fungi by in vitro microbroth dilution assay. Fumitremorgin B and C exhibited a potent fungicidal activity with an MIC in the range of 3.13-25 μg/mL against the test panel fungi, while helvolic acid exhibited a fungistatic activity with an MIC range of 1.56-12.5 μg/mL (Chengliang and Yangmin 2010) . Thus, now a trend has sent in where endophytic Alternaria species are being explored for bioactive compounds with applications in agriculture.

With an upsurge of global disease burden, comprising of new and emerging infections as well as metabolic disorders, there is an increasing demand for newer chemical scaffolds in the drug development pipeline. Studies of fungal endophytes in the last two decades have clearly proven that endophytic fungi serve as excellent biomatrices for novel and untapped chemical entities with potential for new drug development. The variable ecological relationship of these endophytic fungi also makes them amenable sources of a variety of agroactive agents which could replace the currently used harmful synthetic agrochemicals. Alternaria species has a wide range of occurrence in nature, i.e. from a saprobe, a facultative pathogen to an endophyte. Members of Alternaria genus existing as saprobes and as pathogens have been widely explored for their secondary metabolite which is recognized as mycotoxins and phytotoxins. These secondary metabolites could be possibly exploited directly as crop protection agents or could provide leads for developing safer agrochemicals. With increasing number of studies on endophytic fungal diversity, it is expected that newer isolates of Alternaria sp. having endophytic existence shall be identified which shall open up immense opportunities for their exploitation as sources of plant-based medicinals/phytochemicals as well as of novel chemical entities which would possess potential for pharmaceutical and agrochemical development.

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