Endophytic Association: What, Why and How

Endophytic Association: What, Why, and How focuses on the endophytic association of plants, how they have originated inside the host, their importance, and how they are beneficial for the environment, as well as humans. The book discusses how using endophytic microbes in agricultural fields can be enriched without impacting environment negatively, and how they can be utilized for pharmaceutical purposes, including bioremediation. It includes advanced and up-to-date information, as well as future directions for young researchers and scientists who are working in the field of agriculture, pharmaceuticals, bio nanotechnology and bioremediation of environmental contaminants for environmental protection and sustainable development.

• Details the underlying mechanisms of endophyte-host association and their signaling mechanisms
• Describes numerous, successful field studies on the different applications of nanoparticles produced by endophytes (bio-nanotechnology) for sustainable development
• Presents recent advances and challenges in endophyte-associated bio-remediation research and applications for human health
• Provides information on bioactive compounds produced by endophytes for pharmaceutical purposes

• Language: English
• Publisher: Academic Press
• Release date: Nov 1, 2022
• ISBN: 9780323918282

Book preview

Chapter 1

Pathogens control using mangrove endophytic fungi

Rafael Dorighello Cadamuroa, Isabela Maria Agustini da Silveira Bastosa, Catielen Paula Paviab, Isabella Dai Práb, Doris Sobral Marques Souzaac, Mário Steindela, Izabella Thaís da Silvab, Helen Treicheld and Gislaine Fongaroa

aDepartament of Microbioloy, Imunology and Parasitology, UFSC, Federal University of Santa Catarina, Florianópolis, State of Santa Catarina, Brazil

bDepartment of Pharmaceutical Sciences, UFSC, Federal University of Santa Catarina, Florianópolis, State of Santa Catarina, Brazil

cDepartment of Food Science and Technology, UFSC, Food Technology and Bioprocess Research Group, Florianópolis, State of Santa Catarina, Brazil

dLaboratory of Microbiology and Bioprocess, Federal University of Fronteira Sul, Erechim, State of Rio Grande do Sul, Brazil

1.1 An introduction of mangrove and endophytic fungi and natural compounds studies

Natural selection theory's beauty evolves the dispute between organisms, leading to a better adaptation to the delicate balance between energy costs and appropriateness, resulting in higher survival chances. The interactions between organisms propitiate a chemical arms race, claiming a diversity of compounds to survive species in the competition (Cragg and Newman, 2005). The variety of compounds has been elucidated, connecting the interactions between organisms of an ecosystem (Firn and Jones, 2003). In this context, endophytic fungi have an advantageous interaction between plants and fungi established and developed. This relation offers to host nutrients and defensive compounds to help the maintenance (Dias et al., 2012). Fungi are ubiquitous on Earth, and it is estimated that around 1.5 million species, being one million of them endophytic species (Strobel and Daisy, 2003; Krings et al., 2007).

Thus, the mangrove can be considered one of the ecosystems threatened by anthropogenic activities. As an example of this action, we have the occupation, exploitation, and disposal of waste by humans in this environment and the silting up by carelessness in the use of surrounding soils (FAO, 2012). Therefore, the importance of preserving mangroves' high biodiversity is emphasized to maintain the coastal region's productivity; as such, they serve as habitats for several species of animals, plants, and microorganisms (Elturk et al., 2018), which are commonly found in these places. These conditions present low oxygen rates, high temperatures, variable salinity, anaerobic soils, and available carbon (Kodikara et al., 2018).

Mangroves belong to an ecosystem rich in woody plants located between land and sea in tropical or subtropical latitudes (Kathiresan and Bingham, 2001; Kodikara et al. 2018). These trees interact closely with carbon and nitrogen flow, making them the most productive plants in the ocean, with high enzymatic productivity and variety (Alongi, 2002).

The mangrove forest comprises shrubs and trees belonging to the families Rhizophoraceae, Acanthaceae, Lythraceae, Verbenaceae, Combretaceae, and Arecaceae (Rafferty, 2011). In general, in Brazil, mangrove can be classified within different characteristics of families, as red, white, and black mangrove, considering the species Rhizophora mangle (Rhizophoraceae) (Francisco et al., 2018), Laguncularia racemosa (Combretaceae) (Silva et al., 2010), and Avicennia schaueriana (Verbenaceae) (dos Santos et al., 2010), respectively.

The first studies related to mangroves date between 305 BC and 325 BC in the Red Sea and the Persian Gulf, describing the genus Rhizophora (Rejil and Joseph, 2012). These unique ecosystems cover around 15.2 million hectares worldwide (FAO, 2007) and spread across 112 tropical and subtropical countries and territories with about 65 plant species, distributed in 20 genera and 16 families in this ecosystem (Alongi, 2002). Mangrove distribution is crucial to understand the impact of the observed processes of global change and the extension of the mangrove forest (Thomas et al., 2018). In 1980, there were approximately 18 million hectares of mangrove areas worldwide, which was reduced because of climate change to about 3.6 million hectares in 2005, totaling a loss of 20% of the world's mangrove area. Asia presents itself the most significant loss (∼1.9 million hectares), followed by North and Central America (∼690 thousand hectares). This high decrease in mangrove areas has been associated with anthropogenic activity, such as deforestation and urbanization (FAO, 2007).

The term endophyte was applied for the first time by De Barry in 1866, reported later by Freeman (1904) about 1930–1990, counting with several isolations of endophytes' frovarious plants' grasses. The oldest register of endophytes dates from 400 million years ago (Krings et al., 2007). Throughout the transition between aquatic environments to terrestrial, all organisms were challenged with temperature variation of temperature, water disponibility, decrease drients on soil, and a high presence of carbon dioxide. Fungi were selected in these environments, resulting in the adaptation of organisms that allowed their development in this context, granting positive interactions with plants. Also, possible due to genetic variations that are allied to new interactions and production of secondary metabolites with plants.

Fungal endophytes enhance host resistance against abiotic stress, disease attack, insects, and mammalian herbivores by producing a wide range of fungal metabolites. Several metabolites of interest belonging to different chemical classes (alkaloids, steroids, flavonoids, terpenoids, quinones, and phenols) have already been isolated from endophytic fungi (Cagigal, 2016). Discoveries as penicillin in 1928, with Alexander Fleming from Penicillium and paclitaxel in 1993 from an endophytic fungus of the Pacific Yew plant, have drawn particular attention alternative sources of biologically active compounds (Cagigal, 2016). These endophytes derived from natural products also represent a great interest in modern medicine, drug discovery, and biotechnological industry, which has the aim to discover new bioactive molecules with potential application to therapeutic (Rey-Ladino et al., 2011; Chi et al., 2019; Mookherjee et al., 2020; Parthasarathy et al., 2020). The screening of bioactive compounds must consider ecological niches, which drastically influence metabolites produced by endophytes, enhancing the synthesis of molecules required in that context (Schulz et al., 2002). Challenges presented in the environment cooperate with the natural selection of new bioactive, making mangroves a strategic niche option. This intelligent strategy seeks to screen new bioactive compounds from endophytes (Schulz et al., 2002).

Screening of bioactive compounds possess benefits as (a) search for molecules with higher bioactivity against pathogens than synthetic drugs; (b) alternative compounds that cause fewer side effects and less generation of multidrug-resistance over pathogens, and (c) reduction of established synthetic drugs to decrease pollution generated by the presence of residues on soil and water (Gupta et al., 2020). The use of traditional medicine is not seen as new and has been used for many years. A study demonstrated that of a total of 23,000 compounds produced by microorganisms with anti-infectious activity, fungi produced 42%, 32% by filamentous bacteria (actinomycetes) (Demain, 2014).

This chapter aims to explore compounds and his application that elect mangroves as a unique niche to screen bioactive compounds from endophytic fungi, considering antiviral, bactericide, and antiparasitic activity.

1.2 Mangrove endophytic fungi and pathogen control

Among the applications of compounds isolated from endophytic fungi of mangrove plants, the potential application as alternatives to synthetic antibiotics stands out because of the biocontrol of pathogenic bacteria, viruses, and protozoa. Table 1.1 shows the primary host, endophytes, biocompounds, controlled and tested pathogens, and inhibitory concentrations.

Table 1.1

aMIC, minimum inhibitory concentration;

bIC50, 50% inhibitory concentration; NA, not assessable.

1.3 Bacteria control

The discovery of secondary metabolites' activity against pathogenic bacteria during the antibiotic era and forward benefited us by producing new drugs. Secondary metabolites originated from microbes and plants have granted quality and health time to society, reducing deaths and hospitalizations. Antibiotics, in general, are (a) natural products of microorganisms, (b) compounds semisynthetics produced from natural products, (c) structured chemically based on natural products (Pham et al., 2019). Our century also concerns the emergence of antibiotic resistance, influencing the selection of multidrug-resistant (MDR) pathogens (Khawbung et al., 2021). Endophytes were studied, aiming to discover bioactive compounds against pathogenic bacteria related in Table 1.1.

Coelomycetes have related studies showing bioactive compounds produced. One genus in this group highlighted by metabolites relevance was Pestalotiopsis. Strain Pestalotiopsis sp. HHL-101 was isolated from Rhizophora stylosa, a plant present in Dong Zhai Gang-Mangrove, on Hainan Island in China. This genus was a source of isolation of several bioactive compounds as coumarins, lactones, isocoumarin derivatives, chromonescytosporones, alkaloids, and terpenoids. The compounds isolated were pestalotiopisorin B (3S, 4R)-4, 8-dihydroxy-3, 4, 7-trimethylisochroman-1-one, with moderate activity against Escherichia coli and Pseudomonas aeruginosa, with respectively MIC (minimum inhibitory concentration) of 12.5 µg/mL and 50 µg/mL (Xu et al., 2020). Pestalotiopen A (C27H33ClO10) was obtained from endophyte Pestalotiopsis sp. Isolated from the leaves of Rhizophora mucronata, located in Dong Zhai Gang-Mangrove Garden, China. The compound hybrid sesquiterpene-cyclopaldic acid presented activity against pathogenic bacteria Enterococcus faecalis, showing a MIC between 125 and 250 µg/mL (Hemberger et al., 2013). Pestalotia sp. is an endophyte isolated from Heritiera fomes, a plant collected in the mangrove area of Sundarbans, Bangladesh. The compounds oxysporone (4H-furo(2,3-b)pyran-2 (3H)-one), and xylitol (five-carbon sugar alcohol) were obtained using mass spectroscopic analyses. Evaluation of activity against strains Staphylococcus aureus (MRSA) resistant presented a MIC 32–128 µg/mL (Nurunnabi et al., 2018).

The fungi Phomopsis sp. HNY29-2B was found in association with Acanthus ilicifolius, located on the South China Sea. Phomopsis is a genus related to the isolation of bioactive compounds. Known compounds were isolated, acropyrone and ampelanol, MIC values antibacterial activity of acropyrone ranged from 5.60 and 11.21 µg/mL to Bacillus subtilis and Staphylococcus aureus. Ampelanol presented MIC values of 8.50 µg/mL and 17.01 µg/mL against S. aureus and B. subtilis (Cai et al., 2017).

Ascomycetes possess a diversity of metabolites produced by endophytic fungi. Ai et al. (2014) isolated a fungi Guignardia sp. KcF8 from the fruit of tree Kandelia candel is used as a traditional medicine against rheumatoid and arthritis. Three compounds were identified with antibacterial activity using methods of disc essay, Guignardin B (C20H14O5), Guignardin C (C20H12O), and almarumycin BG1 (C20H14O5), being all evaluated against Staphylococcus aureus ATCC 29213. Inhibition zones were 7, 8, and 8 mm, respectively. Guignardin C was evaluated against Enterococcus faecalis ATCC 29212, showing 7 mm, and almarumycin BG1 against Aeromonas hydrophila ATCC 7966 with an inhibition zone of 12 mm. Penicillin was used as control positive with 12, 20, and 7 mm to each compound (Ai et al., 2014).

Pluchea indica is a plant located in Shankou Mangrove Nature Reserve in the mangrove area in Guangxi Province, China. Fungi Ascomycota sp. CYSK-4 was isolated and obtained two known compounds desmethyl-dichlorodiaportin and dichlorodiaportin, and a new isocoumarin dichlorodiaportintone. These compounds were evaluated against S. aureus, B. subtilis, Klebsiella pneumoniae, E. coli, and Acinetobacter calcoaceticus, which demonstrated a MIC value of 25–50 µg/mL (Chen et al., 2018). Two compounds were isolated from Stemphylium sp. 33231, an endophyte of Brguiera sexangula var. Rhynchopetala, collected in South China. The two compounds were a-pyrone derivatives, infectopyrones A and B. Infectopyrones A were evaluated against pathogenic bacteria Staphylococcus albus, E. coli, B. subtilis, Micrococcus tetragenus, and Micrococcus luteus with MIC values of 5.0, 2.5, 10.0, 10.0, 10.0 mg/mL, respectively. Compound B was less effective against the same bacteria's, showing MIC values 10.0, 2.5, 20.0, 10.0, and 10.0 µg/mL. The positive control used ciprofloxacin showing activity against S. albus, E. coli, B. subtilis, M. tetragenus, and M. luteus with values of 0.6, 0.3, 0.6, 0.3, and 0.3 µg/mL, respectively (Zhou et al., 2014).

Three compounds were isolated from endophyte Leptosphaerulina sp. SKS032, living in tissues of Excoecaria agallocha collected Guangxi province, China. Two new compounds, leptospyranonaphthazarin A and leptosnaphthoic acid A, and a known compound diaporthein B. The compounds isolated were evaluated against pathogenic bacteria S. aureus showing MIC values of 25.0, 50.0, and 50.0 µg/mL. The compound Leptosnaphthoic acid A demonstrated weak activity against B. subtilis and K. pneumoniae at a 100 µg/mL concentration. Diaporthein B presented weak activity against B. subtilis, at the same concentration of 100 µg/mL (Cui et al., 2017).

Hyphomycetes are related too as producers of compounds with bioactivity. The endophyte Aspergillus nidulans MA-143 was isolated from leaves of mangrove plant Rhizophora stylosa. A. nidulans was submitted to ethanol stress 0.1%. Compounds isolated where a new anthraquinone derivative isoversicolorin C and versicolorin C which performed bactericidal activity against E. coli, Micrococcus luteus, Vibrio vulnificus, Vibrio anguillarum, Vibrio alginolyticus, Edwardsiella ictaluri, and Vibrio parahaemolyticus with a considerable variation about MIC of compounds, ranging between 1 and 64 µg/mL (Yang et al., 2018). Isolation of endophyte Aspergillus flavipes AIL8 in association with Acanthus ilicifolius collected from Guangdong Province, China. Flavipesin A (C19H16NaO5) is an aromatic butyrolactones, which shows bioactivity against pathogenic bacteria S. aureus and B. subtilis with MIC values 8.0 and 0.25 µg/mL, respectively. The compounds presented the capability to degrade biofilm generated by S. aureus (Bai et al., 2014). Aspergillus sp. YHZ-1 was an endophyte isolated from a mangrove plant located in Hainan Island, China. Two compounds were isolated, Asperphenone A and B, which exhibited moderate activity against four-gram positive bacterias, S. aureus CMCC 26003, Streptococcus pyogenes ATCC19615, B. subtilis CICC 10283, and Micrococcus luteus. luteus with the MIC values range between 0.33 and 21.6 µg/mL (Guo et al., 2018). Aspergillus ochraceus MA-15 was isolated from root parts of mangrove plant Bruguiera gymnorrhiza, located on Hainan Island, China. The compounds obtained were new polyketides asperochrin A, along with known related derivatives, penicillic acid, (R)-7-hydroxymellein, chlorohydroaspyrones A and B, and chlorohydroasperlactones A. The compounds were evaluated against Vibrio harveyi, Vibrio anguillarum, and Aeromonas. hydrophilia with MIC values ranging between 0.5 and 64.0 µg/mL (Liu et al., 2015). These relate demonstrated the advances make about obtaining and evaluating compounds with bioactivity bactericide.

1.4 Viruses control

Contamination and hospitalization caused by viruses have been a concern since the knowledge of entities and pathogenic capabilities. The pandemia by a coronavirus that spread worldwide caused more than 2 million deaths and 98 million hospitalized (World Health Organization, 2020). Beyond that, contamination of environments as an aquatic system by SARS-CoV-2 and other viruses has been related in research, denoting the attention to methods of inactivation of viruses aiming to diminish the contaminations via environments (Bosch et al., 2008; Ihsanullah et al., 2020). The sum of it all stimulates the screening of new alternatives of bioactive compounds with activity virucide and antiviral, related to Table 1.1 (Wang et al., 2015).

The topic aims to increase the knowledge concerning the natural products isolated from endophytic fungi that showed promising antiviral activity against different viruses. Influenza A virus (IAV) is characterized by high morbidity and mortality, circulating in many other animal species. After the 2009 pandemic influenza H1N1 virus and due to the emergence of new drug-resistant viral strains, there is a need for antiviral agents that act by different mechanisms of action. Isolated polyketides of Diaporthe sp. SCSIO 41011, an endophytic fungus from the Rhizophora stylosa plant, was tested against three influenza A virus subtypes: A/Puerto Rico/8/34 H274Y (H1N1), A/FM-1/1/47 (H1N1), and A/Aichi/2/68 (H3N2) (Luo et al., 2018). Using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay, Madin Derby canine kidney (MDCK) cells were cultured and infected with different IAV subtypes. The compounds pestalotiopsone F, pestalotiopsone B, 3,8- dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate, and 5-chloroisorotiorin showed significant anti-IAV activities (IC50 values of 2.52 to 39.97 µM) as well low cytotoxicity. The authors observed that, for compounds pestalotiopsone F and B, the carboxyl group's ethylation at C-1 was essential for anti-IAV activities. Among the xanthones, the compound 3,8-dihydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylate indicated that the hydroxyl group at C-3 probably contribute to the anti-IAV activities.

Mangroves also promote a rich ecosystem for actinomycetes development that produces biologically active secondary metabolites. Jishengella endophytica 161111 had been isolated from the plant Xylocarpus granatum and produced alkaloids in a saline culture medium (Wang et al., 2014b). The compounds named perlolyrine, 1-hydroxy-β-carboline, lumichrome, and 1H-indole-3-carboxaldehyde, showed significant activity against H1N1 cytopathic effect (CPE) inhibition assay (IC50 values ranging from 25.0 to 45.9 µg/mL; SI = 3.0–16.1). The cytotoxic effects were evaluated on Madin-Daby canine kidney (MDCK) cells by MTT assay. The compounds exhibited low cytotoxicity against MDCK cells (CC50 = 116.3 ± 12.1 to 522.5 ± 24.5 µg/mL). The authors showed that the β-carboline alkaloids were active against the H1N1 virus. The unsubstituted H-3 has an important role in the anti-H1N1 activity; thus, the 1-hydroxy-β-carboline is a promising compound for anti-H1N1 drug development.

Trichoderma species are used as a biocontrol against plant diseases because they produce secondary metabolites. It is recognized that its bioactive compounds also have a role as antivirals. The fungus Trichoderma sp. Xy24 was isolated from the leaves, stem, and peels of the plant Xylocarpus granatum (Zhang et al., 2014). Three compounds were isolated and identified as the harzianone (a diterpene), trichoacorenol (sesquiterpene alcohol), and trichodimerol (a polyketide). The compound trichoacorenol exhibited moderate antiviral activity against influenza A subtype H7N9 (IC50 value of 74.6 µM and an inhibition rate of 31.7% at 10–5 mol/L).

From the root of the Aricennia marina, it was possible to isolate an endophytic fungus known as Neosartorya udagawae HDN13-313. After refined studies, four new indole alkaloids containing quinazoline were discovered, known as neosartoryadins A and B, fiscalins E and F, and a biogenetic compound related to fiscalin C. These compounds were then assessed for their antiviral activity against Influenza A (H1N1) by the CPE assay. As a result of this evaluation, the neosartoryadin A and B showed IC50 values of 66 µM and 58 µM respectively, being more active than Ribavirin (IC50 = 94 µM) which was used as a positive control (Yu et al., 2016).

Similarly, indole alkaloids derived from Cladosporium sp. PJX-41 were isolated from ethyl acetate (EtOAc) extracts of both the fermentation broth and mycelia of the fungus and resulted in a novel range of compounds (Peng et al., 2013). They were evaluated against H1N1 by CPE inhibition assay, and the compounds oxoglyantrypine, norquinadoline A, deoxynortryptoquivaline, deoxytryptoquivaline, tryptoquivaline, and quinadoline B exhibited antiviral activities (IC50 = 85, 82, 87, 85, 89, and 82 µM, respectively). All the other compounds were weakly active (IC50 > 100 µM). Frequently isolated from Aspergillus species, this class of alkaloids was isolated from a Cladosporium sp. and allowed the establishment of studies aiming to investigate their antiviral mechanisms to find new drugs against H1N1.

The mangrove rhizosphere soil is also a rich source of endophytic fungi. Gao et al. (2013) isolated the Aspergillus terreus Gwq-48 strain from a mangrove soil sample. They identified three butenolides, the new isoaspulvinone E, together with the well-known aspulvinone E and pulvic acid. Their biological activity was evaluated against H1N1 and showed significant anti-influenza activities (IC50 = 32.3, 56.9, and 29.1 µg/mL) when assessed by CPE inhibition assay and MTT methods. The compounds also exhibited low cytotoxicity (CC50 >250 µg/mL) on A549 and MDCK cell lines. Isoaspulvinone E was the only one with an effective inhibitory effect against the viral neuraminidase (NA), and this provides a new chemotype able to develop new drugs to treat H1N1.

The genus Emericella is one sexual state of Aspergillus and the Emericella sp. HK-ZJ was isolated from the inner bark of Aegiceras corniculatum (Zhang et al., 2011). Chemical studies on Emericella sp. several natural bioactive compounds have been described, such as alkaloids and terpenes, with different biological activities. Eight isoindolone derivatives were isolated from the ethyl acetate extract and tested against H1N1. Isoindolone 1 and 2 showed moderate inhibitory effects with IC50 values of 42.07 µg/mL and 62.05 µg/mL, respectively. They inhibited the replication of Influenza A virus H1N1 in MDCK cells by the CPE inhibition assay.

In addition to the vast content concerning the antiviral activity of compounds isolated from endophytic fungi against the Influenza A Virus (H1N1) virus, there are also studies dedicated to anti-HIV and antihepatitis activities. The mangrove-derived Penicillium sp. Its broad-spectrum antiviral activities were studied IMB17-046 by Li et al. (2019). The anti-HIV assay was performed using 293T cells, and the anti-influenza one assay used the cell-based high-throughput approach with 293T-Gluc cells. An antiviral assay against the hepatitis C (HCV) virus also was improved. Among the isolated compounds, the pyrazine derivative, trypilepyrazinol, and the ergostane analog, 3β-hydroxyergosta-8,14,24(28)-trien-7-one, exhibited broad-spectrum antiviral activities against the different types of viruses (IC50 = 0.5 to 7.7 µM). More specifically, trypilepyrazinol inhibited HIV-1 and HCV with IC50 values of 4.6 and 7.7 µM, respectively. Besides, 3β-hydroxyergosta-8,14,24(28)-trien-7-one also inhibited HIV-1 with an IC50 value of 3.5 µM and demonstrated potent activity against Influenza A with an IC50 value of 0.5 µM, even more potent than ribavirin (used as a positive control).

In a chemical investigation carried out by Hu et al. (2018), a fungal endophyte from the Thai Mangrove, Phomopsis sp. xy21. Six xanthones, called phomoxanthones F-K, were isolated from this endophyte. The phomoxanthones F, G, H, and K were evaluated to assess their anti-HIV potential at 20 µM. Still, only the phomoxanthone F demonstrated a weak inhibition rate (16.48 ± 6.67%) when compared with efavirenz (88.54 ± 0.45%) that was used as a positive control.

Another example was the endophytic fungi Acremonium sp. MERV1 and Chaetomium sp. MERV7 was obtained from A. marina, a plant collected in mangroves in Abu Ghoson, Egypt. In vitro test, performed on Murine MH1 cells, their antiviral activity was evaluated. As a result, it was possible to observe that all samples showed significant HCV inhibition, with values of HCV copy numbers inhibition of −82,483%, −82.4405%, and −97.3724% compared to control cells. The antiviral activity may be due to the samples' ability to inhibit viral replication and antiprotease activity (El-Gendy et al., 2014).

Panax notoginseng is a mangrove plant and medicinal herb with an association with innumerable fungi species, capable of protecting the plant. The fungus Penicillium sp. SYP-ZL1031 was isolated from the roots of P. notoginseng, and five new compounds were identified together with five well-known compounds (Xie et al., 2017). They were tested against Hepatitis C (HCV) genotype 1b and Hepatitis B (HBV) viruses. Two compounds, classified as polyketides, showed potent antiviral activity, named brefeldin A and brefeldin A 7-O-acetate, with ID50 values of 0.013 µM for HCV and 0.022 µM against HBV. The authors indicated that its inhibitory effect of protein translocation could explain a hypothesis of mechanism action from the endoplasmic reticulum to the Golgi complex. Also, the presence of a lactone in the two most potent compounds' structure may influence their antiviral activity.

Enteric viruses are responsible for high morbidity and mortality rates worldwide, especially in children, and are also the target of research with new bioactive compounds isolated from fungi. In 2014, Wang et al. Pestalotiopsis vacinii isolated (cgmcc3.9199) from a mangrove plant known as Kandelia candel (L.) Druce (Rhizophoraceae), popularly used to treat cases of rheumatoid arthritis. The fractionation of the bioactive extract guided by bioassay led to the isolation of eight new metabolites derived from polyketide, vaccines A–G and vaccine A. Tests were performed to evaluate the antiviral potential of these compounds, and only vaccine metabolite A showed antienterovirus 71 (EV71) activity with an IC50 value of 19.2 µM. Three years later, Wang et al. (2017) managed to isolate the same fungus, described in the previous study, obtained from the barks and leaves of Kandelia candel. In this new search, new metabolites were acquired, including vaccinol J, which was the first derivative of saliciloid and showed anti-EV71 activity in vitro, with an IC50 value of 30.7 µM and an inhibition effect higher than Ribavirin (used as a positive control) that showed an IC50 value of 177 µM. These studies evaluated different compounds isolated from other endophytes aiming for antiviral activity.

1.5 Parasites control

Diseases caused by parasites and considered endemic in low-income populations are commonly called neglected tropical diseases (NTDs). Protozoa cause the NTDs such as malaria, leishmaniasis, Chagas disease, and African human trypanosomiasis. Being caused by genera Plasmodium, Leishmania, and Trypanosoma, respectively, with significant morbidity and mortality, mainly in underdeveloped countries worldwide (Ogungbe et al., 2016).

The drugs used against these parasites require a prolonged treatment time, low efficacy in the chronic phase of the disease, high toxicity leading to more significant adverse side effects to patients, and an increase in parasite resistance due to adaptation to these therapeutic drugs. Given the current limited therapeutic arsenal, the epidemiological complexity of these diseases, and the absence of effective vaccines, the search for new active molecules against this parasitosis is imperative and urgent (Varikuti et al., 2018).

Fungi isolated from mangrove plants are rich sources of molecule bioactivity and are reported for their potential in treating different diseases. However, only limited studies have been reported on their antiprotozoal potential. Table 1.1 shows the relation of compounds with antiprozoan activity.

The 1,4-naphthoquinone compounds with naphthazarine structures (solaniol, javanicin, and dihydrojavanicin) were isolated from Fusarium sp. and dihydroisocoumarins (mellein, trans 4,8-dihydroxy-3-methylisochroman1-one, cis 4,8-dihydroxy-3-methylisochroman1-one and 5-hydroxymellein) were isolated from two endophytic fungi of the Avicennia lanata plant. The compounds were evaluated against the bloodstream form of Trypanosoma brucei brucei S427 (Mazlan et al., 2019). The fungal compounds' trypanosomal activity was evaluated by the Alamar blue TM microplate assay (96 wells). The naphthoquinone and dihydroisocoumarin derived compounds showed significant antitripanosomal (IC50 values of 0.047–0.545 µg/mL).

Another compound, oxylipine (9Z, 11E)-13-oxooctadec-9,11-dienoic acid, was isolated from Penicillium herquei, an endophyte Laguncularia racemosa, collected in the Western Region of Ghana. This compound was evaluated against the pathogenic protozoan Plasmodium falciparum 3d7, Trypanosoma brucei, Leishmania donovani, and Leishmania major with weak antiparasitic activity with IC50 values above 100 µM. The positive controls Artesunate, Coptis japonica, Amphotericin B showed activity against P. falciparum, T. brucei, L. donovani, and Leishmania major with IC50 values of 36 nm, 8.20, 0.32, and 0.31 µM, respectively (Hayibor et al., 2019).

1.6 Final considerations

This chapter highlighted the value of biocompounds in medicine addressed the potential of these substances to reduce viruses, bacteria, and parasites. Among biocompounds, endophytic fungi products may provide several substances with activity against pathogenic microorganisms. Some of these substances are due to adaptation that occurs when plants and endophytic fungi interact. Mangrove ecosystems are riches in this type of interaction relationship and have great potential for the search for new substances with antibiotic and antiparasitic effects. Mangrove endophytic fungi represent a valuable alternative collaborating with the screening of active compound for the development of new drugs.

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Chapter 2

Endophytic fungi-mediated synthesis of gold and silver nanoparticles

Khalida Bloch and Sougata Ghosh

Department of Microbiology, School of Science, RK University, Rajkot, Gujarat, India

1.1 Introduction

Nanotechnology-driven solutions have helped in energy generation, food preservation, prevention of disease, environmental monitoring, and numerous other spheres of life. The activity and applications of nanoparticles are largely dependent on the route of fabrication and the reaction conditions (Adersh et al., 2015). Various physical methods like chemical etching, laser ablation, milling, sputtering, and lithography are employed to synthesize gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs). Similarly, numerous chemical processes like vapor deposition, sol–gel, laser pyrolysis, condensation, and thermal deposition are also used for fabrication of tailor-made nanoparticles with specific shape and size (Bloch et al., 2021). However, these methods for synthesis of nanoparticles involve hazardous and corrosive chemicals which render the nanoparticles incompatible and toxic limiting their biomedical applications (Ghosh et al., 2016a). Hence alternative green methods for synthesis of nanoparticles are being continuously explored to ensure better stability and superior applications. Several bacteria, fungi, algae, and medicinal plants are reported to synthesize gold, silver, platinum, palladium, copper, and bimetallic nanoparticles (Ghosh et al., 2016b; Rokade et al., 2017, 2018; Jamdade et al., 2019).

Among various other nanoparticles, AuNPs and AgNPs are most noteworthy due to their wide range of applications that include catalysis, preventing antimicrobial, leishmanicidal, anticancer, drug delivery, sensing, and imaging (Ghosh et al., 2016c, 2016d; Shende et al., 2017, 2018; Ghosh, 2018; Bhagwat et al., 2018; Shinde et al., 2018). Additionally, AgNPs are also used in nanodevices, cosmetics, biosensors, and also as potent antimicrobial agent against multidrug-resistant pathogens (Ranpariya et al., 2021; Robkhob et al., 2020; Karmakar et al., 2020). Endophytes are the unexplored group of organisms which reside within the internal tissues of plants without harming the plants (Hemashekhar et al., 2019). Diverse groups of secondary metabolites, enzymes, alkaloids secreted by the microbial endophytes are reported to exhibit numerous therapeutic properties and industrial applications (Ghosh et al., 2020). Endophytic fungi are most suitable for the synthesis of nanoparticles as they form large biomass under high agitation and pressure in bioreactors (Naik, 2020). Mycogenic synthesis of nanoparticle is advantageous due to ease in scaling up and downstream processing. Fungi can synthesize intracellular as well as extracellular metal nanoparticles. In intracellular synthesis, the reduction process occurs in the lower side of the cell surface due to the positively charged group of enzymes present in the cell membrane. Nanoparticles get entrapped in the cell membrane and reducing sugars participate in the reduction of nanoparticles (Uzma et al., 2018). Extracellular synthesis of nanoparticles is carried out with the help of several metabolites. NADPH-dependent reductase is involved in the reduction of metal ions. Anthraquinones and NADPH-nitrate reductase efficiently reduce AgNPs. Similarly, for synthesis of AuNPs several fungal polypeptides and proteins play vital role in the reduction and stabilization process. In this chapter, the role of endophytic fungi for synthesis of various nanoparticles is discussed in detail.

1.2 Gold nanoparticles

Various endophytic fungi have the ability to produce AuNPs either extracellularly or intracellularly as listed in Table 2.1. Endophytic fungi Alternaria sp. isolated from the root of Rauvolfia tetraphylla was used for synthesis of gold nanoparticles (AuNPs). The collected roots were initially surface sterilized with mercuric chloride and was inoculated in potato dextrose agar (PDA) medium and was allowed to incubate for 7 days at 30°C. A 5 mL of endophytic extract was mixed with 50 mL of 5 mM of gold chloride solution at room temperature. After 30 min, ruby red color was observed which indicated the formation of AuNPs. The reaction mixture was centrifuged at 10,000 rom for 20 min and pellets were dried followed by further characterization. The UV–Vis spectra showed surface plasmon resonance at 530 nm while the energy dispersive X-ray spectroscopy confirmed the elemental Au peak at 3 keV. Transmission electron microscopy (TEM) exhibited that the size of the mycogenic AuNPs was about 28 nm with various shapes such as triangle, circular, and some with sporadic shape. Antibacterial activity of the AuNPs was checked against E. coli, P. aeruginosa, K. pneumoniae, and S. aureus. Maximum zone of inhibition was observed in P. aeruginosa. The bacterial growth kinetics was measured and the effect of AuNPs on growth curve of E. coli and P. aeruginosa was observed which showed alteration in both organisms. The AuNPs affected the cytoplasmic layer of E. coli and P. aeruginosa and caused the release the cellular materials. The AuNPs-mediated cell surface disintegration resulted in quick effluxes of K+ from E. coli and P. aeruginosa, hence influencing the cell permeability. Further antibiofilm activity against E. coli and P. aeruginosa indicated inhibition of biofilm formation at 1 mg/L and 5 mg/L concentration of AuNPs. The mycogenic AuNPs also showed high free radical scavenging activity (Hemashehkhar et al., 2019).

Table 2.1

AuNPs were synthesized from endophytic fungi Aspergillus clavatus isolated from the stem tissues of Azadirachta indica. The host plant was surface sterilized and placed on PDA plates followed by 20 days incubation at 25°C. The fungal strain was grown in 200 mL of MGYP medium (0.3% malt extract, 1% glucose, 0.3% yeast extract, 0.5% peptone, pH, 7.0) under shaking condition at 200 rpm for 8 days at 25°C. The fungal biomass was harvested by centrifugation at 5000 rpm for 20 min after fermentation. The mycelia were then washed thrice with sterile distilled water and both wet mycelia and culture extract were used for the synthesis of AuNPs. A 10 g of mycelial biomass was mixed with 100 mL of 1 mM HAuCl4 and the reaction mixture was kept on shaker at 200 rpm at room temperature that showed rapid color change from fresh white to dark purple indicating the formation of AuNPs. The reaction mixture characterized under UV–Vis spectra showed a peak at 540 nm which was attributed to the surface plasmon resonance (SPR) of the mycogenic AuNPs. The biogenic AuNPs were 20–35 nm in size with nanotriangles, spherical, and hexagonal shape. The atomic force spectroscopy (AFM) analysis revealed that maximum height of nanotriangles was 30 nm with thickness of 2–8 nm. Nanocrystalline nature was examined by X-ray diffraction (XRD) which exhibited four identical peaks at 44.5°, 65.6°, and 78.6° of elemental gold. It was observed that several secretary enzymes such as NADH-dependent reductase of microbes may be responsible for the reduction of metal ions to nanoparticles thereby facilitating their nucleation and growth (Verma et al., 2011).

Biosynthesis of AuNPs was done from endophytic fungi Cladosporium cladosporioides isolated from marine seaweed Sargassum wightii. The seaweed was surface sterilized and a sample of 1 cm was placed on PDA supplemented with chloramphenicol at 28°C. After the formation of mycelia, the fungus was subcultured on PDA media free from antibiotic and was incubated for 30 days at 28°C. The fungal biomass was collected and mixed with 1 mM HAuCl4 that resulted in the gradual color change from yellow to reddish violet indicating formation of AuNPs. The UV–Vis spectra showed a sharp narrow SPR peak at 540 nm. Complete reduction of the metal ions to AuNPs was observed with 2 h. The resulting AuNPs were 60 nm in size with monocrystalline face centered cubic nature. Fourier transform infrared (FTIR) spectroscopic analysis showed strong peak at 3307 cm−1 which indicated the presence of O–H group of phenolics and alcoholic molecules that might have played a vital role in bioreduction and stabilization. Atomic force microscopic (AFM) analysis showed a surface roughness of AuNPs of 26 nm. The average particle size of the AuNPs was found to be 30–60 nm by dynamic light scattering (DLS) analysis while the energy dispersive X-ray spectroscopy (EDX) showed the elemental Au peak at 1.6 and 2.2 keV. It was also concluded that NADPH-dependent enzymes participated and reduction of AuNPs. Antibacterial activity of AuNPs was carried out against Staphylococcus aureus (MTCC 7433) and Bacillus subtilis (MTCC 441). Highest zone of inhibition was observed against S. aureus. The biogenic AuNPs also showed moderate antioxidant activity (Manjunath et al.,