Leishmanicidal, cytotoxic, antimicrobial and enzymatic activities of Diaporthe species, a mangrove-isolated endophytic fungus

This study evaluated the biotechnological potential of the endophytic fungus Diaporthe species, which is isolated from Brazilian mangroves. In vitro studies have investigated the antimicrobial activity of Diaporthe spp. against human pathogens, such as Escherichia coli , Staphylococcus aureus , Salmonella enteritidis , and Candida albicans , as well as phytopathogenic fungi, such as Colletotrichum species, Ceratocystis paradoxa , Fusarium oxysporum , Phythophthora sojae and Rhizopus microspores . Additionally, the enzyme production and leishmanicidal and cytotoxic activities of Diaporthe spp. were tested. Minimal inhibitory concentrations (MICs) of the crude extract of Diaporthe spp. 94 ranged from 756 to 949 µg.mL -1 (MIC 50 ) and from 3.094 to 7.082 μg.mL -1 (MIC90); the antagonism index (AI) ranged from 20 to 62% with type A interactions (“deadlock” with mycelial contact). An assay to detect enzyme production showed that this endophytic fungus produced the enzyme cellulase. The crude extract of Diaporthe spp. also demonstrated activity against the promastigote form of Leishmania infantum chagasi , exhibiting 90% cell death at 6.000 and 10.000 µg.mL -1 and 80% cell death at 4.000 µg.mL -1 . Cytotoxicity tests on cultured human skin fibroblasts (the HFF-1 cell line) indicated cell viability of between 73 and 55% at 4.000 to 10.000 μg.mL -1 at 24 h; between 69 and 56% at 4.000 to 8.000 μg.mL -1 for 48 h, and 59% at 4.000 to 6.000 μg.mL -1 for 72 h. analysis Data were submitted to the Shapiro-Wilk normality test and Levene’s test of homogeneity. Analysis of variance (ANOVA) and Tukey analysis were used to indicate the differences between the experimental groups. The values were considered to be significant when P-values ≤ 0.05 were obtained using GraphPad Prism software (Version 7.04 for Windows). Origin Pro version 9 software was employed to produce graphics and calculate the MIC 50 and MIC 90 (slope of linear regression) values.


INTRODUCTION
Mangroves are ecosystems located in transition areas between terrestrial and marine environments, which has forced them to adapt to such conditions as high salinity, tidal flooding, high temperatures, anaerobic soil, and large amounts of sludge. Thus, mangroves represent a useful ecosystem for identifying plants and microorganisms that have ecological, morphological, biological, and physiological adaptations and survive under these particular conditions (Jalgaonwala et al., 2011;Sebastianes et al., 2013). Endophytes are fungi or bacteria that colonize plants from the inside in symbiotic relationships, causing no visible harm to the host (Strobel, 2018). However, depending on external factors, such as environmental conditions and the host, endophytes may switch between pathogenic and commensal or mutualistic lifestyles (Bacon et al., 2008;Fesel and Zuccaro, 2016). Since the identification of the first endophytic fungus, significant attention has been devoted to the identification of new bioactive compounds that can be synthesized by them (Sebastianes et al., 2012;Sebastianes et al., 2013;Strobel, 2018). This research provides an alternative to plant exploitation, thereby facilitating the conservation of flora diversity, which is increasingly under threat worldwide (Zheng et al., 2015;Gurgel et al., 2020), as well as reducing the market value of these biomolecules (Strobel and Daisy, 2003;Sebastianes et al., 2012;Bibi et al., 2020). The broad spectrum of biological activity exhibited by endophytic fungal isolates enables, among other things, the exploration of substances with potential for antimicrobial and enzyme production, which can be achieved on a large scale through fermentation and presents considerable potential for industrial applications (Dezam et al., 2017;Marques et al., 2018;Yan et al., 2018;Gurgel et al., 2020). Several studies have described endophytic fungi with antimicrobial activities (Ratnaweera et al., 2015;Bezerra et al., 2015;Campos et al., 2015;Chen et al., 2016Chen et al., , 2019Khan et al., 2016;Deshmukh et al., 2020) and enzymatic activities in several plant species and mangroves from different regions (Bezerra et al., 2015;Martinho et al., 2019).
Secondary metabolites produced by endophytic fungi also have promising applications in phytopathogen control (Macías-Rubalcava and Sánchez-Fernández, 2017), as demonstrated by studies employing antagonism tests.
Endophytes may present activity against human parasites that are commonly related to neglected diseases that infect millions of people worldwide, especially in developing countries. Among these parasites, Leishmania infantum chagasi, the aetiological agent of visceral leishmaniasis, causes high morbidity and mortality (Maran et al., 2016).
The most common form of leishmaniasis is the cutaneous form. Cutaneous leishmaniasis is caused by several species of Leishmania, including Leishmania braziliensis and Leishmania amazonensis. However, this leishmaniasis is the least severe and lethal. A number of studies have demonstrated activities against this type of leishmaniasis (Santiago et al., 2011;Cota et al., 2018). To date, there have been no studies investigating the role played by related endophytic fungi from mangroves in producing secondary metabolites with leishmanicidal activity against L. infantum chagasi.
Fungi from marine environments grow under particular conditions that can contribute to the synthesis of new compounds (Sebastianes et al., 2012). In this context, endophytic fungi associated with plants that inhabit mangrove ecosystems may represent a promising source of secondary metabolites that have not been fully elucidated to date. The present study evaluated the in vitro antimicrobial, antagonistic and leishmanicidal activities, enzyme production, and cytotoxicity activities of secondary metabolites produced by the endophytic fungus Diaporthe species 94 strain isolated from Brazilian mangroves.
L. infantum chagasi (MHOM/BR/1972/LD), deposited in the collection of Inflammation and Infectious Diseases Laboratory (LIDI), Federal University of São Paulo, Brazil, and human skin fibroblasts from a cell line (HFF-1) deposited in the collection of the National Institute of Physics of Optics and Photonics Science and Technology, University of São Paulo, Brazil, were tested via cytotoxicity assays.

Culture conditions and obtaining crude extracts
To obtain crude extracts (CE), the Diaporthe spp. 94 strain was grown in Petri dishes containing medium potato dextrose agar (PDA) (KASVI TM , Brazil) for 7 days at 28°C. Eight discs (5 mm diameter) of mycelia from these cultures were taken from the plates and inoculated into 500-mL Erlenmeyer flasks containing 200 mL of potato dextrose liquid medium (PD) (KASVI TM , Brazil) followed by incubation for 7 days at 28°C in the absence of light and static conditions. The culture medium containing metabolites was separated from the mycelium by a 1-µm paper filter and subsequently utilized in biological assays.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License microplates. The inoculum was standardized at 0.5 on the scale of McFarland (1.5 × 10 8 ) (McFarland, 1907) and carried out a dilution of 1:10 in Mueller Hinton (MH) broth (KASVI TM , Brazil), obtaining a bacterial suspension of 1.0 × 10 7 colony forming units (CFU/mL). Serial dilutions of the CE were prepared in MH broth at concentrations of 10.000 to 1.000 μg.mL -1 . Penicillin and streptomycin (GIBCO, USA) were employed as antimicrobial agent standards at a concentration of 2.500 µg.mL -1 was added to 100 µL of bacterial cells adjusted to the concentration 0.5 on the McFarland scale and 100 µL of the crude extract of each concentration, obtaining a final volume of 200 µl per well. The microplates were incubated at 37°C for 24 h. After incubation, 20 µl of Alamar Blue® (Sigma-Aldrich, USA) was added to each well for a final concentration of 0.01%. Subsequently, the microplates were read at 570 nm in a fluorescence spectrophotometer (SPECTRAMAX M3).

Antagonism assays
A mycelial disc (5 mm in diameter) taken from a 7-day culture of the Diaporthe 94 (4) strain on PDA was placed at one edge of a 15-cm Petri dish containing PDA (KASVI TM , Brazil). On the same day, an inoculum of each phytopathogen to be tested (5-mm-diameter mycelial disc) was added to another edge of the plate at a distance of 2 cm from the margin. The plates were incubated at 28°C and observed for 10 consecutive days. Plates inoculated with phytopathogens alone were employed as controls. The tests were performed in triplicate, and the analysis of endophyticphytopathogen interactions was based on the scale of Badalyan et al. (2004) according to 3 types (A, B and C) and 4 subtypes (CA1, CA2, CB1, and CB2), where A means deadlock with mycelial contact, B means deadlock at a distance, C means replacement and overgrowth without initial deadlock, CA1 means partial and complete replacement after initial deadlock with mycelial contact, CA2 means complete replacement after initial deadlock, and CB1 and CB2 mean partial and complete replacement after initial deadlock at a distance, respectively. The antagonism index (AI) was calculated after 10 days of incubation using the formula of Campanile et al. (2007): AI = (RMrm) / RM × 100, where rm is the ray of the colony towards the antagonist, and RM is the average of the three rays of the colony in the other directions.

Enzymatic activity
The Diaporthe 94 (4) strain was evaluated for enzymatic activity using a qualitative method. The following enzymes were analysed: amylase (Buzzini and Martini, 2002), cellulase (Teather and Wood, 1982), protease (Castro et al., 2014), lipase and esterase (Sierra, 1957). For these tests, the endophytic strain was previously grown on a 15-cm Petri dish containing PDA at 28°C for 8 to 12 days, and discs of mycelium (5 mm in diameter) were inoculated onto the centre of plates containing specific medium for each enzyme. The plates were incubated at 28°C, and the enzymatic activity was observed based on the presence or absence of halos surrounding mycelial discs. The tests were performed in triplicate.

Leishmanicidal activity
L. infantum chagasi in its promastigote form was cultured in 25-cm 2 T25 flasks (KASVI, BRA) containing medium 199 (SIGMA-ALDRICH, USA) supplemented with foetal bovine serum (FBS) (LGC, BRA), pen/strep antibiotics (GIBCO, USA) and L-glutamine (SIGMA-ALDRICH, USA). In the assay, each well contained 100 µL of a cell suspension previously adjusted to 1 × 10 5 and 100 µL of the CE diluted into four different concentrations: 10.000, 8.000, 6.000 and 4.000 µg.mL -1 , with 200 µL being the final volume of each well. Amphotericin B (GIBCO, USA) was used as a leishmanicidal agent standard at a concentration of 0.1 mM. The plates were incubated at 23°C for 24 and 48 h. For the cell viability test, the colorimetric Alamar Blue® (Sigma-Aldrich, USA) was employed. After each incubation period, 1.5 mM Alamar Blue® was added and incubated in the incubator, with CO2 levels being maintained between 5 and 10%. Absorbance was read in a microplate reader (TP-READER, NM-THERMO PLATE) with a wavelength of 550 nm.
The absorbance was read in a microplate reader (Thermo Scientific®, USA) at a 570-nm wavelength.

Statistical analysis
Data were submitted to the Shapiro-Wilk normality test and Levene's test of homogeneity. Analysis of variance (ANOVA) and Tukey analysis were used to indicate the differences between the experimental groups. The values were considered to be significant when P-values ≤ 0.05 were obtained using GraphPad Prism software (Version 7.04 for Windows). Origin Pro version 9 software was employed to produce graphics and calculate the MIC50 and MIC90 (slope of linear regression) values.

Antimicrobial activity assays
The MIC 50 and MIC 90 were the minimum concentrations of CE required to inhibit 50 and 90%, respectively, of the pathogens tested. The data showed that CE was effective against the following pathogens: E. coli, MIC 50 = 767 µg.  (Table 1).

Antagonism assays
Diaporthe spp. 94 (4) strain inhibited the mycelial growth of Colletotrichum spp. by 33%, and the endophytephytopathogen interaction observed was of type CA1. For F. oxysporum, the mycelial inhibition was 50%, and the endophyte-phytopathogen interaction observed was of type CA1. For P. sojae, the mycelial inhibition was 20%, and the endophyte-phytopathogen interaction observed was of type A. Finally, for R. microsporus, the mycelia inhibition was 62%, and the endophyte-phytopathogen interaction observed was of type A. There was no activity against C. paradoxa. Type A interaction occurs when there is a deadlock with mycelial contact, and type CA1 interaction occurs when there is a partial replacement after the initial deadlock with mycelial contact (Table 2).

Enzymatic activity
Qualitative enzymatic assays showed that the endophytic Diaporthe spp. 94 (4) strain produced the enzyme cellulase. The assays did not indicate production of the enzymes amylase, protease, lipase or esterase.

Leishmanicidal assays
The CE produced by Diaporthe strain 94 (4) was tested against the promastigote forms of L. infantum chagasi in the periods of 24 and 48 h at 4.000, 6.000, 8.000 and 10.000 µg.mL -1 . In the 24-h period, the concentration of 4.000 µg.mL -1 had a mortality greater than 80%, and in the concentrations of 6.000, 8.000 and 10.000 µg.mL -1 , there was mortality of at least 95% of the cells. In the 48h period, the concentration of 4.000 µg.mL -1 had a mortality greater than 75%, and in the concentrations of 6.000, 8.000 and 10.000 µg.mL -1 , there was mortality of at least 95% of the cells. (Figure 1). In the 24 and 48-h periods, the concentration of 4.000 µg.mL -1 was significantly different from the concentrations of 6.000, 8.000 and 10.000 µg.mL -1 ; the concentrations of 6.000 and 8.000 µg.mL -1 were significantly different from the concentration of 10.000 µg.mL -1 .

Cytotoxicity
Assays for cytotoxicity showed cell viability above 60% at concentrations of 4.000 and 6.000 µg.mL -1 , above 50% at concentrations of 8.000 and 10.000 µg.mL -1 , and less than 10% at a concentration of 20.000 µg.mL -1 in the 24-h period. In the 48-h period, cell viability was above 60% at concentrations of 4.000 and 6.000 µg.mL -1 , above 50% at a concentration of 8.000 µg.mL -1 , above 40% at a concentration of 10.000 µg.mL -1 , and above 15% at a concentration of 20.000 µg.mL -1 . In the 72-h period, the cell viability was above 60% at a concentration of 4.000 µg.mL -1 , above 50% at a concentration of 6.000 µg.mL -1 and up to 20% at concentrations of 8.000, 10.000 and 20.000 µg.mL -1 (Figure 2). In the 24-h period, the concentrations of 4.000 and 6.000 µg.mL -1 were significantly different from the concentrations of 8.000, 10.000 and 20.000, and the concentration of 8.000 µg.mL -1 was significantly different from the concentration of 10.000 µg.mL -1 . In the 48-h period, the concentrations of 4.000, 6.000, 8.000 and 10.000 µg.mL -1 were significantly different from the concentration of 20.000, and the concentration of 8.000 µg.mL -1 was significantly different from the concentration of 10.000 µg.mL   concentrations of EB Diaporthe strain showed means above 60% in the concentrations of 4.000 and 6.000 µg.mL -1 the latter being different statistically from the concentrations of 8.000 and 10.000 µg.mL -1 . ANOVA followed by the Tukeys test were used to determine statistical significance (*p ≤ 0.05).
concentrations of 8.000, 10.000 and 20.000 µg.mL -1 were significantly different from each other.

DISCUSSION
Endophytic fungi isolated from mangrove ecosystems, including fungi belonging to the genus Diaporthe, have been described as rich sources of biologically active compounds with a variety of applications (Lin et al., 2005;Huang et al., 2008;Sebastianes et al., 2012;Brissow et al., 2017;de Medeiros et al., 2018;Huang et al., 2019), opening new avenues for biotechnological research. In this study, we evaluated the biotechnological potential of crude extract from Diaporthe spp. 94 isolates that were isolated from Brazilian mangroves for applications in the fields of medicine, agriculture and industry. Thus, evaluating the various effects of these compounds may enable researchers to identify an antimicrobial or antiparasitic effect to control different diseases.
The emergence of multidrug-resistant microorganisms has generated considerable attention and stimulated the search for new drugs. In this context, studies on the antimicrobial activity of endophytic mangrove-derived fungi against resistant human pathogens have been demonstrated (Prihanto et al., 2011;Sebastianes et al., 2012;Handayani et al., 2017;de Medeiros et al., 2018;Huang et al., 2019). Bezerra et al. (2015) tested 32 endophytic fungi isolated from Bauhinia forficata against 10 pathogenic bacteria in disc diffusion assays. Of these 32 isolates, 11 presented antibacterial activity against one or more bacteria among S. aureus (UFPEDA02), Streptococcus pyogenes (UFPEDA07), Mycobacterium smegmatis (UFPEDA71), Bacillus subtilis (UFPEDA86), Enterococcus faecalis (UFPEDA138), Salmonella Typhi (UFPEDA478), Pseudomonas aeruginosa (UFPEDA735), Enterobacter aerogenes (UFPEDA739), Proteus vulgaris (UFPEDA740), and E. coli (UFPEDA224). Among the bacteria tested in common with this present study are E. coli, where six endophytic fungi presented antimicrobial action, S. aureus and the genus Salmonella, where for each, an endophytic fungus showed activity. Therefore, our results show promise, since the dose exhibiting effectiveness against pathogenic bacteria is ten times lower than the dose showing cytotoxicity in mammalian cells. Thus, our data suggest that this compound could be evaluated in a future study seeking new biomolecules with promising activity for future antibiotics. Campos et al. (2015) isolated 82 endophytic fungi from the Caesalpinia echinata plant and tested their ethyl acetate extracts. Three extracts presented activity against E. coli and S. aureus, where MIC values varied between 32 and 64 µg/mL, and one presented activity against Salmonella bacteria, where MIC was 64 µg/mL. The MIC values were lower than those in the present study, probably due to the purification of the extract.
However, the fact that our compound has a ten-or twelve-fold greater effect on fibroblast cytotoxicity makes it a promising biomolecule for future studies attempting to achieve microbial control. Ratnaweera et al. (2015) isolated eight endophytic fungi of Opuntia dillenii. The eight strains showed antibacterial activity against at least one of the tested bacteria, including E. coli and S. aureus. Additionally, Khan et al. (2016) isolated the endophytic fungus Cladosporium species of Rauwolfia serpentina, and its extract exhibited antibacterial activity against E. coli and S. aureus among other bacteria. The endophytic Diaporthe presented promising antimicrobial activity against S. aureus, E. coli and S. enteritidis. When comparing our results with those of other authors, we must evaluate the antimicrobial effects and the dosedependent effect in killing the studied bacteria. Our data suggest that this activity is dose-dependent and may favour the regulation of the dose used, given the evaluated antimicrobial effect. We suggest that in future tests, after producing these compounds in greater purity, we can amplify this effect and achieve greater bacterial control. Endophytic fungi provide novel opportunities to control phytopathogens (Campanile et al., 2007). The antagonistic activity displayed by Diaporthe spp. 94 in our study could be explained by their secreting diffusible antifungal compounds into the culture medium, thereby inhibiting mycelial growth of Colletotrichum spp., F. oxysporum, P. sojae and R. microsporus. This inhibition could also be due to competing for space and/or nutrient depletion in the medium in the dual-culture assay. Competitive interactions of type A (deadlock with mycelial contact) and type CA1 (partial replacement after initial deadlock with mycelial contact), which were observed in the antagonistic assays, indicate different modes of action of Diaporthe spp. on phytopathogens, supporting our hypotheses regarding the possible mechanisms of control employed by endophytes.
Lytic enzymes secreted by endophytes, including cellulases, are another important mechanism by which plant-associated fungi control pathogens (Tripathi et al., 2008). These enzymes can suppress phytopathogens from breaking down cell walls, thereby restricting their growth (Gao et al., 2010). In this study, Diaporthe spp. 94 secreted cellulase into the culture medium. This potential for extracellular enzyme production by endophytic fungi from mangroves has also been described by other studies (Maria et al., 2005;Thatoi et al., 2013;Bezerra et al., 2015;Maroldi et al., 2018). These microorganisms naturally degrade lignocellulosic plant biomass of sediments in mangrove environments (Behera et al., 2017) and thus also suggest other applications, such as the processing of fruit juices in the textile, food and paper industries (Gao et al., 2008).
Leishmaniasis is a neglected tropical disease, and its prevalence in the poorest populations is not a major stimulus for the pharmaceutical industry. Thus, the search for new medicines to treat leishmaniasis has not been heavily funded. Therefore, contributing studies that may lead to the discovery of a compound or compounds with anti-leishmaniasis activity is of great importance for the control of these diseases. Similar works tested endophytic fungi isolated from plants from different Brazilian biomes, and they obtained leishmanicides that exhibited promise against other species of Leishmania that cause integumentary leishmaniasis, which is less deadly than visceral leishmaniasis (VL) (Marinho et al., 2005;Campos et al., 2008;Rosa et al., 2010;Brissow et al., 2017;Cota et al., 2018). Little is known about the anti-leishmaniasis activity of these isolates against the species that cause VL (Leishmania donovani and L. infantum chagasi). The results obtained for the control of L. chagasi demonstrate that the compound has leishmanicidal activity at a dose similar to the IC50 observed in fibroblasts. However, most of the drugs available and recommended to treat visceral leishmaniasis are highly toxic. Thus, in future research, we will evaluate in vivo whether the toxic effect is capable of compromising homeostasis in a murine model. Thus, we will be able to evaluate these data more globally. However, these results are promising for future studies seeking a new compound for the control of visceral leishmaniasis.
In Brazil, the aetiological agent of VL is L. infantum chagasi, our choice for the testing of inhibition. Our data demonstrated a high mortality rate at a dose of 4.000 µg.mL -1 in different periods of exposure (24 and 48 h) ( Figure 1). These data are promising compared with those found in the literature, where tests for less aggressive leishmaniasis were performed (Marinho et al., 2005;Campos et al., 2008;Santiago et al., 2011;Campos et al., 2015;Brissow et al., 2017).
The search for compounds with biological activity has great importance for research correlating new therapies for existing diseases. However, to continue the studies, it is of great relevance for these studies to demonstrate that for basal cells, the toxicity is considerably lower compared to the toxicity observed on the pathogenic microorganisms ( Figure 2). Thus, we evaluated fibroblast cytotoxicity, an important model to establish these parameters. Thus, our study leads us to new bioprospects from these compounds, since the cytotoxicity in fibroblasts was considerably lower than that found in the pathogens studied. Thus, we suggest that these biomolecules are promising for future studies in which we are searching for new compounds with biological activity.
This study demonstrated the biotechnological potential of the endophytic fungus Diaporthe spp. 94 isolated from Brazilian mangroves. We described for the first time the leishmanicidal activity of the secondary metabolites of this fungus on the promastigote forms of L. infantum chagasi and the cytotoxic effects of metabolites on HFF-1 cells. Our results may provide a new biological source of novel drugs.

Conclusion
The results of this study indicate that the tested CE from Diaporthe spp. 94 has antimicrobial action and antileishmaniasis activity at the two lower concentrations and has low toxicity at those same concentrations. These results suggest a that is fungus has strong biotechnological potential for future applications, as it is important to identify new drugs and antimicrobials because the rapid resistance that microorganisms have acquired is not compatible with new drug production. It is also important to develop new leishmanicidal drugs with lower cost and toxicity and greater effectiveness.